US20070048297A1 - Human secreted proteins - Google Patents

Abstract

The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating diseases, disorders, and/or conditions related to said human secreted proteins. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

Description

STATEMENT UNDER 37 C.F.R. §1.77(b)(4)
• This application refers to a “Sequence Listing” listed below, which is provided as an electronic document on two identical compact discs (CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each contain the file “PS950D1_SeqList.txt” (7,281 bytes, created on Mar. 20, 2002), which is hereby incorporated in its entirety herein. The Sequence Listing may be viewed on an IBM-PC machine running the MS-Windows operating system.
FIELD OF THE INVENTION
• The present invention relates to human secreted proteins/polypeptides, and isolated nucleic acid molecules encoding said proteins/polypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders related to said proteins/polypeptides (relatedness may be by direct or indirect association, by cause, by consequence, or by effect on said diseases and disorders). Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
BACKGROUND OF THE INVENTION
• Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals,” which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.
• One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.
• Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a “linker” holding the protein to the membrane.
• Thus there exists a clear need for identifying and using novel secreted polynucleotides and polypeptides. Identification and sequencing of human genes is a major goal of modern scientific research. For example, by identifying genes and determining their sequences, scientists have been able to make large quantities of valuable human “gene products.” These include human insulin, interferon, Factor VIII, tumor necrosis factor, human growth hormone, tissue plasminogen activator, and numerous other compounds. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis).
SUMMARY OF THE INVENTION
• The present invention relates to human secreted proteins/polypeptides, and isolated nucleic acid molecules encoding said proteins/polypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders related to said proteins/polypeptides (relatedness may be by direct or indirect association, or by cause, consequence, or effect on said diseases and disorders). Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.
DETAILED DESCRIPTION
• Polynucleotides and Polypeptides of the Invention
• Description of Table 1A
• Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC Deposit No: Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
• In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.
• SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein
• Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
• Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods
• The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
• Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.
• Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene
• Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
• The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
• Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
• The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCC Deposit No. Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCC deposit No. Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCC Deposit No. Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCC Deposit No. Z.
• Description of Table 1B (Comprised of Tables 1B.1 and 1B.2)
• Table 1B.1 and Table 1B.2 summarize some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifiers (SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column of Tables 1B.1 and 1B.2 provide the gene numbers in the application for each clone identifier. The second column of Tables 1B.1 and 1B.2 provide unique clone identifiers, “Clone ID:”, for cDNA clones related to each contig sequence disclosed in Table 1A and/or Table 1B. The third column of Tables 1B.1 and 1B.2 provide unique contig identifiers, “Contig ID:” for each of the contig sequences disclosed in these tables. The fourth column of Tables 1B.1 and 1B.2 provide the sequence identifiers, “SEQ ID NO:X”, for each of the contig sequences disclosed in Table 1A and/or 1B.
• Table 1B.1
• The fifth column of Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineates the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1B.1 as SEQ ID NO:Y (column 6). Column 7 of Table 1B.1 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y). Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wis.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1B.1 as “Predicted Epitopes”. In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1B.1. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8 of Table 1B.1 (“Cytologic Band”) provides the chromosomal location of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM identification number is disclosed in Table 1B.1, column 9 labeled “OMIM Disease Reference(s)”. A key to the OMIM reference identification numbers is provided in Table 5.
• Table 1B.2
• Column 5 of Table 1B.2, “Tissue Distribution” shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first code number shown in Table 1B.2 column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested. The second number in column 5 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of ³³P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.
• Description of Table 1C
• Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).
• Description of Table 1D
• Table 1D: In preferred embodiments, the present invention encompasses a method of detecting, preventing, treating, and/or ameliorating a disease or disorder listed as listed in the “Preferred Indications” column of Table 1D (below); comprising administering to a patient (in which such detection, prevention, treatment, and/or amelioration is desired) a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Table 1A and Table 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Table 1D) in an amount effective to detect, prevent, treat, or ameliorate the disease or disorder.
• As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to prevent, treat, or ameliorate the associated disease.
• The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of detecting, diagnosing, treating, preventing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, or ameliorate the disease or disorder. The first and seccond columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.
• In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.
• The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).
• In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.
• The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.
• The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.
• The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.
• The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypematremia, hypokalemia, and hyperkalemia.
• The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.
• The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).
• The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.
• The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.
• The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypemephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.
• The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.
• The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.
• Description of Table 1E
• Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention and indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating.
• Table 1E describes the use of FMAT technology, inter alia, for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound flurophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for highthroughput screening using flourometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).
• Table 1E also describes the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).
• Description of Table 1F
• Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.
• TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.
• To quantify gene expression the Taqman® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a Taqman® probe (distinct from the primers) during PCR. The Taqman® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Fold DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.
• After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.
• For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using Trizol extraction or by using the Ambion RNAqueous™-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using Taqman®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.
• Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).
• Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
• In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
• The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in Taqman® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.
• The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.
• The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).
• The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
• The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
• The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).
• Description of Table 1F
• Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.
• TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.
• To quantify gene expression the Taqman® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a Taqman® probe (distinct from the primers) during PCR. The Taqman® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Fold DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.
• After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.
• For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using Trizol extraction or by using the Ambion RNAqueous™-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using Taqman®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.
• Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).
• Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
• In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
• The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in Taqman® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.
• The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.
• The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).
• The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
• The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
• The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).
• Description of Table 2
• Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, “Clone ID:”, corresponding to a cDNA clone disclosed in Table 1A or 1B. The second column provides the unique contig identifier, “Contig ID:” corresponding to contigs in Table 1B and allowing for correlation with the information in Table 1B. The third column provides the sequence identifier, “SEQ ID NO:X”, for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”) as further described below. The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, “Score/Percent Identity”, provides a quality score or the percent identity, of the hit disclosed in columns five and six. Columns 8 and 9, “NT From” and “NT To” respectively, delineate the polynucleotides in “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
• Description of Table 3
• Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, “Clone ID”, for a cDNA clone related to contig sequences disclosed in Table 1B. The second column provides the sequence identifier, “SEQ ID NO:X”, for contig sequences disclosed in Table 1A and/or 1B. The third column provides the unique contig identifier, “Contig ID:”, for contigs disclosed in Table 1B. The fourth column provides a unique integer ‘a’ where ‘a’ is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth column provides a unique integer ‘b’ where ‘b’ is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a +14. For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be substituted into the general formula of a-b, and used to describe polynucleotides which may be preferably excluded from the invention. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, five, ten, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone).
• Description of Table 4
• Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B.2, Column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.
• Description of Table 5
• Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (Table 5, Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8, as determined using the Morbid Map database.
• Description of Table 6
• Table 6 summarizes some of the ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application. These deposits were made in addition to those described in the Table 1A.
• Description of Table 7
• Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers and vector information relating to these cDNA libraries.
• The first column shows the first four letters indicating the Library from which each library clone was derived. The second column indicates the catalogued tissue description for the corresponding libraries. The third column indicates the vector containing the corresponding clones. The fourth column shows the ATCC deposit designation for each library clone as indicated by the deposit information in Table 6.
• Definitions
• The following definitions are provided to facilitate understanding of certain terms used throughout this specification.
• In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
• In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
• As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof (e.g., the polypeptide delinated in columns fourteen and fifteen of Table 1A); a nucleic acid sequence contained in SEQ ID NO:X (as described in column 5 of Table 1A and/or column 3 of Table 1B) or the complement thereof; a cDNA sequence contained in Clone ID: (as described in column 2 of Table 1A and/or 1B and contained within a library deposited with the ATCC); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 (EXON From-To) of Table 1C or a fragment or variant thereof; or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).
• In the present invention, “SEQ ID NO:X” was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc. (HGS) in a catalogued and archived library. As shown, for example, in column 2 of Table 1B, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID:). Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library. Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID: to determine the library source by reference to Tables 6 and 7. Table 7 lists the deposited cDNA libraries by name and links each library to an ATCC Deposit. Library names contain four characters, for example, “HTWE.” The name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example “HTWEP07”. As mentioned below, Table 1A and/or 1B correlates the Clone ID names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one can use Tables 1A, 1B, 6, 7, and 9 to determine the corresponding Clone ID, which library it came from and which ATCC deposit the library is contained in. Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.
• In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 7 and 8 of Table 1A or the complement thereof, the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID: (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCC, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1C or the complement thereof. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.
• Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).
• Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
• The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
• In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• “SEQ ID NO:X” refers to a polynucleotide sequence described in column 5 of Table 1A, while “SEQ ID NO:Y” refers to a polypeptide sequence described in column 10 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 6 of Table 1A. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. The polynucleotide sequences are shown in the sequence listing immediately followed by all of the polypeptide sequences. Thus, a polypeptide sequence corresponding to polynucleotide sequence SEQ ID NO:2 is the first polypeptide sequence shown in the sequence listing. The second polypeptide sequence corresponds to the polynucleotide sequence shown as SEQ ID NO:3, and so on.
• The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
• “SEQ ID NO:X” refers to a polynucleotide sequence described, for example, in Tables 1A, 1B or 2, while “SEQ ID NO:Y” refers to a polypeptide sequence described in column 11 of Table 1A and or column 6 of Table 1B.1. SEQ ID NO:X is identified by an integer specified in column 4 of Table 1B.1. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. “Clone ID:” refers to a cDNA clone described in column 2 of Table 1A and/or 1B.
• “A polypeptide having functional activity” refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide for binding) to an anti-polypeptide antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.
• The polypeptides of the invention can be assayed for functional activity (e.g. biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay secreted polypeptides (including fragments and variants) of the invention for activity using assays as described in the examples section below.
• “A polypeptide having biological activity” refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
• Tables
• Table 1A
• Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC Deposit No: Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
• In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.
• SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein
• Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
• Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods
• The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.
• Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.
• Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene
• Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
• The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
• Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
• The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCC Deposit No. Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCC deposit No. Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCC Deposit No. Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCC Deposit No. Z.

  LENGTHY TABLE REFERENCED HERE
  US20070048297A1-20070301-T00001
  Please refer to the end of the specification for access instructions.

  
  Table 1B (Comprised of Tables 1B.1 and 1B.2)
• The first column in Table 1B.1 and Table 1B.2 provides the gene number in the application corresponding to the clone identifier. The second column in Table 1B.1 and Table 1B.2 provides a unique “Clone ID:” for the cDNA clone related to each contig sequence disclosed in Table 1B.1 and Table 1B.2. This clone ID references the cDNA clone which contains at least the 5′ most sequence of the assembled contig and at least a portion of SEQ ID NO:X as determined by directly sequencing the referenced clone. The referenced clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein. The third column in Table 1B.1 and Table 1B.2 provides a unique “Contig ID” identification for each contig sequence. The fourth column in Table 1B.1 and Table 1B.2 provides the “SEQ ID NO:” identifier for each of the contig polynucleotide sequences disclosed in Table 1B.
• Table 1B.1
• The fifth column in Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence “SEQ ID NO:X” that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1B.1, column 6, as SEQ ID NO:Y. Where the nucleotide position number “To” is lower than the nucleotide position number “From”, the preferred ORF is the reverse complement of the referenced polynucleotide sequence. The sixth column in Table 1B.1 provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by “ORF (From-To)”. Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. Column 7 in Table 1B.1 lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN (Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table 1B. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.
• Column 8 in Table 1B.1 provides a chromosomal map location for certain polynucleotides of the invention. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Each sequence in the UniGene database is assigned to a “cluster”; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often available for one or more sequence(s) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster.
• A modified version of the computer program BLASTN (Altshul, et al., J. Mol. Biol. 215:403-410 (1990), and Gish, and States, Nat. Genet. 3:266-272) (1993) was used to search the UniGene database for EST or cDNA sequences that contain exact or near-exact matches to a polynucleotide sequence of the invention (the ‘Query’). A sequence from the UniGene database (the ‘Subject’) was said to be an exact match if it contained a segment of 50 nucleotides in length such that 48 of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same UniGene cluster, and mapping data was available for this cluster, it is indicated in Table 1B under the heading “Cytologic Band”. Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.
• Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIM™ and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000. If the putative chromosomal location of a polynucleotide of the invention (Query sequence) was associated with a disease in the Morbid Map database, an OMIM reference identification number was noted in column 9, Table 1B.1, labelled “OMIM Disease Reference(s). Table 5 is a key to the OMIM reference identification numbers (column 1), and provides a description of the associated disease in Column 2.
• Table 1B.2
• Column 5, in Table 1B.2, provides an expression profile and library Code:Count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1B, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention. The first number in Table 1B.2, column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4. The second number in column 5 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of ³³P dCTP, using oligo (dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.

  LENGTHY TABLE REFERENCED HERE
  US20070048297A1-20070301-T00002
  Please refer to the end of the specification for access instructions.

  LENGTHY TABLE REFERENCED HERE
  US20070048297A1-20070301-T00003
  Please refer to the end of the specification for access instructions.

• Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof).

  LENGTHY TABLE REFERENCED HERE
  US20070048297A1-20070301-T00004
  Please refer to the end of the specification for access instructions.

• Tables 1D and 1E: The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.
• The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” columns of Table 1D and Table 1E; comprising administering to a patient (in which such treatment, prevention, or amelioration is desired) a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and seccond columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.
• In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D and Table 1E; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Colum 3 of Table 1D.
• The “Preferred Indications” columns of Table 1D and Table 1E describe diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The recitation of “Cancer” in the “Preferred Indications” columns indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).
• In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.
• The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.
• The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.
• The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.
• The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.
• The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.
• The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).
• The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.
• The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.
• The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypemephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.
• The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.
• The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.

  TABLE 1D
  Gene No.	cDNA Clone ID	Preferred Indication Identifiers

  1	H6BSF56	Cancer
  2	H6EDM64	Cancer
  3	H6EEC72	Cancer
  4	HACAB68	Connective/Epithelial,
  		Immune/Hematopoietic
  5	HACBJ56	Cancer
  6	HACBS22	Cancer
  7	HADDE71	Cancer
  8	HADDJ13	Connective/Epithelial
  9	HADMB15	Cancer
  10	HAGBQ12	Excretory,
  		Neural/Sensory
  11	HAGDW20	Neural/Sensory,
  		Reproductive
  12	HAGEG10	Cancer
  13	HAGEQ79	Digestive,
  		Neural/Sensory
  14	HAGFS57	Cancer
  15	HAGHN57	Cancer
  16	HAHEA15	Cardiovascular
  17	HAJAA47	Immune/Hematopoietic
  18	HAJAY92	Cancer
  19	HAJBV67	Cancer
  20	HAJCH70	Cancer
  21	HAOAG15	Cancer
  22	HAQAI92	Digestive,
  		Mixed Fetal,
  		Reproductive
  23	HAQCE11	Reproductive
  24	HATBI94	Cancer
  25	HATCB45	Cancer
  26	HATCD80	Endocrine,
  		Reproductive
  27	HATCI03	Endocrine,
  		Immune/Hematopoietic,
  		Neural/Sensory
  28	HATEH20	Cancer
  29	HBAGD86	Cancer
  30	HBCJL35	Cancer
  31	HBDAB91	Immune/Hematopoietic
  32	HBDAB91	Immune/Hematopoietic
  33	HBGBC29	Cancer
  34	HBGNC72	Cancer
  35	HBHAA05	Neural/Sensory
  36	HBHAA81	Cancer
  37	HBIAA59	Cancer
  38	HBIAC29	Cancer
  39	HBICW51	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  40	HBJAB02	Cancer
  41	HBJAC65	Cancer
  42	HBJBM12	Immune/Hematopoietic
  43	HBJCR46	Cancer
  44	HBJDS79	Cancer
  45	HBJDW56	Immune/Hematopoietic
  46	HBJEL16	Cancer
  47	HBJFK45	Immune/Hematopoietic
  48	HBJIG20	Cancer
  49	HBJKD16	Cancer
  50	HBMBM96	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  51	HBMBX01	Cancer
  52	HBMTM11	Cancer
  53	HBMTX26	Immune/Hematopoietic
  54	HBMTY48	Immune/Hematopoietic,
  		Reproductive
  55	HBMUH74	Cardiovascular,
  		Immune/Hematopoietic,
  		Reproductive
  56	HBMWE61	Immune/Hematopoietic
  57	HBNAX40	Cancer
  58	HBNBJ76	Cancer
  59	HBQAB79	Neural/Sensory
  60	HBQAC57	Neural/Sensory
  61	HBSAK32	Mixed Fetal,
  		Musculoskeletal,
  		Neural/Sensory
  62	HBXCM66	Cardiovascular,
  		Neural/Sensory,
  		Reproductive
  63	HBXCX15	Immune/Hematopoietic,
  		Neural/Sensory
  64	HCDCY76	Cancer
  65	HCDDL48	Musculoskeletal
  66	HCE1G78	Cancer
  67	HCE2H52	Cancer
  68	HCE3B04	Digestive,
  		Neural/Sensory
  69	HCE5F78	Immune/Hematopoietic,
  		Neural/Sensory
  70	HCEDR26	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  71	HCEEE79	Neural/Sensory
  72	HCEEQ25	Mixed Fetal,
  		Neural/Sensory
  73	HCEEU18	Neural/Sensory
  74	HCEFZ82	Cancer
  75	HCEGX05	Cancer
  76	HCFLN88	Cancer
  77	HCFLT90	Cancer
  78	HCHAB84	Cancer
  79	HCMSX51	Cancer
  80	HCNCO11	Digestive
  81	HCNSD29	Cardiovascular,
  		Digestive,
  		Immune/Hematopoietic
  82	HCQBH72	Digestive,
  		Excretory,
  		Immune/Hematopoietic
  83	HCQCC96	Cancer
  84	HCQCJ56	Cardiovascular,
  		Digestive,
  		Reproductive
  85	HCQCM24	Cancer
  86	HCRAY10	Cancer
  87	HCRBF72	Cancer
  88	HCRNF78	Cancer
  89	HCUAF85	Immune/Hematopoietic
  90	HCUCF89	Immune/Hematopoietic
  91	HCUCK44	Cancer
  92	HCUDD64	Cancer
  93	HCWAE64	Immune/Hematopoietic
  94	HCWFU39	Immune/Hematopoietic,
  		Neural/Sensory
  95	HCWUL09	Immune/Hematopoietic,
  		Neural/Sensory
  96	HDHAA42	Cancer
  97	HDHEB76	Mixed Fetal,
  		Neural/Sensory
  98	HDPCW16	Cancer
  99	HDPDI72	Immune/Hematopoietic
  100	HDPDJ58	Cancer
  101	HDPFF10	Cancer
  102	HDPFU43	Cancer
  103	HDPFY18	Cancer
  104	HDPGE24	Cancer
  105	HDPIU94	Cancer
  106	HDPOC24	Cancer
  107	HDPOL37	Immune/Hematopoietic,
  		Reproductive
  108	HDPOO76	Cancer
  109	HDPPD93	Cancer
  110	HDPPQ30	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Musculoskeletal
  111	HDPPW82	Immune/Hematopoietic
  112	HDPXN20	Immune/Hematopoietic
  113	HDQHM36	Immune/Hematopoietic
  114	HDTAU35	Immune/Hematopoietic
  115	HDTAV54	Cancer
  116	HDTFX18	Immune/Hematopoietic,
  		Reproductive
  117	HDTGW48	Immune/Hematopoietic,
  		Reproductive
  118	HDTLM18	Immune/Hematopoietic
  119	HE2CA60	Cancer
  120	HE2CA60	Cancer
  121	HE2CH58	Digestive,
  		Mixed Fetal
  122	HE2CM39	Cancer
  123	HE2HC60	Cancer
  124	HE2PO93	Cancer
  125	HE6AU52	Mixed Fetal
  126	HE6CS65	Cancer
  127	HE6DO92	Immune/Hematopoietic,
  		Mixed Fetal
  128	HE6EY13	Cancer
  129	HE6FU11	Mixed Fetal,
  		Neural/Sensory,
  		Respiratory
  130	HE6FV29	Cancer
  131	HE8FC45	Cancer
  132	HE8FC45	Cancer
  133	HE8FD92	Cancer
  134	HE8FD92	Cancer
  135	HE8FD92	Cancer
  136	HE8FD92	Cancer
  137	HE8FD92	Cancer
  138	HE8SG96	Mixed Fetal,
  		Neural/Sensory
  139	HE8TY46	Cancer
  140	HE9CY05	Mixed Fetal
  141	HE9EA10	Cancer
  142	HE9GG20	Cancer
  143	HEBCI18	Cancer
  144	HEBCY54	Cancer
  145	HEBDF77	Neural/Sensory
  146	HEBDQ91	Neural/Sensory
  147	HEBFR46	Cancer
  148	HEBGE07	Neural/Sensory
  149	HEGAU15	Excretory,
  		Immune/Hematopoietic,
  		Reproductive
  150	HELAT35	Cardiovascular,
  		Mixed Fetal
  151	HELBU54	Cardiovascular,
  		Digestive
  152	HELGG84	Cancer
  153	HELGG84	Cancer
  154	HEMEY47	Cancer
  155	HEOMC46	Immune/Hematopoietic
  156	HEPBA14	Reproductive
  157	HEQAH80	Cancer
  158	HEQBF89	Reproductive
  159	HETCI16	Cancer
  160	HETDW58	Cancer
  161	HETEY67	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Reproductive
  162	HFCDW95	Cancer
  163	HFCEI04	Neural/Sensory
  164	HFCFD04	Neural/Sensory
  165	HFCFE20	Cancer
  166	HFEAY59	Connective/Epithelial
  167	HFGAJ16	Cancer
  168	HFIHZ75	Cancer
  169	HFIJA29	Cancer
  170	HFIJA68	Musculoskeletal
  171	HFKES05	Cancer
  172	HFKEU12	Excretory
  173	HFPCZ55	Cancer
  174	HFPDR62	Immune/Hematopoietic,
  		Neural/Sensory
  175	HFPDS07	Cancer
  176	HFRAB10	Excretory,
  		Immune/Hematopoietic,
  		Neural/Sensory
  177	HFTBM38	Cancer
  178	HFTDH56	Cancer
  179	HFVGK35	Cancer
  180	HFVHW43	Digestive
  181	HFXAV37	Immune/Hematopoietic,
  		Neural/Sensory
  182	HFXBN86	Neural/Sensory
  183	HFXBT66	Neural/Sensory
  184	HFXFZ46	Neural/Sensory
  185	HGBER72	Cancer
  186	HGBEY14	Cancer
  187	HGBGN34	Cancer
  188	HGBHP91	Digestive
  189	HGCAC19	Cancer
  190	HGCAC19	Cancer
  191	HGCAC19	Cancer
  192	HHEAK45	Cancer
  193	HHEGS55	Immune/Hematopoietic
  194	HHEOW19	Cancer
  195	HHFFF87	Cancer
  196	HHFFL34	Cancer
  197	HHFFS40	Cancer
  198	HHGCS78	Immune/Hematopoietic
  199	HHGDT26	Immune/Hematopoietic,
  		Reproductive
  200	HHPFU28	Cancer
  201	HHPSA85	Cancer
  202	HHSBI06	Cancer
  203	HHSBI65	Cancer
  204	HHSDI53	Cancer
  205	HHSFC09	Cancer
  206	HHSGL28	Cancer
  207	HILCA24	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  208	HILCA24	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  209	HISAT67	Cancer
  210	HJBCU75	Cancer
  211	HJMAA03	Cancer
  212	HJMAV41	Cancer
  213	HJMAY90	Cancer
  214	HJPBE39	Cancer
  215	HJPBK28	Cancer
  216	HJPCH08	Cancer
  217	HKABU43	Cancer
  218	HKACI79	Cancer
  219	HKAFF50	Cancer
  220	HKGBF25	Cancer
  221	HKIXC44	Cancer
  222	HKMLK03	Digestive,
  		Excretory,
  		Immune/Hematopoietic
  223	HKMLM95	Cancer
  224	HKTAB41	Digestive,
  		Excretory
  225	HLDBG17	Cancer
  226	HLDCA54	Cancer
  227	HLDQU79	Cancer
  228	HLDRT09	Cancer
  229	HLHAP05	Immune/Hematopoietic,
  		Neural/Sensory,
  		Respiratory
  230	HLHCS23	Respiratory
  231	HLIBO72	Cancer
  232	HLICE88	Cancer
  233	HLICO10	Cancer
  234	HLJBS28	Cancer
  235	HLMBW89	Cancer
  236	HLMGP50	Digestive,
  		Immune/Hematopoietic
  237	HLMJB64	Cancer
  238	HLMMX62	Cancer
  239	HLQAS12	Cancer
  240	HLQCL64	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  241	HLQCX36	Digestive
  242	HLWAF06	Immune/Hematopoietic,
  		Reproductive
  243	HLWAU42	Cancer
  244	HLWAU42	Cancer
  245	HLWAV47	Cancer
  246	HLWBB73	Cancer
  247	HLWCN37	Cancer
  248	HLWDB73	Cancer
  249	HLYDF73	Immune/Hematopoietic
  250	HLYEU59	Immune/Hematopoietic
  251	HLYGB19	Cancer
  252	HLYGE16	Cancer
  253	HLYGY91	Cancer
  254	HMCAZ04	Cancer
  255	HMCAZ04	Cancer
  256	HMCAZ04	Cancer
  257	HMCAZ04	Cancer
  258	HMCAZ04	Cancer
  259	HMCFH60	Cancer
  260	HMDAB29	Digestive,
  		Neural/Sensory
  261	HMDAD44	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Neural/Sensory
  262	HMEBB82	Cancer
  263	HMEDE24	Cardiovascular
  264	HMEDI90	Cardiovascular,
  		Musculoskeletal,
  		Neural/Sensory
  265	HMELM75	Cancer
  266	HMIAK10	Neural/Sensory
  267	HMIBF07	Neural/Sensory
  268	HMICI80	Cardiovascular,
  		Endocrine,
  		Neural/Sensory
  269	HMICP65	Cancer
  270	HMJAK70	Neural/Sensory
  271	HMSBE04	Immune/Hematopoietic
  272	HMSCL38	Immune/Hematopoietic,
  		Neural/Sensory
  273	HMSCR69	Cancer
  274	HMSHC86	Immune/Hematopoietic
  275	HMSHU20	Immune/Hematopoietic,
  		Reproductive
  276	HMSHY25	Immune/Hematopoietic
  277	HMTAB77	Cancer
  278	HMUAE26	Cancer
  279	HMUAN45	Cancer
  280	HMVBC31	Cancer
  281	HMVDU15	Cancer
  282	HMWBL03	Cancer
  283	HMWJF53	Cancer
  284	HNEAK81	Immune/Hematopoietic
  285	HNECL22	Cancer
  286	HNECW49	Immune/Hematopoietic
  287	HNEDH88	Immune/Hematopoietic
  288	HNFAC50	Cancer
  289	HNFGR08	Immune/Hematopoietic
  290	HNFHF34	Cancer
  291	HNGAK51	Immune/Hematopoietic
  292	HNGAM58	Immune/Hematopoietic
  293	HNGBH53	Immune/Hematopoietic
  294	HNGDQ38	Immune/Hematopoietic
  295	HNGDX18	Cancer
  296	HNGDY34	Immune/Hematopoietic
  297	HNGEA34	Digestive,
  		Immune/Hematopoietic
  298	HNGEQ75	Immune/Hematopoietic,
  		Neural/Sensory
  299	HNGGA68	Immune/Hematopoietic,
  		Musculoskeletal
  300	HNGGP65	Immune/Hematopoietic
  301	HNGHZ69	Immune/Hematopoietic
  302	HNGIV64	Immune/Hematopoietic
  303	HNGJB41	Immune/Hematopoietic
  304	HNGKT41	Immune/Hematopoietic
  305	HNGMW45	Immune/Hematopoietic
  306	HNGNK44	Immune/Hematopoietic
  307	HNGNO53	Immune/Hematopoietic
  308	HNGPJ25	Immune/Hematopoietic,
  		Mixed Fetal,
  		Musculoskeletal
  309	HNHEN82	Cancer
  310	HNHFE71	Immune/Hematopoietic
  311	HNHGK22	Immune/Hematopoietic
  312	HNHHB10	Immune/Hematopoietic,
  		Reproductive
  313	HNHKS19	Immune/Hematopoietic,
  		Reproductive
  314	HNTBT17	Cancer
  315	HNTMH79	Cancer
  316	HOABP31	Cancer
  317	HOABP31	Cancer
  318	HOACG07	Cancer
  319	HODAG07	Reproductive
  320	HODBB70	Reproductive
  321	HODBV05	Cancer
  322	HODCZ32	Reproductive
  323	HOEBK60	Cancer
  324	HOFAA78	Cancer
  325	HOFNB74	Reproductive
  326	HOFNU55	Reproductive
  327	HOGBF01	Reproductive
  328	HORBS82	Cancer
  329	HORBV76	Immune/Hematopoietic,
  		Reproductive
  330	HOSDO75	Cancer
  331	HOSEC25	Musculoskeletal
  332	HOSEI81	Digestive,
  		Musculoskeletal
  333	HOSEJ94	Cancer
  334	HOUCA21	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Musculoskeletal
  335	HOUDE92	Cancer
  336	HOUDR07	Cancer
  337	HOUED72	Connective/Epithelial
  338	HOUFS04	Cancer
  339	HOUHI25	Cancer
  340	HOVBD85	Musculoskeletal,
  		Reproductive
  341	HPCAB41	Immune/Hematopoietic,
  		Reproductive
  342	HPCAL26	Cancer
  343	HPEAD23	Cancer
  344	HPFBA54	Reproductive
  345	HPFCI36	Cancer
  346	HPFDI37	Cancer
  347	HPIAA80	Cancer
  348	HPJBJ51	Immune/Hematopoietic,
  		Reproductive
  349	HPJBJ51	Immune/Hematopoietic,
  		Reproductive
  350	HPJBU43	Reproductive
  351	HPJCW58	Reproductive
  352	HPMBX22	Cancer
  353	HPMCJ84	Reproductive
  354	HPMCV30	Cancer
  355	HPMFH77	Cancer
  356	HPQAX38	Cardiovascular
  357	HPQAX38	Cardiovascular
  358	HPQCB83	Cancer
  359	HPQCC53	Cancer
  360	HPRBH85	Cancer
  361	HPRCA64	Cancer
  362	HPRCD35	Cancer
  363	HPTRM02	Cancer
  364	HPWBA29	Reproductive
  365	HPWDK06	Cancer
  366	HRAAD30	Cancer
  367	HRADA42	Cancer
  368	HRADF49	Cancer
  369	HRADN25	Cancer
  370	HRADT25	Digestive,
  		Excretory
  371	HRDAI17	Cancer
  372	HRDDQ39	Cancer
  373	HRDER22	Cancer
  374	HRDEX93	Cancer
  375	HRDFK37	Cancer
  376	HRGBD54	Cancer
  377	HROEA08	Cancer
  378	HSAVA08	Immune/Hematopoietic
  379	HSAVW42	Cancer
  380	HSAWN53	Immune/Hematopoietic
  381	HSAWZ40	Immune/Hematopoietic
  382	HSAYC41	Excretory,
  		Immune/Hematopoietic,
  		Reproductive
  383	HSDZM54	Neural/Sensory
  384	HSHBF76	Cancer
  385	HSIFG47	Digestive
  386	HSJBY32	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  387	HSKDR27	Cancer
  388	HSLHG78	Cancer
  389	HSLHX15	Musculoskeletal
  390	HSNAP85	Cancer
  391	HSNAZ09	Cancer
  392	HSNBM34	Cancer
  393	HSOAH16	Digestive
  394	HSQBF66	Cancer
  395	HSQDO85	Cancer
  396	HSQES57	Cancer
  397	HSRBE06	Cancer
  398	HSSDI26	Musculoskeletal
  399	HSSEA64	Cancer
  400	HSSEF77	Cancer
  401	HSSFE38	Cancer
  402	HSSGJ58	Musculoskeletal
  403	HSWBE76	Cancer
  404	HSXCP38	Cardiovascular,
  		Neural/Sensory
  405	HSYBI06	Cancer
  406	HT1SC27	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  407	HT3BF49	Immune/Hematopoietic
  408	HT4FV41	Cancer
  409	HT5FX79	Cancer
  410	HT5GR59	Cancer
  411	HTAEI78	Immune/Hematopoietic
  412	HTDAA78	Immune/Hematopoietic
  413	HTEAG62	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  414	HTECB02	Cancer
  415	HTECC15	Cancer
  416	HTEDF18	Reproductive
  417	HTEDJ28	Cancer
  418	HTEDS12	Cardiovascular,
  		Immune/Hematopoietic,
  		Reproductive
  419	HTEED26	Reproductive
  420	HTEED26	Reproductive
  421	HTEEF26	Cancer
  422	HTEEF26	Cancer
  423	HTEEW69	Reproductive
  424	HTEGS07	Reproductive
  425	HTEGS11	Cancer
  426	HTEHA56	Cancer
  427	HTEHU59	Cancer
  428	HTEJD29	Reproductive
  429	HTEKM46	Cancer
  430	HTEMQ17	Cancer
  431	HTENR63	Cancer
  432	HTGGM44	Cancer
  433	HTHBZ06	Cancer
  434	HTLAP64	Cancer
  435	HTLBT80	Cancer
  436	HTLDA84	Reproductive
  437	HTLDN29	Cancer
  438	HTLDU78	Reproductive
  439	HTLEC82	Cancer
  440	HTLEM16	Cancer
  441	HTLEV48	Reproductive
  442	HTLFA13	Musculoskeletal,
  		Reproductive
  443	HTLFI73	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  444	HTLGI89	Cancer
  445	HTLIF11	Cancer
  446	HTLIF12	Excretory,
  		Reproductive
  447	HTLIF12	Excretory,
  		Reproductive
  448	HTLIF12	Excretory,
  		Reproductive
  449	HTLIF12	Excretory,
  		Reproductive
  450	HTLIF12	Excretory,
  		Reproductive
  451	HTLIF12	Excretory,
  		Reproductive
  452	HTNAM63	Endocrine
  453	HTNBK13	Cancer
  454	HTOAI50	Digestive,
  		Immune/Hematopoietic
  455	HTOAM11	Immune/Hematopoietic,
  		Neural/Sensory
  456	HTODH57	Immune/Hematopoietic
  457	HTODH83	Immune/Hematopoietic
  458	HTOEV16	Cancer
  459	HTOGR38	Immune/Hematopoietic
  460	HTOHO21	Immune/Hematopoietic
  461	HTOHQ05	Immune/Hematopoietic
  462	HTOJL95	Cancer
  463	HTOJL95	Cancer
  464	HTPDU17	Cancer
  465	HTSFJ32	Immune/Hematopoietic
  466	HTTCB60	Cancer
  467	HTTEE41	Cancer
  468	HTTEZ02	Cancer
  469	HTWEH94	Immune/Hematopoietic
  470	HTXBD09	Cancer
  471	HTXDB22	Cancer
  472	HTXDC38	Cancer
  473	HTXDC77	Cancer
  474	HTXDD61	Cancer
  475	HTXDG92	Cancer
  476	HTXET11	Digestive,
  		Immune/Hematopoietic
  477	HTXFA72	Immune/Hematopoietic
  478	HTXJY08	Cancer
  479	HTXKF95	Cancer
  480	HTXMZ07	Cancer
  481	HUFCL31	Digestive,
  		Immune/Hematopoietic
  482	HUKBT67	Cancer
  483	HUKDF20	Cardiovascular,
  		Neural/Sensory,
  		Reproductive
  484	HUKDY82	Cancer
  485	HUSCJ14	Cancer
  486	HUSGL67	Cancer
  487	HUSGU40	Cancer
  488	HUSIR18	Cancer
  489	HUVDJ48	Digestive,
  		Reproductive
  490	HWAAI12	Cancer
  491	HWBBQ70	Immune/Hematopoietic,
  		Neural/Sensory
  492	HWBCN36	Immune/Hematopoietic
  493	HWBDJ08	Cancer
  494	HWBFX16	Immune/Hematopoietic
  495	HWDAC26	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Neural/Sensory
  496	HWDAG96	Cancer
  497	HWDAJ01	Connective/Epithelial
  498	HWHPB78	Cancer
  499	HYABC84	Cancer
  500	HYABC84	Cancer
  501	H2CBD20	Digestive
  502	H2CBH91	Cancer
  503	H2LBA54	Cancer
  504	H2LBB09	Cancer
  505	H2LBB09	Cancer
  506	H2MAC63	Digestive,
  		Reproductive
  507	H2MBA76	Cancer
  508	H2MBF60	Cancer
  509	H6BSM88	Cancer
  510	H6EEA48	Cancer
  511	H6EEN71	Cancer
  512	H6EEO05	Cancer
  513	H6EEU40	Cancer
  514	H7TDB54	Cancer
  515	H7TMB95	Cancer
  516	HAAAT06	Cancer
  517	HACAD42	Connective/Epithelial,
  		Mixed Fetal,
  		Neural/Sensory
  518	HACBJ11	Cancer
  519	HACBS86	Cancer
  520	HACBT91	Cancer
  521	HACBZ73	Cancer
  522	HACCK29	Connective/Epithelial
  523	HADAB60	Cancer
  524	HADAM31	Connective/Epithelial
  525	HADCL19	Connective/Epithelial
  526	HADCZ65	Connective/Epithelial,
  		Immune/Hematopoietic
  527	HADDC04	Connective/Epithelial,
  		Reproductive
  528	HADDP23	Cancer
  529	HADDP51	Cancer
  530	HADDR24	Cancer
  531	HADET62	Connective/Epithelial
  532	HADEY08	Cancer
  533	HADEY22	Connective/Epithelial
  534	HADEY22	Connective/Epithelial
  535	HADFB84	Cancer
  536	HADFD01	Cancer
  537	HADFD10	Cancer
  538	HADFK11	Connective/Epithelial
  539	HADFT44	Connective/Epithelial,
  		Mixed Fetal,
  		Neural/Sensory
  540	HADFW20	Connective/Epithelial
  541	HADFX10	Connective/Epithelial,
  		Neural/Sensory
  542	HADFY80	Connective/Epithelial,
  		Digestive
  543	HADGD93	Cardiovascular,
  		Connective/Epithelial
  544	HADMA77	Cancer
  545	HADXA10	Cancer
  546	HADXA10	Cancer
  547	HAFBB15	Cancer
  548	HAFBL14	Cancer
  549	HAGAB62	Cancer
  550	HAGAB83	Neural/Sensory
  551	HAGAE84	Neural/Sensory
  552	HAGAF75	Digestive,
  		Neural/Sensory
  553	HAGAK40	Cancer
  554	HAGAU43	Neural/Sensory
  555	HAGAZ36	Neural/Sensory
  556	HAGBC57	Cancer
  557	HAGBL31	Neural/Sensory
  558	HAGBO09	Mixed Fetal,
  		Neural/Sensory
  559	HAGBO12	Neural/Sensory
  560	HAGBO51	Neural/Sensory
  561	HAGBS89	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  562	HAGBV06	Cancer
  563	HAGBV25	Cancer
  564	HAGBV29	Immune/Hematopoietic,
  		Neural/Sensory
  565	HAGCC87	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  566	HAGCH67	Neural/Sensory
  567	HAGCI69	Neural/Sensory,
  		Reproductive
  568	HAGCT33	Immune/Hematopoietic,
  		Mixed Fetal,
  		Neural/Sensory
  569	HAGCZ70	Neural/Sensory
  570	HAGDC73	Cancer
  571	HAGDG84	Immune/Hematopoietic,
  		Neural/Sensory
  572	HAGDH85	Neural/Sensory
  573	HAGDI69	Neural/Sensory
  574	HAGDJ53	Immune/Hematopoietic,
  		Neural/Sensory
  575	HAGDJ56	Cardiovascular,
  		Endocrine,
  		Neural/Sensory
  576	HAGDL51	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  577	HAGDO70	Cancer
  578	HAGDT30	Cancer
  579	HAGDW68	Endocrine,
  		Neural/Sensory
  580	HAGDX84	Cancer
  581	HAGEK37	Cancer
  582	HAGEK86	Cancer
  583	HAGEP30	Neural/Sensory
  584	HAGEQ58	Neural/Sensory
  585	HAGEQ67	Cancer
  586	HAGEU26	Neural/Sensory
  587	HAGEW83	Neural/Sensory
  588	HAGEX49	Cancer
  589	HAGEX49	Cancer
  590	HAGFD75	Cancer
  591	HAGFF43	Cancer
  592	HAGFJ67	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  593	HAGFM58	Cardiovascular,
  		Neural/Sensory
  594	HAGFT48	Cancer
  595	HAGFU31	Neural/Sensory
  596	HAGFW13	Neural/Sensory
  597	HAGHE85	Cardiovascular,
  		Neural/Sensory
  598	HAGHR18	Neural/Sensory
  599	HAGIB90	Cancer
  600	HAHEM51	Cardiovascular
  601	HAHSA76	Cardiovascular
  602	HAHSD51	Cancer
  603	HAIBR76	Cancer
  604	HAIBT20	Cancer
  605	HAIBV91	Cancer
  606	HAICE62	Cancer
  607	HAICL90	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  608	HAICV44	Cancer
  609	HAIDP45	Cancer
  610	HAJAB88	Cancer
  611	HAJAZ56	Cancer
  612	HAMFC67	Cancer
  613	HAMFQ38	Cancer
  614	HAMGG01	Cancer
  615	HANGB24	Cancer
  616	HANKC93	Musculoskeletal
  617	HAPAD35	Cancer
  618	HAPBR13	Cancer
  619	HAPBU09	Cancer
  620	HAPBU86	Cancer
  621	HAPBU86	Cancer
  622	HAPNJ33	Cancer
  623	HAPNL62	Cancer
  624	HAPNO50	Cancer
  625	HAPNY10	Cancer
  626	HAPPW83	Cancer
  627	HAPQJ73	Cancer
  628	HAPQK26	Reproductive
  629	HAPQU71	Cancer
  630	HAPQU71	Cancer
  631	HAPQW18	Cancer
  632	HAPQX44	Cancer
  633	HAPRK55	Cancer
  634	HAPSH37	Cancer
  635	HAQBG57	Cancer
  636	HAQBY85	Cancer
  637	HAQBZ15	Cancer
  638	HAQCE18	Immune/Hematopoietic,
  		Reproductive
  639	HAQCF94	Cancer
  640	HARAE26	Neural/Sensory
  641	HARAT69	Cancer
  642	HARAZ81	Cancer
  643	HASAU26	Cancer
  644	HASAX57	Cancer
  645	HASAY07	Cancer
  646	HATAE01	Cancer
  647	HATAG52	Endocrine,
  		Neural/Sensory,
  		Reproductive
  648	HATAL05	Cancer
  649	HATBA90	Endocrine
  650	HATBM71	Endocrine
  651	HATCF80	Cancer
  652	HATCI67	Cancer
  653	HATCJ27	Cancer
  654	HATCS79	Endocrine,
  		Immune/Hematopoietic
  655	HATCX03	Cancer
  656	HATDE03	Cancer
  657	HATDF41	Cancer
  658	HATDH23	Cancer
  659	HATDH55	Cancer
  660	HATDO84	Endocrine
  661	HATDU01	Cancer
  662	HATDW05	Endocrine
  663	HATEF13	Digestive,
  		Endocrine
  664	HATEF64	Cancer
  665	HATEH40	Cancer
  666	HATEI22	Cancer
  667	HAUCC84	Cancer
  668	HAWAS41	Connective/Epithelial,
  		Excretory,
  		Immune/Hematopoietic
  669	HAWBA65	Cancer
  670	HBAGH64	Cancer
  671	HBAGV01	Connective/Epithelial,
  		Excretory
  672	HBAMC50	Excretory
  673	HBAMC57	Excretory
  674	HBBBA42	Cancer
  675	HBBBB08	Neural/Sensory
  676	HBBBE83	Cancer
  677	HBBMA11	Neural/Sensory
  678	HBCAK10	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  679	HBCAK80	Cancer
  680	HBCAQ48	Cancer
  681	HBCAY17	Cancer
  682	HBCGE46	Musculoskeletal
  683	HBGBA14	Cancer
  684	HBGBE75	Cancer
  685	HBGBP22	Cancer
  686	HBGFQ34	Reproductive
  687	HBGML95	Reproductive
  688	HBGMT60	Cancer
  689	HBHAA53	Neural/Sensory
  690	HBIAU43	Cancer
  691	HBIAW58	Neural/Sensory
  692	HBIBB20	Cancer
  693	HBIBF26	Cancer
  694	HBIBM33	Neural/Sensory
  695	HBIBN67	Cancer
  696	HBIBQ69	Immune/Hematopoietic,
  		Neural/Sensory
  697	HBIBR38	Neural/Sensory
  698	HBIBR61	Cancer
  699	HBIBS33	Neural/Sensory
  700	HBIBT13	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  701	HBIBZ20	Neural/Sensory
  702	HBICB80	Cancer
  703	HBJAC40	Cancer
  704	HBJAV56	Immune/Hematopoietic,
  		Musculoskeletal
  705	HBJAY14	Immune/Hematopoietic
  706	HBJBQ69	Immune/Hematopoietic
  707	HBJBR40	Immune/Hematopoietic
  708	HBJCH46	Immune/Hematopoietic,
  		Musculoskeletal
  709	HBJCR17	Cancer
  710	HBJCS26	Cancer
  711	HBJCW24	Cancer
  712	HBJDC57	Immune/Hematopoietic,
  		Reproductive
  713	HBJDR18	Immune/Hematopoietic
  714	HBJDR83	Immune/Hematopoietic
  715	HBJEE51	Immune/Hematopoietic
  716	HBJEL21	Cancer
  717	HBJFH84	Cancer
  718	HBJFJ14	Cancer
  719	HBJFJ26	Cancer
  720	HBJFJ83	Immune/Hematopoietic,
  		Mixed Fetal
  721	HBJFJ83	Immune/Hematopoietic,
  		Mixed Fetal
  722	HBJFP47	Immune/Hematopoietic,
  		Reproductive
  723	HBJFR77	Cancer
  724	HBJFU30	Cancer
  725	HBJFX41	Immune/Hematopoietic
  726	HBJHO83	Immune/Hematopoietic,
  		Reproductive
  727	HBJHS92	Immune/Hematopoietic,
  		Neural/Sensory
  728	HBJHT01	Immune/Hematopoietic,
  		Reproductive
  729	HBJHT01	Immune/Hematopoietic,
  		Reproductive
  730	HBJHW06	Immune/Hematopoietic,
  		Reproductive
  731	HBJIR14	Cancer
  732	HBJJA26	Immune/Hematopoietic
  733	HBJJX02	Immune/Hematopoietic
  734	HBJLH78	Immune/Hematopoietic
  735	HBJND04	Cancer
  736	HBJND57	Immune/Hematopoietic
  737	HBKDF66	Cancer
  738	HBKEA94	Cancer
  739	HBKEE60	Digestive
  740	HBKEI41	Endocrine,
  		Mixed Fetal,
  		Reproductive
  741	HBMBD51	Digestive,
  		Immune/Hematopoietic
  742	HBMBD73	Cancer
  743	HBMBE33	Immune/Hematopoietic
  744	HBMBM17	Immune/Hematopoietic,
  		Reproductive
  745	HBMCL59	Immune/Hematopoietic
  746	HBMCM96	Immune/Hematopoietic,
  		Neural/Sensory
  747	HBMCQ74	Cancer
  748	HBMCQ74	Cancer
  749	HBMCT40	Cancer
  750	HBMDM08	Immune/Hematopoietic
  751	HBMSN62	Cancer
  752	HBMSO30	Immune/Hematopoietic
  753	HBMTM50	Cancer
  754	HBMUD59	Cancer
  755	HBMUI10	Cancer
  756	HBMUJ48	Cancer
  757	HBMUR39	Immune/Hematopoietic
  758	HBMVF65	Endocrine,
  		Immune/Hematopoietic,
  		Neural/Sensory
  759	HBMVF65	Endocrine,
  		Immune/Hematopoietic,
  		Neural/Sensory
  760	HBMWC39	Cancer
  761	HBMWJ92	Cancer
  762	HBMWS52	Immune/Hematopoietic
  763	HBMXE34	Cancer
  764	HBMXG01	Immune/Hematopoietic
  765	HBMXG76	Immune/Hematopoietic
  766	HBMXM05	Excretory,
  		Immune/Hematopoietic,
  		Neural/Sensory
  767	HBMXW83	Cancer
  768	HBNAE74	Excretory,
  		Musculoskeletal,
  		Reproductive
  769	HBNAX16	Cancer
  770	HBNAZ35	Endocrine,
  		Reproductive
  771	HBODK40	Cancer
  772	HBODV76	Cancer
  773	HBPAD89	Cancer
  774	HBPAF39	Immune/Hematopoietic,
  		Neural/Sensory
  775	HBQAC45	Neural/Sensory
  776	HBQAC72	Neural/Sensory
  777	HBQAE37	Neural/Sensory
  778	HBSAJ63	Cancer
  779	HBSAJ63	Cancer
  780	HBSDD24	Cancer
  781	HBWBD25	Immune/Hematopoietic,
  		Neural/Sensory
  782	HBXAS93	Neural/Sensory
  783	HBXAT27	Cancer
  784	HBXAW57	Neural/Sensory
  785	HBXBI29	Neural/Sensory
  786	HBXBM24	Neural/Sensory
  787	HBXBM78	Cancer
  788	HBXCD59	Immune/Hematopoietic,
  		Neural/Sensory
  789	HBXCE43	Neural/Sensory
  790	HBXCG08	Cancer
  791	HBXCM52	Cancer
  792	HBXCQ03	Cancer
  793	HBXCR15	Cancer
  794	HBXDL52	Cancer
  795	HBXDL52	Cancer
  796	HBXDN08	Cancer
  797	HBXDN65	Neural/Sensory
  798	HBXFA04	Neural/Sensory
  799	HBXFE64	Neural/Sensory
  800	HBXFI33	Immune/Hematopoietic,
  		Neural/Sensory
  801	HBXFP72	Cancer
  802	HBXFS31	Neural/Sensory
  803	HBXFW01	Neural/Sensory
  804	HBXGE12	Cancer
  805	HBXGL91	Neural/Sensory,
  		Reproductive
  806	HBXGM24	Cancer
  807	HBZAI75	Digestive,
  		Reproductive
  808	HCABP33	Cancer
  809	HCABW10	Cancer
  810	HCACZ65	Cancer
  811	HCBAB34	Cancer
  812	HCDAA24	Cancer
  813	HCDAA24	Cancer
  814	HCDAF17	Cancer
  815	HCDAH02	Immune/Hematopoietic,
  		Musculoskeletal
  816	HCDAP33	Cancer
  817	HCDAR40	Cardiovascular,
  		Immune/Hematopoietic,
  		Musculoskeletal
  818	HCDAS02	Cancer
  819	HCDBE76	Cancer
  820	HCDBO32	Cancer
  821	HCDBW67	Cancer
  822	HCDBZ31	Musculoskeletal
  823	HCDCB03	Cancer
  824	HCDCE51	Cancer
  825	HCDCI42	Immune/Hematopoietic,
  		Musculoskeletal
  826	HCDDB15	Cancer
  827	HCDDX81	Musculoskeletal
  828	HCDDY28	Cardiovascular,
  		Musculoskeletal
  829	HCDEB19	Cancer
  830	HCDEN46	Cancer
  831	HCDES69	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  832	HCE1D45	Cancer
  833	HCE1N56	Cancer
  834	HCE1T53	Neural/Sensory
  835	HCE1Y27	Digestive,
  		Neural/Sensory,
  		Reproductive
  836	HCE1Y34	Immune/Hematopoietic,
  		Neural/Sensory
  837	HCE2B57	Musculoskeletal,
  		Neural/Sensory
  838	HCE2E47	Immune/Hematopoietic,
  		Mixed Fetal,
  		Neural/Sensory
  839	HCE2I23	Neural/Sensory
  840	HCE2P90	Neural/Sensory
  841	HCE3A54	Neural/Sensory
  842	HCE3C46	Immune/Hematopoietic,
  		Neural/Sensory
  843	HCE3D58	Cancer
  844	HCE3D89	Endocrine,
  		Neural/Sensory
  845	HCE3J43	Cancer
  846	HCE3L04	Neural/Sensory
  847	HCE3N23	Cancer
  848	HCE3R01	Cancer
  849	HCE3R01	Cancer
  850	HCE3R01	Cancer
  851	HCE3R46	Cancer
  852	HCE4H32	Cancer
  853	HCE4H32	Cancer
  854	HCE4T64	Cancer
  855	HCE4W88	Cancer
  856	HCE5B62	Neural/Sensory
  857	HCE5H86	Cancer
  858	HCE5J64	Digestive,
  		Neural/Sensory
  859	HCEBF54	Cancer
  860	HCECO77	Cancer
  861	HCEDH42	Neural/Sensory
  862	HCEDJ05	Neural/Sensory
  863	HCEDJ26	Cancer
  864	HCEDN07	Digestive,
  		Mixed Fetal,
  		Neural/Sensory
  865	HCEDO17	Cancer
  866	HCEEG48	Neural/Sensory
  867	HCEEM33	Cancer
  868	HCEEP16	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  869	HCEER60	Cardiovascular,
  		Digestive,
  		Neural/Sensory
  870	HCEFA10	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  871	HCEFA50	Neural/Sensory
  872	HCEFA94	Neural/Sensory
  873	HCEFC27	Cancer
  874	HCEFG93	Neural/Sensory
  875	HCEFH31	Cancer
  876	HCEFK56	Cancer
  877	HCEFN51	Cancer
  878	HCEGG08	Cancer
  879	HCEGH74	Cancer
  880	HCEGK81	Cancer
  881	HCEGS49	Connective/Epithelial,
  		Neural/Sensory,
  		Reproductive
  882	HCEGU75	Cancer
  883	HCEGY33	Cancer
  884	HCEHW24	Neural/Sensory
  885	HCEJL08	Cancer
  886	HCEJP93	Cancer
  887	HCELB04	Cancer
  888	HCEMA08	Cancer
  889	HCENN67	Digestive,
  		Endocrine,
  		Neural/Sensory
  890	HCENQ22	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  891	HCEOF01	Neural/Sensory,
  		Reproductive
  892	HCEOF01	Neural/Sensory,
  		Reproductive
  893	HCEON94	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  894	HCEOQ67	Cancer
  895	HCEOV48	Cancer
  896	HCEPC90	Neural/Sensory
  897	HCEPO08	Cancer
  898	HCESB03	Immune/Hematopoietic,
  		Neural/Sensory
  899	HCESK44	Cancer
  900	HCETE08	Mixed Fetal,
  		Neural/Sensory,
  		Reproductive
  901	HCETL19	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  902	HCEWD90	Cancer
  903	HCEWE62	Neural/Sensory
  904	HCEZW14	Cancer
  905	HCFAT42	Immune/Hematopoietic
  906	HCFAT66	Immune/Hematopoietic
  907	HCFBA30	Cardiovascular,
  		Immune/Hematopoietic,
  		Reproductive
  908	HCFBM77	Immune/Hematopoietic
  909	HCFBV39	Cancer
  910	HCFCB72	Immune/Hematopoietic
  911	HCFCG91	Cancer
  912	HCFCM81	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  913	HCFCW39	Cancer
  914	HCFCY49	Cancer
  915	HCFDD18	Digestive,
  		Immune/Hematopoietic,
  		Mixed Fetal
  916	HCFLB10	Cardiovascular,
  		Immune/Hematopoietic
  917	HCFLC03	Cancer
  918	HCFLJ52	Cancer
  919	HCFLL33	Immune/Hematopoietic,
  		Reproductive
  920	HCFLP48	Immune/Hematopoietic
  921	HCFLQ12	Cancer
  922	HCFLY20	Cancer
  923	HCFLY20	Cancer
  924	HCFMA39	Immune/Hematopoietic
  925	HCFMJ40	Immune/Hematopoietic
  926	HCFML07	Cancer
  927	HCFMR75	Digestive,
  		Immune/Hematopoietic
  928	HCFMX16	Immune/Hematopoietic
  929	HCFMX88	Immune/Hematopoietic,
  		Neural/Sensory
  930	HCFNM40	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  931	HCFNM50	Immune/Hematopoietic
  932	HCFNN16	Cancer
  933	HCFNN75	Cancer
  934	HCFOG17	Immune/Hematopoietic
  935	HCFOH93	Immune/Hematopoietic,
  		Reproductive
  936	HCGBA15	Cancer
  937	HCHAC68	Cancer
  938	HCHBP49	Cancer
  939	HCHCA79	Digestive,
  		Neural/Sensory,
  		Reproductive
  940	HCHCG33	Cancer
  941	HCHMY57	Cancer
  942	HCHOC06	Reproductive
  943	HCHOY52	Cancer
  944	HCHQB93	Cancer
  945	HCHQB93	Cancer
  946	HCLBK61	Cancer
  947	HCLCU75	Respiratory
  948	HCMSA37	Cardiovascular
  949	HCMSR07	Cardiovascular
  950	HCNAI74	Digestive
  951	HCNCT01	Digestive
  952	HCNDR39	Cancer
  953	HCNSD91	Cancer
  954	HCNSF01	Cancer
  955	HCNSG06	Digestive,
  		Reproductive
  956	HCNSG32	Digestive,
  		Reproductive
  957	HCPAE41	Cancer
  958	HCQAK36	Cancer
  959	HCQAQ47	Cancer
  960	HCQAS72	Cancer
  961	HCQBM95	Digestive,
  		Immune/Hematopoietic
  962	HCQCM95	Cancer
  963	HCQCM95	Cancer
  964	HCQCV23	Cancer
  965	HCQCV23	Cancer
  966	HCQDD32	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  967	HCQDD61	Digestive,
  		Immune/Hematopoietic
  968	HCQDT67	Digestive,
  		Immune/Hematopoietic
  969	HCRAI29	Neural/Sensory
  970	HCRBI79	Cancer
  971	HCRBL20	Cancer
  972	HCRBX84	Cancer
  973	HCRMA24	Digestive,
  		Musculoskeletal
  974	HCRMR35	Cancer
  975	HCRMR35	Cancer
  976	HCRMR35	Cancer
  977	HCROC18	Cancer
  978	HCUAE53	Immune/Hematopoietic
  979	HCUAO46	Immune/Hematopoietic
  980	HCUAT74	Cancer
  981	HCUBA28	Cancer
  982	HCUBC45	Cancer
  983	HCUBM41	Immune/Hematopoietic
  984	HCUBN69	Immune/Hematopoietic
  985	HCUBY47	Digestive,
  		Immune/Hematopoietic
  986	HCUCV66	Cancer
  987	HCUDJ41	Immune/Hematopoietic
  988	HCUEC55	Immune/Hematopoietic
  989	HCUEG85	Immune/Hematopoietic
  990	HCUES35	Immune/Hematopoietic,
  		Neural/Sensory
  991	HCUFC77	Cancer
  992	HCUFD17	Cancer
  993	HCUFD46	Immune/Hematopoietic
  994	HCUFE33	Immune/Hematopoietic
  995	HCUFJ09	Cancer
  996	HCUFQ58	Cardiovascular,
  		Immune/Hematopoietic
  997	HCUFQ58	Cardiovascular,
  		Immune/Hematopoietic
  998	HCUFX08	Immune/Hematopoietic
  999	HCUGB76	Immune/Hematopoietic,
  		Reproductive
  1000	HCUGK79	Immune/Hematopoietic
  1001	HCUGQ19	Immune/Hematopoietic
  1002	HCUGR26	Immune/Hematopoietic
  1003	HCUGR86	Immune/Hematopoietic
  1004	HCUHE27	Immune/Hematopoietic
  1005	HCUHL82	Cardiovascular,
  		Immune/Hematopoietic,
  		Reproductive
  1006	HCUHM71	Immune/Hematopoietic,
  		Musculoskeletal
  1007	HCWAK88	Immune/Hematopoietic
  1008	HCWAL10	Cardiovascular,
  		Immune/Hematopoietic
  1009	HCWAT71	Immune/Hematopoietic
  1010	HCWBQ52	Immune/Hematopoietic
  1011	HCWCH16	Immune/Hematopoietic
  1012	HCWDM69	Digestive,
  		Immune/Hematopoietic
  1013	HCWEB38	Immune/Hematopoietic
  1014	HCWEB72	Immune/Hematopoietic
  1015	HCWEF04	Cancer
  1016	HCWEI82	Immune/Hematopoietic
  1017	HCWEM96	Cancer
  1018	HCWFJ16	Immune/Hematopoietic
  1019	HCWFJ16	Immune/Hematopoietic
  1020	HCWFK03	Cancer
  1021	HCWHD30	Immune/Hematopoietic
  1022	HCWHT34	Immune/Hematopoietic,
  		Mixed Fetal
  1023	HCWHT52	Immune/Hematopoietic
  1024	HCWKO32	Immune/Hematopoietic
  1025	HCWLE50	Immune/Hematopoietic
  1026	HCWUF93	Cancer
  1027	HCWUW24	Immune/Hematopoietic
  1028	HCYBA32	Cancer
  1029	HDAAV67	Musculoskeletal,
  		Neural/Sensory
  1030	HDABR74	Cancer
  1031	HDABW45	Immune/Hematopoietic,
  		Musculoskeletal
  1032	HDACJ52	Cancer
  1033	HDCBM09	Immune/Hematopoietic
  1034	HDFAB86	Mixed Fetal,
  		Neural/Sensory
  1035	HDFIB37	Connective/Epithelial,
  		Neural/Sensory
  1036	HDFMB91	Neural/Sensory
  1037	HDHAA55	Immune/Hematopoietic,
  		Neural/Sensory
  1038	HDHEA33	Cancer
  1039	HDHEB12	Immune/Hematopoietic,
  		Neural/Sensory
  1040	HDHEB80	Neural/Sensory
  1041	HDHIA16	Cancer
  1042	HDHIA26	Neural/Sensory
  1043	HDHMA71	Cancer
  1044	HDLAL94	Cancer
  1045	HDPAB86	Cancer
  1046	HDPAE80	Cancer
  1047	HDPAQ86	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1048	HDPBD56	Cancer
  1049	HDPBN48	Digestive,
  		Immune/Hematopoietic
  1050	HDPCG79	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  1051	HDPCV29	Immune/Hematopoietic
  1052	HDPDA36	Immune/Hematopoietic
  1053	HDPDC59	Immune/Hematopoietic,
  		Musculoskeletal
  1054	HDPFG13	Cancer
  1055	HDPFK27	Immune/Hematopoietic,
  		Neural/Sensory
  1056	HDPFZ05	Immune/Hematopoietic,
  		Neural/Sensory
  1057	HDPGA84	Cancer
  1058	HDPGR80	Cancer
  1059	HDPGU14	Cancer
  1060	HDPGX09	Cancer
  1061	HDPIE44	Cancer
  1062	HDPIE73	Immune/Hematopoietic
  1063	HDPIF35	Immune/Hematopoietic
  1064	HDPIF65	Immune/Hematopoietic
  1065	HDPIH25	Cancer
  1066	HDPIY31	Cancer
  1067	HDPJH72	Cancer
  1068	HDPJV53	Immune/Hematopoietic
  1069	HDPJV75	Cancer
  1070	HDPKC55	Cardiovascular,
  		Immune/Hematopoietic,
  		Reproductive
  1071	HDPKD16	Cancer
  1072	HDPMC52	Digestive,
  		Immune/Hematopoietic,
  		Musculoskeletal
  1073	HDPML04	Connective/Epithelial,
  		Immune/Hematopoietic
  1074	HDPMM22	Immune/Hematopoietic
  1075	HDPNC21	Cancer
  1076	HDPNJ26	Cancer
  1077	HDPOD73	Immune/Hematopoietic
  1078	HDPOT33	Cancer
  1079	HDPPB70	Cancer
  1080	HDPPC19	Immune/Hematopoietic
  1081	HDPPE05	Cancer
  1082	HDPSA70	Cancer
  1083	HDPSS56	Cancer
  1084	HDPSZ07	Immune/Hematopoietic
  1085	HDPSZ07	Immune/Hematopoietic
  1086	HDPSZ07	Immune/Hematopoietic
  1087	HDPTI49	Immune/Hematopoietic,
  		Neural/Sensory
  1088	HDPTP22	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  1089	HDPYE25	Immune/Hematopoietic,
  		Neural/Sensory
  1090	HDQGD06	Cancer
  1091	HDQGD06	Cancer
  1092	HDQGD06	Cancer
  1093	HDQGN08	Immune/Hematopoietic
  1094	HDQGO62	Cancer
  1095	HDQPM16	Cancer
  1096	HDRAA17	Cancer
  1097	HDRAC68	Cancer
  1098	HDSAC78	Cancer
  1099	HDSAH37	Connective/Epithelial
  1100	HDSAM57	Immune/Hematopoietic
  1101	HDSAO14	Cancer
  1102	HDSAO64	Cancer
  1103	HDSAP15	Cancer
  1104	HDTAR39	Cancer
  1105	HDTAS57	Cancer
  1106	HDTBP62	Cancer
  1107	HDTBQ77	Cancer
  1108	HDTDA48	Immune/Hematopoietic,
  		Neural/Sensory
  1109	HDTDE66	Cancer
  1110	HDTDG75	Immune/Hematopoietic
  1111	HDTDS09	Cancer
  1112	HDTFF53	Cancer
  1113	HDTGW76	Cancer
  1114	HDTGZ56	Immune/Hematopoietic
  1115	HDTHZ85	Cancer
  1116	HDTIM39	Cancer
  1117	HDTKJ29	Cancer
  1118	HDUAB12	Cancer
  1119	HDUAD68	Cancer
  1120	HE2AC74	Cancer
  1121	HE2AC74	Cancer
  1122	HE2AC75	Mixed Fetal
  1123	HE2AI94	Cancer
  1124	HE2AT61	Cancer
  1125	HE2AX36	Cancer
  1126	HE2AY96	Cancer
  1127	HE2BD72	Cancer
  1128	HE2BH50	Cancer
  1129	HE2CB53	Cancer
  1130	HE2CC17	Excretory,
  		Mixed Fetal
  1131	HE2CJ53	Cancer
  1132	HE2CK47	Cancer
  1133	HE2CM34	Cancer
  1134	HE2DG46	Mixed Fetal
  1135	HE2DI16	Mixed Fetal
  1136	HE2DJ84	Cancer
  1137	HE2DY23	Cancer
  1138	HE2DY25	Cancer
  1139	HE2EE80	Cancer
  1140	HE2EH45	Mixed Fetal
  1141	HE2FE89	Cardiovascular,
  		Digestive,
  		Mixed Fetal
  1142	HE2FR49	Cancer
  1143	HE2GB19	Cancer
  1144	HE2GO81	Cancer
  1145	HE2HB61	Mixed Fetal
  1146	HE2HB64	Cancer
  1147	HE2HF76	Cancer
  1148	HE2ID09	Mixed Fetal
  1149	HE2IE66	Cancer
  1150	HE2NW57	Mixed Fetal
  1151	HE2OA95	Cancer
  1152	HE2OC39	Mixed Fetal,
  		Musculoskeletal
  1153	HE2PB61	Cancer
  1154	HE2PI43	Cancer
  1155	HE2PJ56	Cancer
  1156	HE6CJ41	Immune/Hematopoietic,
  		Mixed Fetal
  1157	HE6DC37	Digestive,
  		Mixed Fetal,
  		Reproductive
  1158	HE6DN83	Cancer
  1159	HE6EI30	Immune/Hematopoietic,
  		Mixed Fetal
  1160	HE6ET70	Mixed Fetal,
  		Reproductive
  1161	HE6GO65	Mixed Fetal,
  		Neural/Sensory
  1162	HE8AN83	Mixed Fetal,
  		Musculoskeletal
  1163	HE8AU68	Cancer
  1164	HE8BE20	Cancer
  1165	HE8BP05	Mixed Fetal
  1166	HE8BP64	Cancer
  1167	HE8BQ49	Mixed Fetal
  1168	HE8BR18	Cancer
  1169	HE8BR30	Mixed Fetal
  1170	HE8BT58	Cancer
  1171	HE8BU60	Cancer
  1172	HE8CA13	Cancer
  1173	HE8CC34	Cardiovascular,
  		Digestive,
  		Mixed Fetal
  1174	HE8CH08	Cancer
  1175	HE8DG02	Mixed Fetal
  1176	HE8DK52	Cancer
  1177	HE8DZ94	Cancer
  1178	HE8EN79	Cancer
  1179	HE8EX86	Cancer
  1180	HE8FC10	Immune/Hematopoietic,
  		Mixed Fetal,
  		Reproductive
  1181	HE8FG15	Cancer
  1182	HE8FG24	Cancer
  1183	HE8FK78	Cancer
  1184	HE8FL24	Mixed Fetal
  1185	HE8FL68	Mixed Fetal
  1186	HE8FR53	Cancer
  1187	HE8MA27	Cancer
  1188	HE8MG56	Mixed Fetal
  1189	HE8MQ01	Cancer
  1190	HE8MS43	Cancer
  1191	HE8MY77	Cancer
  1192	HE8NC81	Cancer
  1193	HE8NO09	Cancer
  1194	HE8QU21	Immune/Hematopoietic,
  		Mixed Fetal
  1195	HE8SH74	Immune/Hematopoietic,
  		Mixed Fetal,
  		Musculoskeletal
  1196	HE8UX34	Mixed Fetal
  1197	HE9AE05	Cancer
  1198	HE9BJ14	Mixed Fetal,
  		Musculoskeletal
  1199	HE9CI81	Cancer
  1200	HE9CJ38	Mixed Fetal
  1201	HE9CM11	Mixed Fetal
  1202	HE9CN58	Cancer
  1203	HE9CV59	Cancer
  1204	HE9DG54	Cancer
  1205	HE9DH59	Cancer
  1206	HE9DZ47	Endocrine,
  		Immune/Hematopoietic,
  		Mixed Fetal
  1207	HE9EC36	Cancer
  1208	HE9EM54	Immune/Hematopoietic,
  		Mixed Fetal
  1209	HE9FH28	Mixed Fetal
  1210	HE9HE13	Cancer
  1211	HE9HE13	Cancer
  1212	HE9HF59	Mixed Fetal
  1213	HE9HV71	Cancer
  1214	HE9NB82	Cancer
  1215	HE9NE43	Mixed Fetal
  1216	HE9RN58	Cancer
  1217	HE9TA42	Cancer
  1218	HEAAC21	Cancer
  1219	HEAAC39	Neural/Sensory,
  		Reproductive
  1220	HEAAC48	Reproductive
  1221	HEAAD63	Neural/Sensory,
  		Reproductive
  1222	HEAAE19	Immune/Hematopoietic,
  		Reproductive
  1223	HEAAM54	Reproductive
  1224	HEAAM96	Reproductive
  1225	HEAAN52	Cancer
  1226	HEAAU28	Reproductive
  1227	HEAAW54	Reproductive
  1228	HEAAW94	Cancer
  1229	HEBAP51	Cancer
  1230	HEBAT05	Cancer
  1231	HEBBF78	Cancer
  1232	HEBBK04	Cancer
  1233	HEBCN80	Neural/Sensory
  1234	HEBCW57	Mixed Fetal,
  		Neural/Sensory
  1235	HEBDF90	Cancer
  1236	HEBDW31	Cancer
  1237	HEBFL36	Neural/Sensory
  1238	HEBGC01	Neural/Sensory
  1239	HEBGE23	Cancer
  1240	HEBGE85	Digestive,
  		Neural/Sensory,
  		Reproductive
  1241	HEBGJ94	Cancer
  1242	HEBGM06	Cancer
  1243	HEEAB58	Cancer
  1244	HEEAF49	Cancer
  1245	HEEAJ46	Mixed Fetal,
  		Reproductive
  1246	HEGAI20	Reproductive
  1247	HEIAC52	Cancer
  1248	HELAC55	Cardiovascular,
  		Immune/Hematopoietic,
  		Musculoskeletal
  1249	HELAT58	Cardiovascular
  1250	HELAW94	Cancer
  1251	HELDF80	Cancer
  1252	HELDH39	Cancer
  1253	HELDK79	Cardiovascular
  1254	HELDQ42	Cancer
  1255	HELEE85	Cancer
  1256	HELEL76	Cancer
  1257	HELEL76	Cancer
  1258	HELEO45	Cancer
  1259	HELFA57	Cancer
  1260	HELFO30	Cancer
  1261	HELGF28	Cancer
  1262	HELGP60	Cardiovascular,
  		Excretory,
  		Immune/Hematopoietic
  1263	HELHN47	Cancer
  1264	HELHP11	Cardiovascular,
  		Immune/Hematopoietic
  1265	HELHP11	Cardiovascular,
  		Immune/Hematopoietic
  1266	HEMAE30	Cancer
  1267	HEMBV40	Cancer
  1268	HEMCJ80	Cancer
  1269	HEMCL55	Cardiovascular
  1270	HEMDB07	Cardiovascular
  1271	HEMDR05	Cardiovascular,
  		Digestive,
  		Immune/Hematopoietic
  1272	HEMGK71	Cardiovascular,
  		Immune/Hematopoietic,
  		Musculoskeletal
  1273	HEOMF59	Immune/Hematopoietic
  1274	HEOMJ73	Cancer
  1275	HEOMR67	Cancer
  1276	HEOMU25	Connective/Epithelial,
  		Immune/Hematopoietic
  1277	HEOMU44	Cancer
  1278	HEONI85	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  1279	HEONK04	Cancer
  1280	HEONP08	Immune/Hematopoietic
  1281	HEPAD15	Endocrine,
  		Reproductive
  1282	HEPBC23	Cancer
  1283	HEPBV09	Reproductive
  1284	HEPCF35	Neural/Sensory,
  		Reproductive
  1285	HEPCU48	Cancer
  1286	HEQAH47	Cancer
  1287	HEQAP92	Cancer
  1288	HEQAV53	Cancer
  1289	HEQBJ01	Cancer
  1290	HEQBJ01	Cancer
  1291	HEQBJ01	Cancer
  1292	HEQBM94	Cancer
  1293	HEQCB93	Cancer
  1294	HERAI63	Connective/Epithelial
  1295	HERAQ22	Connective/Epithelial
  1296	HERAS61	Connective/Epithelial
  1297	HESAG57	Cancer
  1298	HETAA62	Cancer
  1299	HETBB70	Immune/Hematopoietic,
  		Reproductive
  1300	HETBJ88	Cancer
  1301	HETCM67	Cancer
  1302	HETDD61	Reproductive
  1303	HETDD61	Reproductive
  1304	HETDJ34	Cancer
  1305	HETDM73	Cancer
  1306	HETDP76	Cancer
  1307	HETFO57	Cancer
  1308	HETGZ31	Cancer
  1309	HETHD26	Cancer
  1310	HETHM27	Cancer
  1311	HETIN36	Cancer
  1312	HFAAI17	Neural/Sensory
  1313	HFAAJ45	Immune/Hematopoietic,
  		Neural/Sensory
  1314	HFADF41	Neural/Sensory
  1315	HFADM09	Cancer
  1316	HFAUA23	Cancer
  1317	HFCAG75	Cancer
  1318	HFCAI40	Cancer
  1319	HFCAQ17	Cancer
  1320	HFCBC16	Neural/Sensory
  1321	HFCBL53	Cancer
  1322	HFCBL53	Cancer
  1323	HFCBL53	Cancer
  1324	HFCBT29	Cancer
  1325	HFCCZ31	Cancer
  1326	HFCDN13	Cancer
  1327	HFCDT67	Cancer
  1328	HFCDY36	Neural/Sensory
  1329	HFCEC45	Cancer
  1330	HFCET43	Cancer
  1331	HFEAG55	Cancer
  1332	HFEAU63	Connective/Epithelial
  1333	HFEBA88	Cancer
  1334	HFEBK75	Connective/Epithelial
  1335	HFEBO15	Cancer
  1336	HFEBO17	Cancer
  1337	HFFAE46	Neural/Sensory
  1338	HFFAH01	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1339	HFFAL70	Cancer
  1340	HFFAV61	Neural/Sensory
  1341	HFGAB50	Cancer
  1342	HFGAE28	Cancer
  1343	HFGAN63	Cancer
  1344	HFHDN80	Cardiovascular,
  		Digestive,
  		Immune/Hematopoietic
  1345	HFIAB78	Cancer
  1346	HFIAD23	Cancer
  1347	HFIAK06	Musculoskeletal,
  		Reproductive
  1348	HFICH70	Musculoskeletal
  1349	HFIHQ57	Musculoskeletal,
  		Reproductive
  1350	HFIIK29	Cancer
  1351	HFIIK29	Cancer
  1352	HFIIK29	Cancer
  1353	HFIIK29	Cancer
  1354	HFIIQ27	Cancer
  1355	HFIIQ64	Cancer
  1356	HFIIZ61	Cancer
  1357	HFIJD81	Cancer
  1358	HFIJF44	Cancer
  1359	HFITA02	Immune/Hematopoietic,
  		Musculoskeletal
  1360	HFITF80	Cancer
  1361	HFIUK66	Cancer
  1362	HFIUT21	Cancer
  1363	HFIVB04	Cancer
  1364	HFIXC39	Cancer
  1365	HFIXC69	Cancer
  1366	HFIXE39	Cancer
  1367	HFIYP15	Immune/Hematopoietic,
  		Musculoskeletal
  1368	HFIZE10	Cancer
  1369	HFIZF51	Musculoskeletal
  1370	HFIZK42	Immune/Hematopoietic,
  		Musculoskeletal
  1371	HFIZM89	Musculoskeletal
  1372	HFKBA62	Digestive,
  		Excretory,
  		Neural/Sensory
  1373	HFKBC47	Cancer
  1374	HFKDX53	Cancer
  1375	HFKEB14	Cancer
  1376	HFKEG63	Excretory
  1377	HFKES35	Cancer
  1378	HFKES35	Cancer
  1379	HFKEU17	Cancer
  1380	HFKEV77	Cancer
  1381	HFKFI15	Cancer
  1382	HFKFI35	Excretory
  1383	HFKFK49	Cancer
  1384	HFKFV88	Cancer
  1385	HFKFV88	Cancer
  1386	HFKFV88	Cancer
  1387	HFKFX64	Excretory
  1388	HFOXD49	Cancer
  1389	HFOXE28	Cancer
  1390	HFOYH74	Musculoskeletal
  1391	HFOYP02	Musculoskeletal
  1392	HFOYR24	Musculoskeletal
  1393	HFOYR54	Cancer
  1394	HFOZB26	Cancer
  1395	HFPBF54	Cancer
  1396	HFPBF54	Cancer
  1397	HFPBI93	Cancer
  1398	HFPBJ64	Musculoskeletal,
  		Neural/Sensory
  1399	HFPBQ55	Musculoskeletal,
  		Neural/Sensory,
  		Reproductive
  1400	HFPCK22	Cancer
  1401	HFPCM32	Neural/Sensory
  1402	HFPCM36	Cancer
  1403	HFPCS84	Neural/Sensory
  1404	HFPCU47	Neural/Sensory
  1405	HFPCY66	Digestive,
  		Neural/Sensory
  1406	HFPDC65	Cancer
  1407	HFPDE42	Musculoskeletal,
  		Neural/Sensory
  1408	HFPDE88	Neural/Sensory
  1409	HFPDO25	Neural/Sensory
  1410	HFPDP70	Neural/Sensory
  1411	HFPDR39	Cancer
  1412	HFPDX08	Cancer
  1413	HFPEP69	Neural/Sensory
  1414	HFRAU40	Cancer
  1415	HFRAY90	Cancer
  1416	HFSAY91	Cancer
  1417	HFSBC10	Immune/Hematopoietic,
  		Mixed Fetal
  1418	HFSBE94	Immune/Hematopoietic
  1419	HFTAN11	Cancer
  1420	HFTAR27	Cancer
  1421	HFTAR30	Cancer
  1422	HFTAS49	Cancer
  1423	HFTBB50	Cancer
  1424	HFTBL17	Cancer
  1425	HFTBL17	Cancer
  1426	HFTCF02	Digestive,
  		Musculoskeletal,
  		Neural/Sensory
  1427	HFTCI85	Neural/Sensory
  1428	HFTCJ32	Neural/Sensory
  1429	HFTCO17	Cancer
  1430	HFTCW07	Neural/Sensory
  1431	HFTDF32	Cancer
  1432	HFTDF79	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  1433	HFTDK11	Cancer
  1434	HFTDU08	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  1435	HFVGK67	Digestive,
  		Immune/Hematopoietic
  1436	HFVHD38	Cancer
  1437	HFVHY57	Cancer
  1438	HFVIC33	Cancer
  1439	HFXAK32	Cancer
  1440	HFXAK59	Cancer
  1441	HFXBI64	Neural/Sensory
  1442	HFXBL05	Mixed Fetal,
  		Neural/Sensory
  1443	HFXBM52	Neural/Sensory
  1444	HFXBR58	Neural/Sensory
  1445	HFXBV67	Digestive,
  		Neural/Sensory
  1446	HFXBY20	Neural/Sensory
  1447	HFXCB70	Neural/Sensory
  1448	HFXCI42	Neural/Sensory
  1449	HFXCL59	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  1450	HFXCM22	Neural/Sensory,
  		Reproductive
  1451	HFXCN18	Neural/Sensory
  1452	HFXCS53	Cancer
  1453	HFXDB37	Neural/Sensory
  1454	HFXDI32	Neural/Sensory
  1455	HFXDJ43	Neural/Sensory
  1456	HFXDL76	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  1457	HFXDM75	Neural/Sensory
  1458	HFXDO18	Neural/Sensory
  1459	HFXDP44	Neural/Sensory
  1460	HFXDR08	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  1461	HFXDR28	Neural/Sensory
  1462	HFXDR28	Neural/Sensory
  1463	HFXDR47	Cancer
  1464	HFXDZ03	Immune/Hematopoietic,
  		Neural/Sensory
  1465	HFXED33	Neural/Sensory
  1466	HFXEE88	Neural/Sensory
  1467	HFXGR32	Neural/Sensory
  1468	HFXGT51	Neural/Sensory
  1469	HFXGW16	Neural/Sensory
  1470	HFXHC15	Neural/Sensory
  1471	HFXHI33	Immune/Hematopoietic,
  		Neural/Sensory
  1472	HFXHL21	Neural/Sensory
  1473	HFXHL83	Neural/Sensory
  1474	HFXHM49	Neural/Sensory
  1475	HFXHM93	Neural/Sensory
  1476	HFXHN89	Immune/Hematopoietic,
  		Neural/Sensory
  1477	HFXJB21	Neural/Sensory
  1478	HFXJN93	Neural/Sensory
  1479	HFXJS15	Cancer
  1480	HFXJT53	Cancer
  1481	HFXKG56	Neural/Sensory
  1482	HFXKL60	Cancer
  1483	HFXLG08	Neural/Sensory
  1484	HFXLK91	Cancer
  1485	HFXLM32	Neural/Sensory,
  		Reproductive
  1486	HGBAX83	Digestive
  1487	HGBBR29	Cancer
  1488	HGBDL51	Cancer
  1489	HGBDV35	Cancer
  1490	HGBDX28	Cancer
  1491	HGBGX31	Cancer
  1492	HGBHE23	Cancer
  1493	HGBHI15	Digestive
  1494	HGCMW39	Cancer
  1495	HGLAG32	Cancer
  1496	HGLAH08	Cancer
  1497	HGLAH86	Immune/Hematopoietic
  1498	HGLBC33	Cancer
  1499	HGLBG15	Cancer
  1500	HGLBM55	Cancer
  1501	HGLDA95	Cancer
  1502	HGLDB06	Cancer
  1503	HGLDE15	Digestive,
  		Immune/Hematopoietic
  1504	HHBEI14	Cancer
  1505	HHBGL33	Cardiovascular,
  		Digestive
  1506	HHEAW44	Immune/Hematopoietic
  1507	HHEBP28	Cancer
  1508	HHECK41	Cancer
  1509	HHECR10	Immune/Hematopoietic
  1510	HHEMC55	Immune/Hematopoietic
  1511	HHEMM20	Immune/Hematopoietic
  1512	HHEMM80	Immune/Hematopoietic
  1513	HHEMP35	Cancer
  1514	HHEMZ08	Cancer
  1515	HHENC17	Cancer
  1516	HHENF95	Immune/Hematopoietic
  1517	HHENR74	Immune/Hematopoietic
  1518	HHENU33	Immune/Hematopoietic
  1519	HHENY07	Cancer
  1520	HHEOK77	Cancer
  1521	HHEPE72	Immune/Hematopoietic
  1522	HHEPE81	Cancer
  1523	HHEPM64	Cancer
  1524	HHEQI04	Connective/Epithelial,
  		Excretory,
  		Immune/Hematopoietic
  1525	HHEQY60	Immune/Hematopoietic
  1526	HHFBA31	Cancer
  1527	HHFCI81	Cancer
  1528	HHFCN78	Cardiovascular,
  		Immune/Hematopoietic
  1529	HHFCT95	Cancer
  1530	HHFDN16	Cardiovascular
  1531	HHFEB79	Connective/Epithelial
  1532	HHFEC39	Cancer
  1533	HHFEN34	Cardiovascular
  1534	HHFFZ01	Cancer
  1535	HHFGI71	Cardiovascular
  1536	HHFGJ54	Cancer
  1537	HHFGL38	Cardiovascular,
  		Immune/Hematopoietic
  1538	HHFGR75	Cardiovascular,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1539	HHFGZ23	Cardiovascular,
  		Digestive,
  		Endocrine
  1540	HHFHG26	Cardiovascular,
  		Neural/Sensory
  1541	HHFHM47	Cardiovascular,
  		Immune/Hematopoietic
  1542	HHGAA76	Immune/Hematopoietic,
  		Reproductive
  1543	HHGAD46	Cancer
  1544	HHGAT09	Cancer
  1545	HHGBC21	Cancer
  1546	HHGBF91	Cancer
  1547	HHGBG63	Cancer
  1548	HHGBV02	Immune/Hematopoietic,
  		Reproductive
  1549	HHGBW55	Immune/Hematopoietic,
  		Reproductive
  1550	HHGBX88	Cancer
  1551	HHGCA26	Reproductive
  1552	HHGDA81	Cancer
  1553	HHGDI12	Neural/Sensory
  1554	HHGDR05	Neural/Sensory
  1555	HHGDR92	Cancer
  1556	HHGDS56	Cancer
  1557	HHGDW65	Cancer
  1558	HHLBA86	Digestive
  1559	HHNAC56	Digestive
  1560	HHPBG90	Cancer
  1561	HHPDE28	Cancer
  1562	HHPDJ11	Cancer
  1563	HHPDX86	Neural/Sensory
  1564	HHPEA17	Neural/Sensory
  1565	HHPEB61	Immune/Hematopoietic,
  		Neural/Sensory,
  		Respiratory
  1566	HHPFP26	Cancer
  1567	HHPFS11	Cardiovascular,
  		Neural/Sensory
  1568	HHPFS15	Cancer
  1569	HHPFS18	Cancer
  1570	HHPGH34	Neural/Sensory,
  		Reproductive
  1571	HHPGU74	Neural/Sensory
  1572	HHPGU87	Cancer
  1573	HHPSD42	Immune/Hematopoietic,
  		Neural/Sensory
  1574	HHPSE03	Neural/Sensory
  1575	HHPSE55	Cancer
  1576	HHPSF70	Cancer
  1577	HHPSH74	Cancer
  1578	HHPSL14	Cancer
  1579	HHPSM40	Neural/Sensory
  1580	HHPTF26	Mixed Fetal,
  		Musculoskeletal,
  		Neural/Sensory
  1581	HHSAD31	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  1582	HHSAE74	Neural/Sensory
  1583	HHSAG62	Cancer
  1584	HHSAK17	Neural/Sensory
  1585	HHSBJ92	Cancer
  1586	HHSBN84	Cancer
  1587	HHSCL24	Cancer
  1588	HHSCQ67	Cancer
  1589	HHSCU12	Cancer
  1590	HHSDB43	Cancer
  1591	HHSDL07	Neural/Sensory
  1592	HHSDX07	Cancer
  1593	HHSFF54	Cancer
  1594	HHSGB85	Cancer
  1595	HHSGL84	Neural/Sensory
  1596	HHTLH79	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  1597	HIABC70	Cancer
  1598	HIATG10	Endocrine
  1599	HIBCO70	Cancer
  1600	HIBCR82	Mixed Fetal,
  		Neural/Sensory
  1601	HIBDA41	Cancer
  1602	HIBEC45	Cancer
  1603	HILBW03	Cancer
  1604	HISAE16	Digestive
  1605	HISAG53	Cancer
  1606	HISAN63	Digestive
  1607	HISAT78	Cancer
  1608	HISBA38	Digestive,
  		Immune/Hematopoietic
  1609	HISBB66	Cancer
  1610	HISCJ20	Cancer
  1611	HISCK41	Digestive
  1612	HISCO45	Cancer
  1613	HISEJ52	Cancer
  1614	HJABC58	Cancer
  1615	HJABG59	Immune/Hematopoietic
  1616	HJABR75	Immune/Hematopoietic
  1617	HJABS31	Cancer
  1618	HJABT12	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1619	HJACE25	Cancer
  1620	HJACK21	Cancer
  1621	HJBCG74	Cancer
  1622	HJBCO21	Cancer
  1623	HJBCQ40	Immune/Hematopoietic
  1624	HJBDM36	Cancer
  1625	HJMAF30	Cancer
  1626	HJMAM72	Cancer
  1627	HJMAZ60	Cancer
  1628	HJMBB20	Cancer
  1629	HJMBB20	Cancer
  1630	HJMBB20	Cancer
  1631	HJMBK59	Cancer
  1632	HJMBP01	Cancer
  1633	HJMBQ17	Cancer
  1634	HJMBW62	Reproductive
  1635	HJMBX54	Musculoskeletal,
  		Reproductive
  1636	HJPAF69	Immune/Hematopoietic
  1637	HJPAQ19	Cancer
  1638	HJPAZ35	Cancer
  1639	HJPBI77	Immune/Hematopoietic,
  		Musculoskeletal,
  		Neural/Sensory
  1640	HJPBN96	Cancer
  1641	HJPBU47	Cancer
  1642	HJPCQ19	Cancer
  1643	HJPDJ08	Immune/Hematopoietic
  1644	HJPDK61	Cancer
  1645	HKABI53	Cancer
  1646	HKABN63	Cancer
  1647	HKACA25	Cancer
  1648	HKACO64	Cancer
  1649	HKACP50	Cancer
  1650	HKACX90	Cancer
  1651	HKADI27	Cancer
  1652	HKADN26	Cancer
  1653	HKADP79	Cancer
  1654	HKADT55	Cancer
  1655	HKAEK58	Cancer
  1656	HKAEK72	Connective/Epithelial
  1657	HKAFJ47	Cancer
  1658	HKAFQ41	Cancer
  1659	HKAHH71	Cancer
  1660	HKAJA95	Cancer
  1661	HKAKU90	Cancer
  1662	HKCSZ54	Digestive
  1663	HKFAA15	Cancer
  1664	HKFBB08	Immune/Hematopoietic
  1665	HKGAG59	Cancer
  1666	HKGAJ81	Cancer
  1667	HKGAK45	Musculoskeletal,
  		Reproductive
  1668	HKGAP04	Cancer
  1669	HKGAP57	Immune/Hematopoietic
  1670	HKGAW41	Cancer
  1671	HKGBA21	Connective/Epithelial,
  		Mixed Fetal,
  		Musculoskeletal
  1672	HKGBC33	Immune/Hematopoietic
  1673	HKGBC73	Cancer
  1674	HKGBF61	Cancer
  1675	HKGBH54	Cancer
  1676	HKGBP52	Cancer
  1677	HKGCE23	Cancer
  1678	HKGCE62	Immune/Hematopoietic
  1679	HKGCK41	Cancer
  1680	HKGCK41	Cancer
  1681	HKGCN96	Cancer
  1682	HKGCX05	Cancer
  1683	HKGDA95	Cancer
  1684	HKGDO12	Cancer
  1685	HKIME53	Cancer
  1686	HKIMG23	Cancer
  1687	HKIXB73	Excretory
  1688	HKIXD68	Cancer
  1689	HKIXR91	Cancer
  1690	HKIXS19	Cancer
  1691	HKIXW45	Cancer
  1692	HKIYU90	Excretory,
  		Neural/Sensory
  1693	HKIMLB81	Excretory
  1694	HKMLF77	Excretory
  1695	HKMLM32	Excretory,
  		Neural/Sensory
  1696	HKMLR17	Cancer
  1697	HKMLT89	Excretory,
  		Immune/Hematopoietic,
  		Reproductive
  1698	HKMLV05	Excretory,
  		Immune/Hematopoietic
  1699	HKMLV25	Cancer
  1700	HKMMB79	Cancer
  1701	HKMMC69	Excretory,
  		Immune/Hematopoietic
  1702	HKMMD91	Cancer
  1703	HKMMP90	Excretory,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1704	HKMMU76	Cancer
  1705	HKPAC10	Excretory
  1706	HKPAC50	Cancer
  1707	HKPMA08	Cancer
  1708	HKTAC18	Cancer
  1709	HL1SA89	Cancer
  1710	HL2AB60	Cancer
  1711	HL3AE69	Cancer
  1712	HL3AF32	Cancer
  1713	HLDAV70	Digestive,
  		Immune/Hematopoietic
  1714	HLDBL62	Cancer
  1715	HLDBV18	Cancer
  1716	HLDBV54	Cancer
  1717	HLDCR26	Cancer
  1718	HLDDM27	Cancer
  1719	HLDDM27	Cancer
  1720	HLDNF18	Cancer
  1721	HLDNN84	Digestive,
  		Mixed Fetal
  1722	HLDOD77	Digestive
  1723	HLDOL74	Cancer
  1724	HLDPB24	Cardiovascular,
  		Digestive
  1725	HLDRU08	Cancer
  1726	HLDXF43	Cancer
  1727	HLEAA10	Immune/Hematopoietic
  1728	HLEAA24	Immune/Hematopoietic
  1729	HLHAE14	Neural/Sensory,
  		Respiratory
  1730	HLHAE14	Neural/Sensory,
  		Respiratory
  1731	HLHBS54	Cancer
  1732	HLHCB33	Digestive,
  		Reproductive,
  		Respiratory
  1733	HLHCF14	Connective/Epithelial,
  		Respiratory
  1734	HLHCG24	Cancer
  1735	HLHCH20	Cancer
  1736	HLHCN51	Digestive,
  		Immune/Hematopoietic,
  		Respiratory
  1737	HLHCT96	Cancer
  1738	HLHDC33	Immune/Hematopoietic,
  		Reproductive,
  		Respiratory
  1739	HLHDF92	Cancer
  1740	HLHDJ05	Respiratory
  1741	HLHDL37	Respiratory
  1742	HLHDL69	Cancer
  1743	HLHDL69	Cancer
  1744	HLHDL69	Cancer
  1745	HLHDL69	Cancer
  1746	HLHDM38	Cancer
  1747	HLHDR92	Neural/Sensory,
  		Respiratory
  1748	HLHDY94	Cancer
  1749	HLHEE27	Cancer
  1750	HLHEE38	Connective/Epithelial,
  		Neural/Sensory,
  		Respiratory
  1751	HLHEI72	Musculoskeletal,
  		Respiratory
  1752	HLHEX62	Excretory,
  		Immune/Hematopoietic,
  		Respiratory
  1753	HLHFK59	Digestive,
  		Respiratory
  1754	HLHFP09	Cancer
  1755	HLHGG78	Cancer
  1756	HLHSG15	Cancer
  1757	HLHSQ35	Respiratory
  1758	HLHTB92	Immune/Hematopoietic,
  		Respiratory
  1759	HLHTP55	Cancer
  1760	HLIBD74	Digestive
  1761	HLIBE41	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  1762	HLIBO16	Digestive,
  		Immune/Hematopoietic
  1763	HLJBI22	Cancer
  1764	HLJEE16	Cancer
  1765	HLLAX64	Cancer
  1766	HLLAX95	Immune/Hematopoietic
  1767	HLLCD67	Immune/Hematopoietic
  1768	HLMBX89	Cancer
  1769	HLMBZ14	Immune/Hematopoietic
  1770	HLMCT51	Immune/Hematopoietic,
  		Reproductive
  1771	HLMCT95	Cancer
  1772	HLMDD65	Cancer
  1773	HLMDH01	Immune/Hematopoietic
  1774	HLMDU23	Immune/Hematopoietic
  1775	HLMFB62	Immune/Hematopoietic
  1776	HLMFG52	Immune/Hematopoietic
  1777	HLMFU53	Cancer
  1778	HLMHG68	Cancer
  1779	HLMHN06	Immune/Hematopoietic,
  		Neural/Sensory
  1780	HLMHS15	Immune/Hematopoietic
  1781	HLMIM84	Cancer
  1782	HLMIN52	Cancer
  1783	HLMIQ83	Immune/Hematopoietic
  1784	HLMIW76	Immune/Hematopoietic
  1785	HLMMA65	Immune/Hematopoietic
  1786	HLMMT12	Digestive,
  		Immune/Hematopoietic
  1787	HLMNA19	Cardiovascular,
  		Immune/Hematopoietic
  1788	HLQAD72	Cancer
  1789	HLQAM30	Cancer
  1790	HLQAM59	Digestive
  1791	HLQBB23	Cancer
  1792	HLQBF05	Digestive,
  		Reproductive
  1793	HLQBX64	Cancer
  1794	HLQCY09	Digestive
  1795	HLQCZ43	Cancer
  1796	HLQCZ80	Digestive
  1797	HLQDK45	Digestive
  1798	HLQDM47	Digestive
  1799	HLQDU77	Cancer
  1800	HLSAD72	Connective/Epithelial
  1801	HLTCJ67	Immune/Hematopoietic
  1802	HLTCM28	Immune/Hematopoietic,
  		Respiratory
  1803	HLTCO22	Cancer
  1804	HLTDA14	Immune/Hematopoietic
  1805	HLTDC26	Cancer
  1806	HLTDC26	Cancer
  1807	HLTDI20	Cancer
  1808	HLTDI65	Immune/Hematopoietic
  1809	HLTDK30	Cancer
  1810	HLTDL37	Cancer
  1811	HLTDU35	Cancer
  1812	HLTDX04	Cancer
  1813	HLTEH84	Cancer
  1814	HLTEL39	Cardiovascular,
  		Immune/Hematopoietic
  1815	HLTEN11	Cancer
  1816	HLTEW52	Immune/Hematopoietic
  1817	HLTEZ36	Cancer
  1818	HLTGG14	Cancer
  1819	HLUAF94	Immune/Hematopoietic
  1820	HLWAH33	Mixed Fetal,
  		Reproductive
  1821	HLWAO11	Cancer
  1822	HLWAW73	Cancer
  1823	HLWAX50	Cancer
  1824	HLWBJ93	Cancer
  1825	HLWBK16	Cardiovascular,
  		Connective/Epithelial,
  		Reproductive
  1826	HLWCC11	Reproductive
  1827	HLYAH81	Immune/Hematopoietic
  1828	HLYAH92	Immune/Hematopoietic
  1829	HLYAJ79	Cancer
  1830	HLYAL28	Immune/Hematopoietic
  1831	HLYAR30	Cancer
  1832	HLYAT54	Immune/Hematopoietic
  1833	HLYBC81	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Reproductive
  1834	HLYBD09	Immune/Hematopoietic
  1835	HLYBL67	Immune/Hematopoietic
  1836	HLYBM38	Digestive,
  		Immune/Hematopoietic
  1837	HLYBN23	Immune/Hematopoietic
  1838	HLYBN71	Immune/Hematopoietic
  1839	HLYBS25	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  1840	HLYBT28	Immune/Hematopoietic
  1841	HLYBU15	Immune/Hematopoietic
  1842	HLYBY04	Immune/Hematopoietic
  1843	HLYCE15	Digestive,
  		Immune/Hematopoietic
  1844	HLYCH04	Immune/Hematopoietic
  1845	HLYCY48	Immune/Hematopoietic
  1846	HLYDE38	Immune/Hematopoietic
  1847	HLYDG55	Immune/Hematopoietic
  1848	HLYDO73	Immune/Hematopoietic
  1849	HLYEA60	Cancer
  1850	HLYEJ14	Cancer
  1851	HLYEJ44	Cancer
  1852	HLYEU51	Immune/Hematopoietic
  1853	HLYGV19	Cancer
  1854	HMABK52	Immune/Hematopoietic
  1855	HMACF34	Immune/Hematopoietic
  1856	HMACL77	Cancer
  1857	HMACT74	Immune/Hematopoietic
  1858	HMADJ14	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Musculoskeletal
  1859	HMADJ74	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Musculoskeletal
  1860	HMAEA58	Cancer
  1861	HMAGF01	Cancer
  1862	HMAJS26	Cancer
  1863	HMCED78	Cancer
  1864	HMCFN86	Cancer
  1865	HMCGJ47	Cancer
  1866	HMCGK88	Cancer
  1867	HMCIH27	Cancer
  1868	HMCIQ20	Cardiovascular,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1869	HMCJC19	Immune/Hematopoietic
  1870	HMDAB44	Neural/Sensory
  1871	HMDAE88	Neural/Sensory
  1872	HMDAG62	Cancer
  1873	HMDAK20	Neural/Sensory
  1874	HMDAM08	Neural/Sensory
  1875	HMDAM39	Neural/Sensory
  1876	HMEAA41	Cancer
  1877	HMECM77	Cardiovascular
  1878	HMEEH21	Cardiovascular
  1879	HMEET36	Cancer
  1880	HMEEZ07	Cardiovascular,
  		Reproductive
  1881	HMEFB15	Cardiovascular
  1882	HMEIH57	Cardiovascular,
  		Immune/Hematopoietic
  1883	HMEIJ21	Cancer
  1884	HMEIX79	Cancer
  1885	HMEJC96	Cancer
  1886	HMEJD36	Cardiovascular,
  		Endocrine,
  		Immune/Hematopoietic
  1887	HMEJK28	Cancer
  1888	HMEKH55	Cancer
  1889	HMEKW44	Cardiovascular,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1890	HMEKW71	Cancer
  1891	HMELW26	Cancer
  1892	HMGBT32	Cancer
  1893	HMHBI09	Cancer
  1894	HMHBI93	Cancer
  1895	HMHBP74	Cancer
  1896	HMIAC52	Cancer
  1897	HMIAD75	Neural/Sensory
  1898	HMIAG42	Cancer
  1899	HMIAG55	Cancer
  1900	HMIAG72	Cancer
  1901	HMIAL39	Cancer
  1902	HMIAO82	Cancer
  1903	HMIAR42	Cancer
  1904	HMLAV33	Immune/Hematopoietic,
  		Mixed Fetal,
  		Neural/Sensory
  1905	HMIAZ24	Cancer
  1906	HMIBD93	Cancer
  1907	HMIBE95	Neural/Sensory
  1908	HMIBG57	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  1909	HMJAC12	Neural/Sensory
  1910	HMKAN71	Cancer
  1911	HMKBA33	Neural/Sensory
  1912	HMKCI22	Cancer
  1913	HMKCK32	Neural/Sensory
  1914	HMKCP81	Cancer
  1915	HMKCY49	Immune/Hematopoietic,
  		Neural/Sensory
  1916	HMKDD51	Neural/Sensory
  1917	HMKDG69	Cardiovascular,
  		Neural/Sensory
  1918	HMKDM80	Neural/Sensory
  1919	HMKEG88	Neural/Sensory
  1920	HMMAA09	Immune/Hematopoietic
  1921	HMMAK92	Immune/Hematopoietic
  1922	HMMAL32	Immune/Hematopoietic
  1923	HMMBD19	Immune/Hematopoietic,
  		Reproductive
  1924	HMMBF22	Immune/Hematopoietic,
  		Reproductive
  1925	HMMBH91	Immune/Hematopoietic
  1926	HMMBH94	Immune/Hematopoietic
  1927	HMMBK55	Immune/Hematopoietic
  1928	HMMBQ31	Cardiovascular,
  		Immune/Hematopoietic
  1929	HMMBR63	Cancer
  1930	HMMBS55	Immune/Hematopoietic,
  		Reproductive
  1931	HMMBT47	Immune/Hematopoietic
  1932	HMMCD35	Immune/Hematopoietic
  1933	HMMCD95	Immune/Hematopoietic,
  		Neural/Sensory
  1934	HMPAB26	Cancer
  1935	HMPAP48	Immune/Hematopoietic
  1936	HMQAI38	Immune/Hematopoietic,
  		Reproductive
  1937	HMQAT69	Cancer
  1938	HMQBL90	Digestive,
  		Immune/Hematopoietic
  1939	HMQBV82	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  1940	HMQCA75	Cancer
  1941	HMQCB37	Cancer
  1942	HMQCL80	Immune/Hematopoietic
  1943	HMQCX41	Immune/Hematopoietic
  1944	HMQDM09	Cancer
  1945	HMQDU07	Digestive,
  		Immune/Hematopoietic,
  		Musculoskeletal
  1946	HMSAP33	Immune/Hematopoietic
  1947	HMSAZ48	Immune/Hematopoietic
  1948	HMSBN18	Cancer
  1949	HMSBS25	Immune/Hematopoietic
  1950	HMSBU14	Immune/Hematopoietic
  1951	HMSBZ10	Immune/Hematopoietic
  1952	HMSCB94	Immune/Hematopoietic,
  		Reproductive
  1953	HMSCK12	Immune/Hematopoietic
  1954	HMSCP63	Immune/Hematopoietic
  1955	HMSCV75	Immune/Hematopoietic
  1956	HMSCV85	Immune/Hematopoietic,
  		Reproductive
  1957	HMSCW44	Immune/Hematopoietic,
  		Mixed Fetal,
  		Neural/Sensory
  1958	HMSCZ19	Cancer
  1959	HMSDI67	Digestive,
  		Immune/Hematopoietic
  1960	HMSDI79	Cancer
  1961	HMSDR28	Cancer
  1962	HMSFT25	Immune/Hematopoietic
  1963	HMSFW52	Immune/Hematopoietic
  1964	HMSGT73	Immune/Hematopoietic
  1965	HMSGU30	Cancer
  1966	HMSHB42	Cancer
  1967	HMSHB42	Cancer
  1968	HMSHN72	Immune/Hematopoietic,
  		Reproductive
  1969	HMSHT29	Immune/Hematopoietic
  1970	HMSHW73	Immune/Hematopoietic
  1971	HMSIC48	Cardiovascular,
  		Immune/Hematopoietic
  1972	HMSII36	Immune/Hematopoietic
  1973	HMSIT42	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1974	HMSJB08	Cancer
  1975	HMSJI69	Immune/Hematopoietic
  1976	HMSJM20	Immune/Hematopoietic
  1977	HMSJR44	Immune/Hematopoietic
  1978	HMSKQ91	Immune/Hematopoietic
  1979	HMSKY45	Immune/Hematopoietic
  1980	HMTAF92	Cancer
  1981	HMTAT36	Cancer
  1982	HMUAB93	Cancer
  1983	HMUAD65	Immune/Hematopoietic,
  		Musculoskeletal
  1984	HMUAT23	Cancer
  1985	HMUBA47	Cancer
  1986	HMUBJ22	Cancer
  1987	HMUBK53	Cancer
  1988	HMUBN24	Musculoskeletal
  1989	HMUBO15	Cancer
  1990	HMUBX48	Musculoskeletal,
  		Reproductive
  1991	HMUBY57	Cancer
  1992	HMUBZ15	Cancer
  1993	HMVAL15	Cancer
  1994	HMVBC84	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  1995	HMVBD68	Cancer
  1996	HMVCG17	Immune/Hematopoietic
  1997	HMVCS92	Cancer
  1998	HMVCS92	Cancer
  1999	HMVDB45	Immune/Hematopoietic
  2000	HMVDJ71	Cancer
  2001	HMVDT89	Cancer
  2002	HMVDT89	Cancer
  2003	HMWAO65	Cancer
  2004	HMWAO82	Immune/Hematopoietic
  2005	HMWBD74	Cancer
  2006	HMWBK35	Cancer
  2007	HMWBK86	Immune/Hematopoietic,
  		Mixed Fetal
  2008	HMWBL38	Connective/Epithelial,
  		Immune/Hematopoietic
  2009	HMWBM48	Cancer
  2010	HMWCG28	Cancer
  2011	HMWCP85	Digestive,
  		Immune/Hematopoietic
  2012	HMWDG30	Cancer
  2013	HMWDU20	Cancer
  2014	HMWDX57	Digestive,
  		Immune/Hematopoietic,
  		Respiratory
  2015	HMWDZ63	Immune/Hematopoietic
  2016	HMWEA77	Immune/Hematopoietic
  2017	HMWEC03	Cancer
  2018	HMWEF46	Immune/Hematopoietic
  2019	HMWEK43	Immune/Hematopoietic
  2020	HMWEM23	Cancer
  2021	HMWEM23	Cancer
  2022	HMWER46	Cancer
  2023	HMWEU96	Cancer
  2024	HMWEX02	Cancer
  2025	HMWFB65	Cancer
  2026	HMWFD77	Immune/Hematopoietic
  2027	HMWFO25	Immune/Hematopoietic
  2028	HMWFO89	Cancer
  2029	HMWGM41	Cancer
  2030	HMWGO95	Immune/Hematopoietic
  2031	HMWGV85	Cancer
  2032	HMWGZ42	Immune/Hematopoietic
  2033	HMWHR36	Immune/Hematopoietic
  2034	HMWIM55	Immune/Hematopoietic
  2035	HMWIQ26	Cancer
  2036	HMWIU49	Cancer
  2037	HMWJJ62	Cancer
  2038	HMWJJ64	Cancer
  2039	HNAAD76	Digestive,
  		Immune/Hematopoietic
  2040	HNAAE24	Digestive
  2041	HNALD94	Cancer
  2042	HNALE44	Cancer
  2043	HNDAC35	Cancer
  2044	HNEAA04	Immune/Hematopoietic
  2045	HNEAH26	Immune/Hematopoietic,
  		Neural/Sensory
  2046	HNEAK38	Immune/Hematopoietic
  2047	HNEAK65	Cancer
  2048	HNEBX72	Immune/Hematopoietic,
  		Neural/Sensory
  2049	HNEBY79	Immune/Hematopoietic
  2050	HNECD52	Immune/Hematopoietic,
  		Neural/Sensory
  2051	HNECL75	Cancer
  2052	HNECX90	Cancer
  2053	HNECX90	Cancer
  2054	HNEDA05	Immune/Hematopoietic
  2055	HNEDP75	Immune/Hematopoietic
  2056	HNEDQ02	Cancer
  2057	HNEDU46	Cancer
  2058	HNFAD50	Cancer
  2059	HNFAD50	Cancer
  2060	HNFAG67	Cancer
  2061	HNFCJ77	Immune/Hematopoietic,
  		Reproductive
  2062	HNFCO56	Cancer
  2063	HNFCY57	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Respiratory
  2064	HNFDL89	Digestive,
  		Immune/Hematopoietic
  2065	HNFDT73	Excretory,
  		Immune/Hematopoietic,
  		Reproductive
  2066	HNFDU92	Immune/Hematopoietic
  2067	HNFDY09	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  2068	HNFDY31	Cancer
  2069	HNFEA17	Cancer
  2070	HNFEP55	Cancer
  2071	HNFET12	Immune/Hematopoietic
  2072	HNFFR59	Immune/Hematopoietic
  2073	HNFGC51	Cancer
  2074	HNFGR15	Immune/Hematopoietic
  2075	HNFGW37	Immune/Hematopoietic
  2076	HNFGW53	Cancer
  2077	HNFHA34	Cancer
  2078	HNFHD58	Cancer
  2079	HNFHV68	Immune/Hematopoietic
  2080	HNFIE15	Cancer
  2081	HNFIE29	Immune/Hematopoietic
  2082	HNFIG49	Immune/Hematopoietic
  2083	HNFJE27	Immune/Hematopoietic
  2084	HNFJG16	Immune/Hematopoietic
  2085	HNGAC71	Digestive,
  		Immune/Hematopoietic
  2086	HNGAK42	Immune/Hematopoietic
  2087	HNGAL25	Immune/Hematopoietic
  2088	HNGAT83	Immune/Hematopoietic
  2089	HNGAX06	Cancer
  2090	HNGBB09	Immune/Hematopoietic
  2091	HNGBC53	Immune/Hematopoietic
  2092	HNGBD94	Immune/Hematopoietic
  2093	HNGBE44	Digestive,
  		Immune/Hematopoietic
  2094	HNGBE63	Immune/Hematopoietic
  2095	HNGBI83	Immune/Hematopoietic
  2096	HNGBJ74	Immune/Hematopoietic
  2097	HNGBP30	Immune/Hematopoietic
  2098	HNGBQ61	Immune/Hematopoietic
  2099	HNGBS35	Immune/Hematopoietic
  2100	HNGBW25	Immune/Hematopoietic
  2101	HNGCF29	Immune/Hematopoietic
  2102	HNGCF64	Immune/Hematopoietic
  2103	HNGDF54	Cancer
  2104	HNGDH22	Immune/Hematopoietic
  2105	HNGDH27	Immune/Hematopoietic
  2106	HNGDN07	Immune/Hematopoietic,
  		Reproductive
  2107	HNGDO65	Cancer
  2108	HNGDR39	Immune/Hematopoietic
  2109	HNGDW78	Immune/Hematopoietic
  2110	HNGEA90	Immune/Hematopoietic
  2111	HNGEC17	Immune/Hematopoietic
  2112	HNGEE06	Immune/Hematopoietic
  2113	HNGEF70	Immune/Hematopoietic
  2114	HNGEF72	Immune/Hematopoietic
  2115	HNGEI64	Immune/Hematopoietic
  2116	HNGEJ33	Cancer
  2117	HNGEK64	Immune/Hematopoietic
  2118	HNGEN32	Immune/Hematopoietic
  2119	HNGER85	Immune/Hematopoietic
  2120	HNGES90	Immune/Hematopoietic
  2121	HNGET33	Immune/Hematopoietic
  2122	HNGEX18	Immune/Hematopoietic
  2123	HNGEY45	Immune/Hematopoietic
  2124	HNGEZ02	Immune/Hematopoietic,
  		Reproductive
  2125	HNGEZ90	Immune/Hematopoietic
  2126	HNGFA25	Immune/Hematopoietic
  2127	HNGFB05	Immune/Hematopoietic
  2128	HNGFD30	Immune/Hematopoietic,
  		Mixed Fetal
  2129	HNGFD31	Immune/Hematopoietic
  2130	HNGFD61	Excretory,
  		Immune/Hematopoietic
  2131	HNGFG04	Immune/Hematopoietic
  2132	HNGFG74	Immune/Hematopoietic
  2133	HNGFH32	Immune/Hematopoietic
  2134	HNGFH83	Immune/Hematopoietic
  2135	HNGFI21	Cancer
  2136	HNGFM31	Immune/Hematopoietic
  2137	HNGFN77	Immune/Hematopoietic
  2138	HNGFQ18	Immune/Hematopoietic
  2139	HNGFR54	Immune/Hematopoietic
  2140	HNGFT70	Immune/Hematopoietic
  2141	HNGFU70	Immune/Hematopoietic
  2142	HNGFV39	Immune/Hematopoietic
  2143	HNGGF13	Immune/Hematopoietic
  2144	HNGGK63	Immune/Hematopoietic
  2145	HNGGK65	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  2146	HNGGL11	Immune/Hematopoietic
  2147	HNGGO05	Immune/Hematopoietic
  2148	HNGGS92	Immune/Hematopoietic
  2149	HNGGT10	Cancer
  2150	HNGGT74	Immune/Hematopoietic
  2151	HNGHB89	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2152	HNGHD07	Immune/Hematopoietic
  2153	HNGHK37	Immune/Hematopoietic
  2154	HNGHM47	Immune/Hematopoietic
  2155	HNGHT01	Immune/Hematopoietic,
  		Neural/Sensory
  2156	HNGHT86	Immune/Hematopoietic
  2157	HNGIH40	Immune/Hematopoietic
  2158	HNGIK07	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Musculoskeletal
  2159	HNGIM40	Immune/Hematopoietic
  2160	HNGIM83	Immune/Hematopoietic
  2161	HNGIO93	Cancer
  2162	HNGIS27	Immune/Hematopoietic
  2163	HNGIU16	Immune/Hematopoietic,
  		Reproductive
  2164	HNGIX91	Immune/Hematopoietic
  2165	HNGJA68	Immune/Hematopoietic
  2166	HNGJB57	Immune/Hematopoietic
  2167	HNGJE86	Immune/Hematopoietic
  2168	HNGJH26	Immune/Hematopoietic
  2169	HNGJJ61	Immune/Hematopoietic
  2170	HNGJL07	Immune/Hematopoietic,
  		Neural/Sensory
  2171	HNGJS66	Immune/Hematopoietic,
  		Reproductive
  2172	HNGJU60	Immune/Hematopoietic
  2173	HNGKB09	Immune/Hematopoietic,
  		Reproductive
  2174	HNGKW35	Immune/Hematopoietic
  2175	HNGKY94	Immune/Hematopoietic
  2176	HNGLD28	Immune/Hematopoietic
  2177	HNGOY36	Immune/Hematopoietic
  2178	HNHAB38	Immune/Hematopoietic,
  		Musculoskeletal
  2179	HNHAC43	Cancer
  2180	HNHAD34	Immune/Hematopoietic
  2181	HNHAG83	Immune/Hematopoietic,
  		Mixed Fetal,
  		Musculoskeletal
  2182	HNHAH06	Immune/Hematopoietic
  2183	HNHAJ65	Immune/Hematopoietic
  2184	HNHAL61	Immune/Hematopoietic
  2185	HNHAP58	Cancer
  2186	HNHAW34	Immune/Hematopoietic
  2187	HNHAW35	Immune/Hematopoietic
  2188	HNHAY26	Immune/Hematopoietic
  2189	HNHAY86	Immune/Hematopoietic,
  		Neural/Sensory
  2190	HNHAZ20	Immune/Hematopoietic
  2191	HNHBE21	Immune/Hematopoietic
  2192	HNHBE38	Cancer
  2193	HNHBG18	Immune/Hematopoietic
  2194	HNHBI65	Immune/Hematopoietic
  2195	HNHBM16	Immune/Hematopoietic,
  		Neural/Sensory
  2196	HNHCH78	Immune/Hematopoietic
  2197	HNHCP14	Immune/Hematopoietic
  2198	HNHCQ44	Immune/Hematopoietic
  2199	HNHCT22	Cardiovascular,
  		Immune/Hematopoietic
  2200	HNHCT47	Cardiovascular,
  		Immune/Hematopoietic
  2201	HNHCV48	Immune/Hematopoietic
  2202	HNHCZ54	Cancer
  2203	HNHDC52	Immune/Hematopoietic,
  		Neural/Sensory
  2204	HNHDD95	Immune/Hematopoietic
  2205	HNHDE58	Cancer
  2206	HNHDI17	Immune/Hematopoietic
  2207	HNHDL37	Immune/Hematopoietic
  2208	HNHDM21	Immune/Hematopoietic
  2209	HNHDR57	Immune/Hematopoietic
  2210	HNHDR96	Immune/Hematopoietic
  2211	HNHDU62	Immune/Hematopoietic
  2212	HNHDW34	Immune/Hematopoietic
  2213	HNHDX28	Immune/Hematopoietic
  2214	HNHDZ06	Immune/Hematopoietic
  2215	HNHDZ42	Immune/Hematopoietic
  2216	HNHEF37	Immune/Hematopoietic
  2217	HNHEF49	Immune/Hematopoietic
  2218	HNHEF70	Immune/Hematopoietic
  2219	HNHEG30	Immune/Hematopoietic
  2220	HNHEH38	Immune/Hematopoietic
  2221	HNHEL22	Immune/Hematopoietic
  2222	HNHEN70	Cancer
  2223	HNHEP21	Immune/Hematopoietic
  2224	HNHEP41	Immune/Hematopoietic
  2225	HNHES33	Immune/Hematopoietic,
  		Reproductive
  2226	HNHET16	Immune/Hematopoietic
  2227	HNHEY29	Immune/Hematopoietic
  2228	HNHEZ76	Immune/Hematopoietic
  2229	HNHFF60	Immune/Hematopoietic
  2230	HNHFF81	Immune/Hematopoietic,
  		Neural/Sensory
  2231	HNHFJ49	Immune/Hematopoietic
  2232	HNHFR42	Immune/Hematopoietic
  2233	HNHFX25	Immune/Hematopoietic,
  		Musculoskeletal
  2234	HNHGD95	Cardiovascular,
  		Immune/Hematopoietic
  2235	HNHGR82	Immune/Hematopoietic
  2236	HNHGS62	Immune/Hematopoietic
  2237	HNHGY77	Cancer
  2238	HNHHA47	Digestive,
  		Immune/Hematopoietic,
  		Respiratory
  2239	HNHHN22	Immune/Hematopoietic
  2240	HNHHW53	Immune/Hematopoietic
  2241	HNHIB40	Immune/Hematopoietic
  2242	HNHKI74	Immune/Hematopoietic
  2243	HNHKV56	Immune/Hematopoietic
  2244	HNHLD80	Immune/Hematopoietic
  2245	HNHLS76	Immune/Hematopoietic
  2246	HNHLZ47	Immune/Hematopoietic
  2247	HNHMP15	Immune/Hematopoietic
  2248	HNHMP62	Immune/Hematopoietic
  2249	HNHMY76	Immune/Hematopoietic,
  		Reproductive
  2250	HNHMZ01	Immune/Hematopoietic
  2251	HNHND14	Immune/Hematopoietic
  2252	HNHND94	Immune/Hematopoietic
  2253	HNHOF09	Immune/Hematopoietic
  2254	HNKAA76	Cancer
  2255	HNTAF42	Cancer
  2256	HNTCG32	Cancer
  2257	HNTNY89	Cancer
  2258	HNTRB25	Cancer
  2259	HNTRQ40	Cancer
  2260	HNTSQ23	Cancer
  2261	HOAAH51	Cancer
  2262	HOAAI76	Cancer
  2263	HOAAJ09	Cancer
  2264	HOAAL10	Musculoskeletal
  2265	HOAAU13	Mixed Fetal,
  		Musculoskeletal
  2266	HOABC12	Cancer
  2267	HOABH36	Cancer
  2268	HOBNA89	Musculoskeletal
  2269	HOBNF51	Cancer
  2270	HODAH24	Reproductive
  2271	HODAH46	Cancer
  2272	HODAV25	Cancer
  2273	HODAW64	Cardiovascular,
  		Neural/Sensory,
  		Reproductive
  2274	HODAY17	Cancer
  2275	HODBA45	Reproductive
  2276	HODBC79	Cancer
  2277	HODBD79	Immune/Hematopoietic,
  		Reproductive
  2278	HODBF12	Cancer
  2279	HODBF86	Digestive,
  		Reproductive
  2280	HODBF91	Cancer
  2281	HODBW34	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2282	HODBX93	Reproductive
  2283	HODBZ06	Cancer
  2284	HODCA73	Cancer
  2285	HODCV86	Immune/Hematopoietic,
  		Reproductive
  2286	HODCY44	Reproductive
  2287	HODDB58	Neural/Sensory,
  		Reproductive
  2288	HODDG72	Cancer
  2289	HODDJ25	Cancer
  2290	HODDN21	Reproductive
  2291	HODDO31	Reproductive
  2292	HODDQ06	Cancer
  2293	HODEA20	Cancer
  2294	HODEM38	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2295	HODET37	Reproductive
  2296	HOEBI94	Cancer
  2297	HOEBJ70	Cancer
  2298	HOECB33	Cancer
  2299	HOECX21	Cancer
  2300	HOEDE27	Musculoskeletal
  2301	HOEEK81	Cancer
  2302	HOEEZ62	Musculoskeletal
  2303	HOEFJ26	Cancer
  2304	HOEFL74	Cardiovascular,
  		Digestive,
  		Musculoskeletal
  2305	HOFMF63	Cancer
  2306	HOFMJ65	Cancer
  2307	HOFMK02	Immune/Hematopoietic,
  		Reproductive
  2308	HOFMO16	Reproductive
  2309	HOFMP62	Reproductive
  2310	HOFMT59	Reproductive
  2311	HOFMV22	Reproductive
  2312	HOFND06	Digestive,
  		Reproductive
  2313	HOFNY15	Reproductive
  2314	HOFNY28	Reproductive
  2315	HOFOC41	Reproductive
  2316	HOGAA41	Cancer
  2317	HOGAB51	Immune/Hematopoietic,
  		Reproductive
  2318	HOGAH40	Cancer
  2319	HOGAP06	Immune/Hematopoietic,
  		Reproductive
  2320	HOGAR36	Reproductive
  2321	HOGAR71	Cancer
  2322	HOGCC26	Cancer
  2323	HOGCD78	Reproductive
  2324	HOGCK03	Cancer
  2325	HOGCL01	Cancer
  2326	HOHBB36	Cancer
  2327	HOHBC57	Cancer
  2328	HOHBO66	Cancer
  2329	HOHBZ10	Cancer
  2330	HOHCH71	Cancer
  2331	HOHEB48	Musculoskeletal
  2332	HONAH67	Digestive,
  		Excretory,
  		Reproductive
  2333	HOOAC84	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  2334	HOPBP13	Cancer
  2335	HOQBG21	Cancer
  2336	HORBI80	Cancer
  2337	HORBL77	Cancer
  2338	HOSBX14	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  2339	HOSCZ41	Cancer
  2340	HOSEM81	Cancer
  2341	HOSEO83	Cancer
  2342	HOSFR35	Cancer
  2343	HOUAZ32	Cancer
  2344	HOUBC29	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Reproductive
  2345	HOUBG39	Connective/Epithelial,
  		Immune/Hematopoietic,
  		Musculoskeletal
  2346	HOUCD12	Connective/Epithelial
  2347	HOUDB17	Connective/Epithelial
  2348	HOUDX40	Connective/Epithelial
  2349	HOUEF84	Cancer
  2350	HOUEJ43	Connective/Epithelial
  2351	HOUGS36	Connective/Epithelial
  2352	HOUHQ36	Connective/Epithelial
  2353	HOUIG68	Cancer
  2354	HOUIG92	Cancer
  2355	HOVAD93	Reproductive
  2356	HOVAE10	Cancer
  2357	HOVAE36	Reproductive
  2358	HOVAE82	Immune/Hematopoietic,
  		Reproductive
  2359	HOVAJ68	Reproductive
  2360	HOVAW46	Musculoskeletal,
  		Reproductive
  2361	HOVBB19	Musculoskeletal,
  		Reproductive
  2362	HOVBD31	Cancer
  2363	HOVBE81	Reproductive
  2364	HOVBI16	Cancer
  2365	HOVBS68	Cancer
  2366	HOVCC73	Immune/Hematopoietic,
  		Reproductive
  2367	HOVCF30	Immune/Hematopoietic,
  		Reproductive
  2368	HOVCJ71	Reproductive
  2369	HOVCN53	Reproductive
  2370	HOVCO53	Reproductive
  2371	HPASF94	Cancer
  2372	HPBCG26	Cancer
  2373	HPBCT11	Reproductive
  2374	HPBDE33	Cancer
  2375	HPBDE33	Cancer
  2376	HPBDF31	Cancer
  2377	HPCAG17	Immune/Hematopoietic,
  		Reproductive
  2378	HPCAG17	Immune/Hematopoietic,
  		Reproductive
  2379	HPCAM02	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  2380	HPDDQ17	Endocrine
  2381	HPDDQ28	Endocrine,
  		Musculoskeletal
  2382	HPDDT14	Cancer
  2383	HPEAA65	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2384	HPEAG24	Cancer
  2385	HPEBA84	Immune/Hematopoietic,
  		Reproductive
  2386	HPEBF91	Cancer
  2387	HPEBI09	Digestive,
  		Reproductive
  2388	HPFCJ75	Cancer
  2389	HPFCP75	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  2390	HPFDB66	Cancer
  2391	HPFDD28	Reproductive
  2392	HPFDI47	Digestive,
  		Reproductive
  2393	HPIAF35	Reproductive
  2394	HPIAK27	Cancer
  2395	HPIAL55	Cancer
  2396	HPIAT18	Cancer
  2397	HPIAZ52	Reproductive
  2398	HPIBA07	Cancer
  2399	HPIBA24	Cancer
  2400	HPIBI40	Cancer
  2401	HPIBT19	Reproductive
  2402	HPJAA82	Reproductive
  2403	HPJAB75	Cancer
  2404	HPJAN76	Cancer
  2405	HPJAN76	Cancer
  2406	HPJAU94	Immune/Hematopoietic,
  		Reproductive
  2407	HPJAW78	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  2408	HPJBS16	Connective/Epithelial,
  		Reproductive
  2409	HPJBU04	Immune/Hematopoietic,
  		Reproductive
  2410	HPJCN83	Reproductive
  2411	HPJCP75	Reproductive
  2412	HPJCV35	Reproductive
  2413	HPJCX13	Cancer
  2414	HPLAW13	Cancer
  2415	HPMAI31	Cancer
  2416	HPMBI91	Reproductive
  2417	HPMBT05	Reproductive
  2418	HPMBW95	Cancer
  2419	HPMCW10	Cancer
  2420	HPMCZ18	Cancer
  2421	HPMDA80	Cancer
  2422	HPMDD27	Cancer
  2423	HPMDF45	Excretory,
  		Immune/Hematopoietic,
  		Reproductive
  2424	HPMDP57	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  2425	HPMEG72	Cancer
  2426	HPMFM70	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  2427	HPMFP48	Cancer
  2428	HPMFW01	Cancer
  2429	HPMGM06	Digestive,
  		Neural/Sensory,
  		Reproductive
  2430	HPMGW43	Cancer
  2431	HPMGY89	Cancer
  2432	HPMKB09	Cancer
  2433	HPMSH26	Cancer
  2434	HPMSH96	Mixed Fetal,
  		Reproductive
  2435	HPQAJ25	Cardiovascular,
  		Digestive,
  		Mixed Fetal
  2436	HPQAJ27	Cancer
  2437	HPQAN50	Reproductive
  2438	HPQAO80	Cancer
  2439	HPQAW27	Cancer
  2440	HPQBC90	Cancer
  2441	HPQBJ48	Cancer
  2442	HPQBJ48	Cancer
  2443	HPQBL67	Cancer
  2444	HPQBT17	Cancer
  2445	HPQCF94	Cancer
  2446	HPQCI62	Cancer
  2447	HPQRS74	Cancer
  2448	HPRAD30	Cancer
  2449	HPRCC91	Cancer
  2450	HPRCF40	Cancer
  2451	HPRCF50	Cancer
  2452	HPRCL58	Reproductive
  2453	HPRCM72	Cancer
  2454	HPRCS59	Reproductive
  2455	HPRCT73	Cancer
  2456	HPRTH56	Cancer
  2457	HPTRE80	Cancer
  2458	HPTRI42	Cancer
  2459	HPTRL95	Cancer
  2460	HPTRQ52	Cancer
  2461	HPTTH35	Cancer
  2462	HPTTI65	Endocrine,
  		Reproductive
  2463	HPTTT62	Cancer
  2464	HPTVH24	Cancer
  2465	HPTVH59	Endocrine,
  		Neural/Sensory
  2466	HPTVI04	Cancer
  2467	HPTVI96	Cancer
  2468	HPVAA15	Cancer
  2469	HPVAB20	Cancer
  2470	HPVAB63	Cancer
  2471	HPVAF86	Reproductive
  2472	HPWAH55	Digestive
  2473	HPWAO89	Immune/Hematopoietic,
  		Reproductive
  2474	HPWAS27	Cancer
  2475	HPWAT86	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  2476	HPWAV82	Reproductive
  2477	HPWBA36	Reproductive
  2478	HPWTF23	Cancer
  2479	HPWTF23	Cancer
  2480	HPWTF53	Cancer
  2481	HPXAB56	Immune/Hematopoietic
  2482	HPZAB75	Digestive,
  		Reproductive
  2483	HRAAB26	Excretory
  2484	HRAAC36	Excretory,
  		Immune/Hematopoietic
  2485	HRAAF59	Excretory
  2486	HRAAG89	Cancer
  2487	HRAAO40	Excretory
  2488	HRAAZ12	Cancer
  2489	HRABA19	Excretory,
  		Reproductive
  2490	HRABP28	Excretory,
  		Immune/Hematopoietic
  2491	HRABU56	Cardiovascular,
  		Excretory,
  		Musculoskeletal
  2492	HRABZ80	Excretory,
  		Immune/Hematopoietic,
  		Musculoskeletal
  2493	HRACB01	Excretory
  2494	HRACI39	Excretory
  2495	HRADU15	Excretory
  2496	HRDAH04	Immune/Hematopoietic,
  		Mixed Fetal,
  		Musculoskeletal
  2497	HRDBA20	Musculoskeletal
  2498	HRDBD32	Musculoskeletal
  2499	HRDBL01	Mixed Fetal,
  		Musculoskeletal,
  		Neural/Sensory
  2500	HRDDM85	Musculoskeletal
  2501	HRDDS22	Cancer
  2502	HRDEJ86	Cancer
  2503	HRDEQ34	Musculoskeletal
  2504	HRDFE30	Cancer
  2505	HRDFT83	Musculoskeletal
  2506	HRGCA01	Musculoskeletal
  2507	HRGCA06	Cancer
  2508	HRGSE38	Cancer
  2509	HRLAT43	Cancer
  2510	HRLME03	Cancer
  2511	HROAN20	Cardiovascular,
  		Digestive
  2512	HROAP64	Digestive
  2513	HROAS35	Digestive
  2514	HROAY16	Digestive
  2515	HROBJ10	Cancer
  2516	HROBW46	Digestive,
  		Immune/Hematopoietic
  2517	HRODG86	Cancer
  2518	HRSAL26	Cancer
  2519	HRTAE88	Digestive
  2520	HRTAP63	Cancer
  2521	HRTAR24	Digestive,
  		Immune/Hematopoietic
  2522	HSAAN03	Cancer
  2523	HSAAS05	Immune/Hematopoietic,
  		Neural/Sensory
  2524	HSAAW13	Cancer
  2525	HSABA15	Cancer
  2526	HSABG81	Cancer
  2527	HSATA50	Cardiovascular,
  		Immune/Hematopoietic,
  		Musculoskeletal
  2528	HSATA61	Cancer
  2529	HSATG66	Cancer
  2530	HSATI91	Immune/Hematopoietic
  2531	HSATR50	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2532	HSATT82	Immune/Hematopoietic
  2533	HSATW19	Immune/Hematopoietic,
  		Musculoskeletal
  2534	HSATW67	Excretory,
  		Immune/Hematopoietic,
  		Reproductive
  2535	HSATZ02	Immune/Hematopoietic,
  		Musculoskeletal
  2536	HSAUA95	Immune/Hematopoietic
  2537	HSAUB89	Cancer
  2538	HSAUI53	Immune/Hematopoietic
  2539	HSAUV74	Cancer
  2540	HSAUX39	Immune/Hematopoietic
  2541	HSAVA58	Immune/Hematopoietic,
  		Mixed Fetal
  2542	HSAVE52	Immune/Hematopoietic
  2543	HSAVH32	Immune/Hematopoietic
  2544	HSAVM49	Immune/Hematopoietic
  2545	HSAVO11	Immune/Hematopoietic,
  		Neural/Sensory
  2546	HSAVO17	Immune/Hematopoietic,
  		Musculoskeletal
  2547	HSAVQ13	Immune/Hematopoietic
  2548	HSAVR85	Cancer
  2549	HSAVY92	Immune/Hematopoietic,
  		Neural/Sensory
  2550	HSAVZ05	Digestive,
  		Immune/Hematopoictic
  2551	HSAWB58	Immune/Hematopoietic
  2552	HSAWH36	Immune/Hematopoietic
  2553	HSAWM20	Immune/Hematopoietic
  2554	HSAWM74	Cancer
  2555	HSAWX70	Cancer
  2556	HSAXC22	Immune/Hematopoietic
  2557	HSAXI10	Digestive,
  		Immune/Hematopoietic
  2558	HSAXL49	Immune/Hematopoietic
  2559	HSAXL82	Immune/Hematopoietic
  2560	HSAXN57	Immune/Hematopoietic
  2561	HSAXO45	Immune/Hematopoictic
  2562	HSAXS06	Immune/Hematopoietic,
  		Reproductive
  2563	HSAXS22	Immune/Hematopoietic
  2564	HSAYL24	Immune/Hematopoietic
  2565	HSAYO82	Endocrine,
  		Immune/Hematopoietic
  2566	HSAYR62	Cancer
  2567	HSAZP90	Immune/Hematopoietic
  2568	HSBAJ47	Cancer
  2569	HSDBI90	Digestive,
  		Endocrine,
  		Neural/Sensory
  2570	HSDDC55	Neural/Sensory
  2571	HSDEA26	Neural/Sensory
  2572	HSDEY39	Neural/Sensory
  2573	HSDFF72	Cancer
  2574	HSDFO08	Neural/Sensory
  2575	HSDFR10	Digestive,
  		Neural/Sensory,
  		Reproductive
  2576	HSDGB20	Neural/Sensory
  2577	HSDGH56	Cancer
  2578	HSDGM01	Neural/Sensory
  2579	HSDGM42	Cancer
  2580	HSDGM42	Cancer
  2581	HSDGM42	Cancer
  2582	HSDGM42	Cancer
  2583	HSDGM42	Cancer
  2584	HSDGM42	Cancer
  2585	HSDHD05	Neural/Sensory
  2586	HSDIE51	Cancer
  2587	HSDIK31	Cancer
  2588	HSDIV37	Cancer
  2589	HSDJC96	Cancer
  2590	HSDJE77	Cardiovascular,
  		Immune/Hematopoietic,
  		Neural/Sensory
  2591	HSDJF04	Cancer
  2592	HSDJG47	Cancer
  2593	HSDJH72	Connective/Epithelial,
  		Excretory,
  		Neural/Sensory
  2594	HSDJL07	Neural/Sensory,
  		Reproductive
  2595	HSDJR49	Neural/Sensory
  2596	HSDJV24	Cancer
  2597	HSDJV40	Immune/Hematopoietic,
  		Neural/Sensory
  2598	HSDKA64	Immune/Hematopoietic,
  		Neural/Sensory
  2599	HSDKE82	Neural/Sensory
  2600	HSDKF96	Neural/Sensory
  2601	HSDZO08	Cancer
  2602	HSDZQ96	Neural/Sensory
  2603	HSEBB18	Cancer
  2604	HSFAM19	Cancer
  2605	HSHAG54	Cancer
  2606	HSHAS72	Cancer
  2607	HSHAX04	Cancer
  2608	HSHBT15	Cancer
  2609	HSHCE85	Cancer
  2610	HSIAC81	Digestive
  2611	HSIAF66	Digestive
  2612	HSIAP01	Digestive,
  		Reproductive
  2613	HSIDA33	Cancer
  2614	HSIDA39	Digestive
  2615	HSIDZ25	Cancer
  2616	HSIEB64	Digestive
  2617	HSIEM18	Cancer
  2618	HSIFO61	Cancer
  2619	HSIFO61	Cancer
  2620	HSIGC63	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2621	HSIGM95	Digestive,
  		Immune/Hematopoietic
  2622	HSJAE76	Cancer
  2623	HSJAN83	Digestive,
  		Musculoskeletal
  2624	HSJAQ10	Cancer
  2625	HSJAR59	Musculoskeletal
  2626	HSJAU93	Cancer
  2627	HSJAY14	Cancer
  2628	HSJAY14	Cancer
  2629	HSJAY14	Cancer
  2630	HSJAY14	Cancer
  2631	HSJBB27	Musculoskeletal
  2632	HSKBU03	Musculoskeletal,
  		Neural/Sensory
  2633	HSKCQ51	Cancer
  2634	HSKDE13	Cancer
  2635	HSKDS47	Cancer
  2636	HSKHV81	Musculoskeletal
  2637	HSKXB14	Cancer
  2638	HSKYR49	Cancer
  2639	HSKYU81	Cancer
  2640	HSKYY92	Musculoskeletal
  2641	HSLAB11	Cancer
  2642	HSLAS96	Immune/Hematopoietic,
  		Musculoskeletal
  2643	HSLAW59	Immune/Hematopoietic,
  		Musculoskeletal
  2644	HSLCH54	Cancer
  2645	HSLCH57	Cancer
  2646	HSLCI86	Endocrine,
  		Mixed Fetal,
  		Musculoskeletal
  2647	HSLCS31	Cancer
  2648	HSLCS34	Cancer
  2649	HSLCV16	Cancer
  2650	HSLDW54	Cancer
  2651	HSLEC18	Cancer
  2652	HSLEG59	Musculoskeletal
  2653	HSLFR59	Cancer
  2654	HSLGD91	Cancer
  2655	HSLGF66	Cancer
  2656	HSLGF70	Musculoskeletal,
  		Neural/Sensory
  2657	HSLGP68	Musculoskeletal,
  		Neural/Sensory
  2658	HSNAB88	Cancer
  2659	HSNAH56	Cancer
  2660	HSNAN38	Cancer
  2661	HSNAO19	Cancer
  2662	HSNAQ52	Cancer
  2663	HSNAT08	Cancer
  2664	HSNAW06	Immune/Hematopoietic
  2665	HSNAW37	Cancer
  2666	HSNBJ05	Cancer
  2667	HSNBO90	Cancer
  2668	HSNBQ36	Cancer
  2669	HSNBS39	Cancer
  2670	HSOAE34	Digestive,
  		Immune/Hematopoietic
  2671	HSOAT44	Cancer
  2672	HSOBB94	Cancer
  2673	HSOBH11	Digestive
  2674	HSOBP75	Cancer
  2675	HSOBW65	Digestive
  2676	HSPAA89	Digestive
  2677	HSPAC13	Cancer
  2678	HSPAG75	Digestive
  2679	HSPAI20	Digestive,
  		Neural/Sensory
  2680	HSPAL59	Digestive,
  		Immune/Hematopoietic
  2681	HSPAY90	Cancer
  2682	HSPMF63	Cancer
  2683	HSQAC69	Cancer
  2684	HSQAH14	Cancer
  2685	HSQAX94	Cancer
  2686	HSQBL20	Cancer
  2687	HSQCQ45	Cancer
  2688	HSQCY74	Cancer
  2689	HSQDM74	Cancer
  2690	HSQEG23	Cancer
  2691	HSQEG47	Cancer
  2692	HSQFE72	Cancer
  2693	HSQFE76	Cancer
  2694	HSQFV12	Cancer
  2695	HSRAA81	Cancer
  2696	HSRAO56	Cancer
  2697	HSRAV28	Digestive,
  		Musculoskeletal
  2698	HSRDM56	Cancer
  2699	HSRDW57	Cancer
  2700	HSREC72	Immune/Hematopoietic,
  		Musculoskeletal
  2701	HSREG42	Cancer
  2702	HSRFD18	Cancer
  2703	HSRGZ11	Cancer
  2704	HSRHB59	Cancer
  2705	HSSAN03	Cancer
  2706	HSSCC66	Musculoskeletal
  2707	HSSDI13	Musculoskeletal
  2708	HSSDQ20	Musculoskeletal,
  		Neural/Sensory
  2709	HSSDX38	Musculoskeletal
  2710	HSSED57	Cancer
  2711	HSSEL28	Cancer
  2712	HSSFP88	Cancer
  2713	HSSGS62	Musculoskeletal,
  		Reproductive
  2714	HSSJA23	Cancer
  2715	HSSJF26	Musculoskeletal
  2716	HSSJF96	Musculoskeletal
  2717	HSSJM47	Cancer
  2718	HSSJW30	Cancer
  2719	HSSJW30	Cancer
  2720	HSSJW30	Cancer
  2721	HSSMY35	Cancer
  2722	HSTAL93	Connective/Epithelial
  2723	HSTBG23	Connective/Epithelial
  2724	HSUAF06	Immune/Hematopoietic
  2725	HSUBX67	Immune/Hematopoietic
  2726	HSUSB73	Immune/Hematopoietic,
  		Reproductive
  2727	HSVAC05	Cancer
  2728	HSVAE42	Connective/Epithelial,
  		Neural/Sensory
  2729	HSVAL83	Cancer
  2730	HSVAT36	Immune/Hematopoietic
  2731	HSVAV02	Cancer
  2732	HSVBA83	Endocrine,
  		Mixed Fetal
  2733	HSVBD37	Cancer
  2734	HSVBN46	Cancer
  2735	HSVBY62	Cancer
  2736	HSVBZ53	Cancer
  2737	HSVCF53	Cardiovascular,
  		Neural/Sensory,
  		Reproductive
  2738	HSWAZ17	Connective/Epithelial,
  		Reproductive,
  		Respiratory
  2739	HSWBI16	Cancer
  2740	HSXAI44	Neural/Sensory
  2741	HSXAJ07	Neural/Sensory
  2742	HSXAS59	Neural/Sensory
  2743	HSXAX20	Digestive,
  		Mixed Fetal,
  		Neural/Sensory
  2744	HSXAY60	Cancer
  2745	HSXBB78	Neural/Sensory
  2746	HSXCA83	Cancer
  2747	HSXCX20	Cancer
  2748	HSXFG21	Cancer
  2749	HSXFH82	Cancer
  2750	HSYBD33	Immune/Hematopoietic
  2751	HSYBR79	Cancer
  2752	HSYBV44	Immune/Hematopoietic
  2753	HSYBZ94	Cancer
  2754	HT3AB13	Cancer
  2755	HT4SB02	Immune/Hematopoietic
  2756	HT4SB37	Cardiovascular,
  		Immune/Hematopoietic,
  		Reproductive
  2757	HT4SB81	Cancer
  2758	HT4SB81	Cancer
  2759	HT4SB81	Cancer
  2760	HT5FX76	Cancer
  2761	HTABF81	Cancer
  2762	HTACX63	Immune/Hematopoietic
  2763	HTADC63	Cancer
  2764	HTADO61	Cancer
  2765	HTADQ22	Cancer
  2766	HTAEC59	Cancer
  2767	HTAED89	Immune/Hematopoietic
  2768	HTAEF02	Immune/Hematopoietic
  2769	HTAEH58	Immune/Hematopoietic
  2770	HTAEO35	Immune/Hematopoietic
  2771	HTDAF68	Immune/Hematopoietic
  2772	HTDAI38	Cancer
  2773	HTEAJ87	Mixed Fetal,
  		Neural/Sensory,
  		Reproductive
  2774	HTEAN76	Cancer
  2775	HTEBL56	Cancer
  2776	HTECE87	Cancer
  2777	HTEDF78	Reproductive
  2778	HTEDT87	Cancer
  2779	HTEDX05	Cancer
  2780	HTEEC19	Cancer
  2781	HTEGH03	Cancer
  2782	HTEGH03	Cancer
  2783	HTEGS48	Reproductive
  2784	HTEGY81	Cancer
  2785	HTEHB11	Reproductive
  2786	HTEHB49	Immune/Hematopoietic,
  		Reproductive
  2787	HTEHS91	Cancer
  2788	HTEHV60	Reproductive
  2789	HTEHW80	Reproductive
  2790	HTEID25	Reproductive
  2791	HTEIJ23	Cancer
  2792	HTEIM62	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2793	HTEIV33	Reproductive
  2794	HTEIV65	Reproductive
  2795	HTEJC50	Reproductive
  2796	HTEJD20	Cancer
  2797	HTEJD61	Reproductive
  2798	HTEJF31	Reproductive
  2799	HTEJI29	Reproductive
  2800	HTEJL16	Reproductive
  2801	HTEJP65	Cancer
  2802	HTEJY20	Cancer
  2803	HTEKD35	Reproductive
  2804	HTEKP82	Cardiovascular,
  		Mixed Fetal,
  		Reproductive
  2805	HTEKV69	Reproductive
  2806	HTEKZ52	Reproductive
  2807	HTEQG28	Immune/Hematopoietic,
  		Reproductive
  2808	HTFOB75	Cancer
  2809	HTGAA35	Immune/Hematopoietic
  2810	HTGAD74	Immune/Hematopoietic,
  		Reproductive
  2811	HTGAP05	Immune/Hematopoietic,
  		Neural/Sensory
  2812	HTGAQ29	Immune/Hematopoietic
  2813	HTGAR21	Immune/Hematopoietic
  2814	HTGAS70	Cancer
  2815	HTGAT65	Immune/Hematopoietic,
  		Neural/Sensory,
  		Reproductive
  2816	HTGAU17	Immune/Hematopoietic
  2817	HTGBF47	Immune/Hematopoietic
  2818	HTGBK95	Cancer
  2819	HTGCC01	Immune/Hematopoietic
  2820	HTGCK43	Cancer
  2821	HTGDS43	Immune/Hematopoietic,
  		Neural/Sensory
  2822	HTGDS92	Cancer
  2823	HTGEX34	Digestive,
  		Immune/Hematopoietic
  2824	HTGFM31	Immune/Hematopoietic
  2825	HTGGM37	Digestive,
  		Immune/Hematopoietic
  2826	HTGGN22	Immune/Hematopoietic
  2827	HTHAA41	Cancer
  2828	HTHBC58	Digestive,
  		Immune/Hematopoietic
  2829	HTHBO72	Cancer
  2830	HTHBQ29	Immune/Hematopoietic
  2831	HTHBT76	Cancer
  2832	HTHBZ91	Immune/Hematopoietic
  2833	HTHCA30	Cancer
  2834	HTHCM60	Immune/Hematopoietic
  2835	HTHDB20	Immune/Hematopoietic
  2836	HTHDF45	Immune/Hematopoietic
  2837	HTHDF86	Immune/Hematopoietic
  2838	HTHDH18	Immune/Hematopoietic
  2839	HTHDP65	Cancer
  2840	HTHDT25	Immune/Hematopoietic
  2841	HTHDV50	Immune/Hematopoietic
  2842	HTJMA64	Cancer
  2843	HTJMJ72	Connective/Epithelial,
  		Immune/Hematopoietic
  2844	HTLAD74	Reproductive
  2845	HTLAF81	Cancer
  2846	HTLBF46	Cancer
  2847	HTLBF63	Cancer
  2848	HTLCX82	Cancer
  2849	HTLDD89	Reproductive
  2850	HTLDN34	Reproductive
  2851	HTLDP19	Cancer
  2852	HTLDY30	Cancer
  2853	HTLEJ24	Cancer
  2854	HTLEJ75	Cancer
  2855	HTLEJ75	Cancer
  2856	HTLEP55	Cancer
  2857	HTLEV80	Cancer
  2858	HTLEZ57	Cancer
  2859	HTLFA90	Cancer
  2860	HTLGL33	Reproductive
  2861	HTLGQ25	Reproductive
  2862	HTLGS72	Reproductive
  2863	HTLGY50	Cancer
  2864	HTLHN86	Cancer
  2865	HTLHN86	Cancer
  2866	HTLHN86	Cancer
  2867	HTLHN86	Cancer
  2868	HTLIW29	Cancer
  2869	HTLJC15	Cancer
  2870	HTNAL14	Cancer
  2871	HTNAL34	Endocrine,
  		Reproductive
  2872	HTNBJ15	Cancer
  2873	HTNBJ15	Cancer
  2874	HTNBJ15	Cancer
  2875	HTNBJ15	Cancer
  2876	HTOAC65	Immune/Hematopoietic
  2877	HTOAE47	Immune/Hematopoietic
  2878	HTOAK03	Cancer
  2879	HTOAO58	Immune/Hematopoietic
  2880	HTOAT56	Cancer
  2881	HTOBG07	Immune/Hematopoietic,
  		Musculoskeletal
  2882	HTOBG62	Immune/Hematopoietic
  2883	HTODA92	Cancer
  2884	HTODN35	Immune/Hematopoietic
  2885	HTODO45	Immune/Hematopoietic
  2886	HTOEA53	Digestive,
  		Excretory,
  		Immune/Hematopoietic
  2887	HTOEB55	Cancer
  2888	HTOEB76	Immune/Hematopoietic
  2889	HTOET03	Cancer
  2890	HTOET03	Cancer
  2891	HTOEV01	Immune/Hematopoietic,
  		Reproductive
  2892	HTOFA11	Cancer
  2893	HTOFC33	Immune/Hematopoietic
  2894	HTOGB79	Cancer
  2895	HTOHE22	Immune/Hematopoietic
  2896	HTOHG63	Cancer
  2897	HTOHJ93	Immune/Hematopoietic
  2898	HTOHM12	Immune/Hematopoietic,
  		Neural/Sensory
  2899	HTOHM82	Cancer
  2900	HTOHN40	Immune/Hematopoietic,
  		Neural/Sensory
  2901	HTOHR59	Digestive,
  		Immune/Hematopoietic,
  		Neural/Sensory
  2902	HTOHS29	Cancer
  2903	HTOID65	Cancer
  2904	HTOIE17	Excretory,
  		Immune/Hematopoietic
  2905	HTOIG16	Immune/Hematopoietic,
  		Reproductive
  2906	HTOIH39	Immune/Hematopoietic
  2907	HTOIH51	Immune/Hematopoietic
  2908	HTOJB02	Immune/Hematopoietic
  2909	HTOJJ26	Connective/Epithelial,
  		Digestive,
  		Immune/Hematopoietic
  2910	HTOJP25	Immune/Hematopoietic
  2911	HTOJS23	Immune/Hematopoietic
  2912	HTOJY56	Cancer
  2913	HTOJZ18	Immune/Hematopoietic
  2914	HTPCG10	Cancer
  2915	HTPCO75	Cancer
  2916	HTPCW21	Digestive,
  		Neural/Sensory
  2917	HTPDD68	Cancer
  2918	HTPDV75	Digestive,
  		Reproductive
  2919	HTSER28	Cancer
  2920	HTSET62	Cancer
  2921	HTSFV18	Cancer
  2922	HTSGO13	Cancer
  2923	HTSGO88	Immune/Hematopoietic
  2924	HTTAH05	Reproductive
  2925	HTTAP37	Immune/Hematopoietic,
  		Reproductive
  2926	HTTBJ38	Cancer
  2927	HTTDB11	Cancer
  2928	HTTDG27	Reproductive
  2929	HTTDN24	Cancer
  2930	HTTDO33	Cancer
  2931	HTTDT67	Cancer
  2932	HTTEO25	Cancer
  2933	HTTEP11	Neural/Sensory,
  		Reproductive
  2934	HTTES77	Cancer
  2935	HTTFD29	Reproductive
  2936	HTTFG15	Cancer
  2937	HTWAM19	Immune/Hematopoietic
  2938	HTWBF58	Immune/Hematopoietic
  2939	HTWBO30	Cancer
  2940	HTWBZ57	Cancer
  2941	HTWCC10	Immune/Hematopoietic
  2942	HTWCE14	Cancer
  2943	HTWCT76	Digestive,
  		Immune/Hematopoietic
  2944	HTWDJ17	Cancer
  2945	HTWDM89	Immune/Hematopoietic
  2946	HTWEA05	Immune/Hematopoietic
  2947	HTWEG06	Immune/Hematopoietic
  2948	HTWEQ36	Cancer
  2949	HTWFA21	Immune/Hematopoietic
  2950	HTWFA88	Digestive,
  		Immune/Hematopoietic
  2951	HTWFM85	Cancer
  2952	HTWFO43	Cancer
  2953	HTWLG39	Cancer
  2954	HTXAA20	Cancer
  2955	HTXAD75	Cancer
  2956	HTXAR92	Immune/Hematopoietic
  2957	HTXBS38	Cancer
  2958	HTXBU88	Immune/Hematopoietic
  2959	HTXCP27	Cancer
  2960	HTXCU30	Excretory,
  		Immune/Hematopoietic
  2961	HTXCV44	Immune/Hematopoietic,
  		Neural/Sensory
  2962	HTXDJ21	Immune/Hematopoietic
  2963	HTXDJ75	Digestive,
  		Immune/Hematopoietic,
  		Mixed Fetal
  2964	HTXDJ85	Immune/Hematopoietic
  2965	HTXDK09	Cancer
  2966	HTXDO17	Immune/Hematopoietic,
  		Neural/Sensory,
  		Respiratory
  2967	HTXDT72	Cancer
  2968	HTXDU08	Cancer
  2969	HTXDZ68	Immune/Hematopoietic,
  		Musculoskeletal
  2970	HTXEN33	Immune/Hematopoietic,
  		Reproductive
  2971	HTXES13	Cancer
  2972	HTXFD86	Cancer
  2973	HTXGK12	Cancer
  2974	HTXGL32	Immune/Hematopoietic
  2975	HTXJD08	Digestive,
  		Immune/Hematopoietic
  2976	HTXJD85	Immune/Hematopoietic
  2977	HTXJE12	Cancer
  2978	HTXJI59	Cancer
  2979	HTXJJ92	Cancer
  2980	HTXJM94	Cancer
  2981	HTXJV54	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  2982	HTXJW06	Cancer
  2983	HTXKB57	Cancer
  2984	HTXKH22	Immune/Hematopoietic
  2985	HTXKH40	Cancer
  2986	HTXKK76	Immune/Hematopoietic
  2987	HTXKL53	Cancer
  2988	HTXKS11	Immune/Hematopoietic
  2989	HTXKS27	Cancer
  2990	HTXLC05	Digestive,
  		Immune/Hematopoietic,
  		Respiratory
  2991	HTXLC45	Immune/Hematopoietic
  2992	HTXLT36	Cancer
  2993	HTXLY94	Cancer
  2994	HTXNV66	Cancer
  2995	HTXOL30	Immune/Hematopoietic
  2996	HTXOW27	Cancer
  2997	HTXPD86	Cancer
  2998	HTXPT57	Digestive,
  		Immune/Hematopoietic
  2999	HTYSJ88	Endocrine,
  		Immune/Hematopoietic
  3000	HUDBE20	Reproductive
  3001	HUDBK47	Immune/Hematopoietic,
  		Reproductive
  3002	HUFAB57	Cancer
  3003	HUFAL17	Digestive
  3004	HUFAO92	Digestive,
  		Reproductive
  3005	HUFAO94	Cancer
  3006	HUFAP33	Cancer
  3007	HUFAU71	Cancer
  3008	HUFBK95	Digestive,
  		Reproductive
  3009	HUFBP77	Cancer
  3010	HUFBV62	Cancer
  3011	HUFBY96	Cancer
  3012	HUFCN72	Digestive
  3013	HUFEF79	Cancer
  3014	HUKAD46	Endocrine,
  		Immune/Hematopoietic,
  		Reproductive
  3015	HUKAI28	Cardiovascular,
  		Reproductive
  3016	HUKAO50	Cancer
  3017	HUKCS86	Cancer
  3018	HUKCS86	Cancer
  3019	HUKEA22	Cancer
  3020	HUKEL79	Cancer
  3021	HUKEX37	Reproductive
  3022	HUKFC71	Cancer
  3023	HUKFV37	Cancer
  3024	HUKFY09	Cancer
  3025	HUNAL39	Reproductive
  3026	HUSAO04	Cancer
  3027	HUSAO04	Cancer
  3028	HUSCA09	Cancer
  3029	HUSCJ01	Cancer
  3030	HUSGB23	Cancer
  3031	HUSGJ09	Cardiovascular,
  		Neural/Sensory
  3032	HUSGQ57	Cancer
  3033	HUSGY15	Cancer
  3034	HUSHD41	Cancer
  3035	HUSHK65	Cancer
  3036	HUSIK45	Cancer
  3037	HUSIO57	Cancer
  3038	HUSIP17	Cardiovascular
  3039	HUSIR70	Cancer
  3040	HUSXP50	Cardiovascular,
  		Reproductive
  3041	HUSXY93	Cancer
  3042	HUSYG26	Cancer
  3043	HUVCQ68	Cancer
  3044	HUVDG58	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  3045	HUVEG53	Cancer
  3046	HWAAH11	Cancer
  3047	HWAAQ28	Cancer
  3048	HWAAY60	Cancer
  3049	HWABR43	Digestive,
  		Immune/Hematopoietic
  3050	HWACH06	Cancer
  3051	HWACZ33	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  3052	HWADV90	Immune/Hematopoietic
  3053	HWAEB52	Cancer
  3054	HWBAK71	Immune/Hematopoietic
  3055	HWBBU75	Cancer
  3056	HWBCN81	Immune/Hematopoietic,
  		Reproductive
  3057	HWBCP16	Immune/Hematopoietic
  3058	HWBCX93	Cancer
  3059	HWBEF34	Immune/Hematopoietic,
  		Neural/Sensory
  3060	HWFBB23	Cancer
  3061	HWFBI40	Connective/Epithelial,
  		Digestive,
  		Immune/Hematopoietic
  3062	HWHGV77	Connective/Epithelial
  3063	HWHGW09	Cancer
  3064	HWHHA21	Connective/Epithelial
  3065	HWHPU44	Connective/Epithelial
  3066	HWHRC51	Cancer
  3067	HWLAT50	Cancer
  3068	HWLBO67	Digestive
  3069	HWLGP26	Cancer
  3070	HWLHO31	Cardiovascular,
  		Digestive
  3071	HWLIL31	Cancer
  3072	HWLJN08	Cancer
  3073	HWLRE03	Cancer
  3074	HWTAM38	Digestive,
  		Immune/Hematopoietic,
  		Reproductive
  3075	HWTAW58	Cancer
  3076	HWTBB42	Cancer
  3077	HWTBC75	Cancer
  3078	HWTBI25	Cancer
  3079	HWTBL86	Cancer
  3080	HWTBX66	Cancer
  3081	HYAAC74	Immune/Hematopoietic,
  		Musculoskeletal,
  		Reproductive
  3082	HYAAD61	Immune/Hematopoietic
  3083	HYACC21	Immune/Hematopoietic
  3084	HYBAP75	Cancer
  3085	HYBAQ24	Cancer
  3086	HYBAW56	Musculoskeletal
  3087	HYBBD81	Musculoskeletal

• Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indictions”) describes particular embodiments of the invention as well as indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.
• Table 1E describes the use of, inter alia, FMAT technology for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound flurophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for highthroughput screening using flourometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).
• Table 1E also describes the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).

  LENGTHY TABLE REFERENCED HERE
  US20070048297A1-20070301-T00005
  Please refer to the end of the specification for access instructions.

  
  Table 1F:
• Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.
• TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.
• To quantify gene expression the Taqman® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a Taqman® probe (distinct from the primers) during PCR. The Taqman® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Fold DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.
• After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.
• For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using Trizol extraction or by using the Ambion RNAqueous™-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using Taqman®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.
• Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).
• Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
• In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.
• The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in Taqman® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.
• The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.
• The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).
• The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
• The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
• The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).

  TABLE 1F
  Gene	cDNA	Disease			Exemplary
  No.	Clone ID	Class	Preferred Indications	Cell Line	Targets	Exemplary Accessions
  423	HTEEW69	Immune	Highly preferred indications include	AOSMC	CCR7	gb|X84702|HSDNABLR2
  			immunological disorders such as described herein		CXCR3	gb|Z79783|HSCKRL2
  			under the heading “Immune Activity” and/or		Rag2	gb|AY011962|AY011962
  			“Blood-Related Disorders” (particularly		VLA4	gb|X16983|HSINTAL4
  			including, but not limited to, immune disorders
  			involving muscle tissues and the cardiovascular
  			system (e.g. heart, lungs, circulatory system)).
  			Highly preferred embodiments of the invention
  			include methods of preventing, detecting,
  			diagnosing, treating and/or ameliorating disorders
  			of the immune system (particularly including, but
  			not limited to, immune disorders involving
  			muscle tissue or the cardiovascular system).
  			(AOSMC cells are human aortic smooth muscle
  			cells).
  423	HTEEW69	Immune	Highly preferred indications include	Caco-2	TNF	gb|AJ270944|HSA27094
  			immunological disorders such as described herein
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving the cells of the gastrointestinal tract).
  			Highly preferred embodiments of the invention
  			include methods of preventing, detecting,
  			diagnosing, treating and/or ameliorating disorders
  			of the immune system (particularly including, but
  			not limited to, immune disorders involving cells
  			of the gastrointestinal tract). (The Caco-2 cell line
  			is a human colorectal adenocarcinoma cell line
  			available through the ATCC as cell line number
  			HTB-37).
  423	HTEEW69	Immune	Highly preferred indications include	Daudi	GATA3	gb|X55037|HSGATA3
  			immunological disorders such as described herein		ICAM	gb|X06990|HSICAM1
  			under the heading “Immune Activity” and/or		TNF	gb|AJ270944|HSA27094
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving the B-cells). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving B-cells). (The Daudi
  			cell line is a human B lymphoblast cell line
  			available through the ATCC as cell line number
  			CCL-213).
  423	HTEEW69	Immune	Highly preferred indications include	HEK293	TNF	gb|AJ270944|HSA27094
  			immunological disorders such as described herein
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving epithelial cells or the renal system).
  			Highly preferred embodiments of the invention
  			include methods of preventing, detecting,
  			diagnosing, treating and/or ameliorating disorders
  			of the immune system (particularly including, but
  			not limited to, immune disorders involving
  			epithelial cells or the renal system). (The 293 cell
  			line is a human embryonal kidney epithelial cell
  			line available through the ATCC as cell line
  			number CRL-1573).
  423	HTEEW69	Immune	Highly preferred indications include	Liver	ICAM	gb|X06990|HSICAM1
  			immunological disorders such as described herein
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving cells of the hepatic system). Highly
  			preferred embodiments of the invention include
  			methods of preventing, detecting, diagnosing,
  			treating and/or ameliorating disorders of the
  			immune system (particularly including, but not
  			limited to, immune disorders involving cells of
  			the hepatic system).
  423	HTEEW69	Immune	Highly preferred indications include	NHDF	CIS3	gb|AB006967|AB006967
  			immunological disorders such as described herein		TNF	gb|AJ270944|HSA27094
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving the skin). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving the skin). (NHDF
  			cells are normal human dermal fibroblasts).
  423	HTEEW69	Immune	Highly preferred indications include	SK-N-MC	TNF	gb|AJ270944|HSA27094
  			immunological disorders such as described herein	neuroblastoma	VCAM	gb|A30922|A30922
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving the central nervous system). Highly
  			preferred embodiments of the invention include
  			methods of preventing, detecting, diagnosing,
  			treating and/or ameliorating disorders of the
  			immune system (particularly including, but not
  			limited to, immune disorders involving the
  			central nervous sytem). (The SK-N-MC
  			neuroblastoma cell line is a cell line derived from
  			human brain tissue and is available through the
  			ATCC as cell line number HTB-10).
  423	HTEEW69	Immune	Highly preferred indications include	THP1	CD25	gb|X03137|HSIL2RG7
  			immunological disorders such as described herein		CD40	gb|AJ300189|HSA30018
  			under the heading “Immune Activity” and/or		GATA3	gb|X55037|HSGATA3
  			“Blood-Related Disorders” (particularly		LTBR	gb|AK027080|AK027080
  			including, but not limited to, immune disorders		Rag1	gb|M29474|HUMRAG1
  			involving monocytes). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving monocytes). (The
  			THP1 cell line is a human monocyte cell line
  			available through the ATCC as cell line number
  			TIB-202).
  423	HTEEW69	Immune	Highly preferred indications include	U937	IL1B	gb|X02532|HSIL1BR
  			immunological disorders such as described herein		TNF	gb|AJ270944|HSA27094
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving monocytes). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving monocytes). (The
  			U937 cell line is a human monocyte cell line
  			available through the ATCC; cell #CRL-1593.2).
  878	HCEGG08	Immune	Highly preferred indications include	AOSMC	CIS3	gb|AB006967|AB006967
  			immunological disorders such as described herein		Granzyme	gb|J04071|HUMCSE
  			under the heading “Immune Activity” and/or		B
  			“Blood-Related Disorders” (particularly		IL1B	gb|X02532|HSIL1BR
  			including, but not limited to, immune disorders		IL5	gb|X12705|HSBCDFIA
  			involving muscle tissues and the cardiovascular
  			system (e.g. heart, lungs, circulatory system)).
  			Highly preferred embodiments of the invention
  			include methods of preventing, detecting,
  			diagnosing, treating and/or ameliorating disorders
  			of the immune system (particularly including, but
  			not limited to, immune disorders involving
  			muscle tissue or the cardiovascular system).
  			(AOSMC cells are human aortic smooth muscle
  			cells).
  878	HCEGG08	Immune	Highly preferred indications include	HEK293	ICAM	gb|X06990|HSICAM1
  			immunological disorders such as described herein
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving epithelial cells or the renal system).
  			Highly preferred embodiments of the invention
  			include methods of preventing, detecting,
  			diagnosing, treating and/or ameliorating disorders
  			of the immune system (particularly including, but
  			not limited to, immune disorders involving
  			epithelial cells or the renal system). (The 293 cell
  			line is a human embryonal kidney epithelial cell
  			line available through the ATCC as cell line
  			number CRL-1573).
  878	HCEGG08	Immune	Highly preferred indications include	HUVEC	CCR7	gb|X84702|HSDNABLR2
  			immunological disorders such as described herein		TNF	gb|AJ270944|HSA27094
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving endothelial cells). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving endothelial cells).
  			(HUVEC cells are human umbilical vein
  			endothelial cells).
  878	HCEGG08	Immune	Highly preferred indications include	Jurkat	GATA1	gb|X17254|HSERYF1
  			immunological disorders such as described herein		Rag1	gb|M29474|HUMRAG1
  			under the heading “Immune Activity” and/or		Rag2	gb|AY011962|AY011962
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving T-cells). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving T-cells). (The Jurkat
  			cell line is a human T lymphocyte cell line
  			available through the ATCC; cell line #TIB-152).
  878	HCEGG08	Immune	Highly preferred indications include	Liver	ICAM	gb|X06990|HSICAM1
  			immunological disorders such as described herein
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving cells of the hepatic system). Highly
  			preferred embodiments of the invention include
  			methods of preventing, detecting, diagnosing,
  			treating and/or ameliorating disorders of the
  			immune system (particularly including, but not
  			limited to, immune disorders involving cells of
  			the hepatic system).
  878	HCEGG08	Immune	Highly preferred indications include	NHDF	HLA-c
  			immunological disorders such as described herein
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving the skin). Highly preferred
  			embodiments of the invention include methods of
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving the skin). (NHDF
  			cells are normal human dermal fibroblasts).
  878	HCEGG08	Immune	Highly preferred indications include	SK-N-MC	HLA-c
  			immunological disorders such as described herein	neuroblastoma	VCAM	gb|A30922|A30922
  			under the heading “Immune Activity” and/or
  			“Blood-Related Disorders” (particularly
  			including, but not limited to, immune disorders
  			involving the central nervous system). Highly
  			preferred embodiments of the invention include
  			methods of preventing, detecting, diagnosing,
  			treating and/or ameliorating disorders of the
  			immune system (particularly including, but not
  			limited to, immune disorders involving the
  			central nervous sytem). (The SK-N-MC
  			neuroblastoma cell line is a cell line derived from
  			human brain tissue and is available through the
  			ATCC as cell line number HTB-10).
  878	HCEGG08	Immune	Highly preferred indications include	THP1	CCR3	gb|AB023887|AB023887
  			immunological disorders such as described herein		CCR4	gb|AB023888|AB023888
  			under the heading “Immune Activity” and/or		CTLA4	gb|AF316875|AF316875
  			“Blood-Related Disorders” (particularly		Granzyme	gb|J04071|HUMCSE
  			including, but not limited to, immune disorders		B
  			involving monocytes). Highly preferred		Rag2	gb|AY011962|AY011962
  			embodiments of the invention include methods of		VCAM	gb|A30922|A30922
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving monocytes). (The
  			THP1 cell line is a human monocyte cell line
  			available through the ATCC as cell line number
  			TIB-202).
  878	HCEGG08	Immune	Highly preferred indications include	U937	CCR5	gb|AF161918|AF161918
  			immunological disorders such as described herein		CCR7	gb|X84702|HSDNABLR2
  			under the heading “Immune Activity” and/or		CD25	gb|X03137|HSIL2RG7
  			“Blood-Related Disorders” (particularly		CD30
  			including, but not limited to, immune disorders		CXCR3	gb|Z79783|HSCKRL2
  			involving monocytes). Highly preferred		Rag1	gb|M29474|HUMRAG1
  			embodiments of the invention include methods of		Rag2	gb|AY011962|AY011962
  			preventing, detecting, diagnosing, treating and/or
  			ameliorating disorders of the immune system
  			(particularly including, but not limited to,
  			immune disorders involving monocytes). (The
  			U937 cell line is a human monocyte cell line
  			available through the ATCC as cell line number
  			CRL-1593.2).

• Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases. The first column provides a unique clone identifier, “Clone ID NO:”, corresponding to a cDNA clone disclosed in Table 1A and/or Table 1B. The second column provides the unique contig identifier, “Contig ID:” which allows correlation with the information in Table 1B. The third column provides the sequence identifier, “SEQ ID NO:”, for the contig polynucleotide sequences. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. The fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five. Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”), as described below.
• The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Table 1B, column 3 (e.g., SEQ ID NO:X or the ‘Query’ sequence) was used to search against the NR database. The computer program BLASTX was used to compare a 6-frame translation of the Query sequence to the NR database (for information about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403-410 (1990), and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring ‘Subject’) is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring ‘Subject’ is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than 1.0 e-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of Query amino acids in the HSP and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.
• The PFAM database, PFAM version 2.1, (Sonnhammer, Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each multiple sequence alignment is converted into a probability model called a Hidden Markov Model, or HMM, that represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin, et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1B.1) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6. Column 7 provides the score returned by HMMER version 1.8 for the alignment. Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.
• As mentioned, columns 8 and 9 in Table 2, “NT From” and “NT To”, delineate the polynucleotides of “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.
• The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in ATCC Deposit No: Z. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A and/or 1B.
• Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
• Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing cDNA ATCC Deposit No: Z (e.g., as set forth in columns 2 and 3 of Table 1A and/or as set forth, for example, in Table 1B, 6, and 7). The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X.
• The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

  TABLE 2
  		SEQ			PFam/NR	Score/
  cDNA		ID	Analysis		Accession	Percent	NT
  Clone ID	Contig ID:	NO: X	Method	PFam/NR Description	Number	Identity	From	NT To

  H6BSF56	762968	11	HMMER	PFAM: Zinc-binding dehydrogenases	PF00107	35.6	176	415
  			2.1.1
  			WUblastx.64	(Q9BV79) SIMILAR TO CGI-63	Q9BV79	100%	25	42
  				PROTEIN.		92%	53	427
  H6EDM64	841331	12	WUblastx.64	(Q9UID3) ANG2.	Q9UID3	90%	928	2451
  						36%	203	310
  						36%	931	1038
  						95%	191	871
  H6EEC72	889401	13	WUblastx.64	hypothetical protein DKFZp434L061.1 -	pir|T43456|T43456	80%	1484	1203
  				human		41%	1277	1080
  						35%	973	845
  						34%	659	549
  						57%	991	365
  HACBJ56	847112	15	WUblastx.64	(Q9D2Q2) 2310079F23RIK	Q9D2Q2	65%	98	286
  				PROTEIN.
  HACBS22	847113	16	WUblastx.64	(O60266) ADENYLATE CYCLASE	CYA3_HUMAN	89%	6	416
  				TYPE III (EC 4.6.1.1) (ADENYLATE		25%	1547	2299
  						18%	917	1111
  						93%	416	2449
  HADMB15	847116	19	WUblastx.64	(Q9BVH1) SIMILAR TO DLXIN-1.	Q9BVH1	100%	8	109
  HAGEG10	823543	22	WUblastx.64	(Q9NWT5) CDNA FLJ20618 FIS,	Q9NWT5	100%	1237	1377
  				CLONE KAT05049.		96%	1	156
  HAGFS57	847120	24	WUblastx.64	(Q9Y485) X-LIKE 1 PROTEIN.	Q9Y485	58%	9	872
  HAGHN57	773286	25	WUblastx.64	(O60416) WUGSC: H_RG276O03.2	O60416	98%	65	1444
  				PROTEIN.
  HAHEA15	847013	26	WUblastx.64	(Q9NWD5) HYPOTHETICAL 31.4 KDA	Q9NWD5	76%	455	832
  				PROTEIN.		99%	30	560
  HAJAA47	534670	27	WUblastx.64	(Q9NZA3) CDA14.	Q9NZA3	100%	17	157
  HAJAY92	845601	28	WUblastx.64	(O00549) ORF2-LIKE PROTEIN	O00549	53%	2226	2318
  				(FRAGMENT).		26%	769	915
  						38%	1653	1769
  						31%	1721	2242
  HAJBV67	866415	29	WUblastx.64	(Q9HD45) TRANSMEMBRANE 9	T9S3_HUMAN	100%	13	126
  				SUPERFAMILY PROTEIN		93%	116	1681
  				MEMBER 3 PRECU
  HAOAG15	852204	31	HMMER	PFAM: von Willebrand factor type A	PF00092	180.1	506	1057
  			2.1.1	domain
  			WUblastx.64	(O75578) INTEGRIN ALPHA-10	ITAG_HUMAN	90%	8	3463
  				PRECURSOR.
  HAQCE11	633730	33	WUblastx.64	(Q24333) ELASTIN LIKE PROTEIN	Q24333	95%	61	132
  				(FRAGMENT).
  HATCB45	631172	35	WUblastx.64	(Q9D0I6) 2610014F08RIK PROTEIN.	Q9D0I6	88%	490	645
  HATCI03	580805	37	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	71%	906	688
  				CLONE COL03536.
  HBAGD86	838799	39	WUblastx.64	(Q14287) HYPOTHETICAL	Q14287	37%	801	559
  				PROTEIN (FRAGMENT).
  HBDAB91	789532	41	WUblastx.64	(O00370) PUTATIVE P150.	O00370	40%	587	513
  						34%	529	5
  HBDAB91	864374	42	WUblastx.64	(O00370) PUTATIVE P150.	O00370	40%	907	833
  						35%	849	307
  HBGBC29	691473	43	WUblastx.64	(O60513) BETA-1,4-	B4G4_HUMAN	61%	1	78
  				GALACTOSYLTRANSFERASE 4		98%	65	1021
  				(EC 2.4.1.—) (BET
  HBHAA05	603174	45	WUblastx.64	(Q9H387) PRO2550.	Q9H387	71%	676	386
  HBHAA81	846465	46	WUblastx.64	(Q9D1G3) 1110011D13RIK	Q9D1G3	89%	1329	1502
  				PROTEIN.		79%	28	1329
  HBIAC29	831751	48	WUblastx.64	(Q9D7J5) 2310005N01RIK	Q9D7J5	78%	25	492
  				PROTEIN.		93%	883	927
  HBJAB02	837309	50	WUblastx.64	(Q9NXT6) CDNA FLJ20062 FIS,	Q9NXT6	70%	2	1210
  				CLONE COL01508.
  HBJCR46	815649	53	HMMER	PFAM: WD domain, G-beta repeat	PF00400	36.6	790	867
  			2.1.1
  			WUblastx.64	(Q9DC22) 1200006M05RIK	Q9DC22	96%	207	611
  				PROTEIN.		73%	568	2763
  HBJDS79	813588	54	WUblastx.64	(Q9CY11) 2510039O18RIK	Q9CY11	92%	1119	1325
  				PROTEIN.		89%	1322	1519
  						93%	1032	1127
  						100%	1509	1532
  						66%	2	1075
  HBJEL16	847030	56	WUblastx.64	(O95297) PROTEIN ZERO	O95297	98%	285	491
  				RELATED PROTEIN.
  HBJIG20	866159	58	HMMER	PFAM: Cytochrome c oxidase subunit	PF00510	162.6	321	551
  			2.1.1	III
  			WUblastx.64	(BAA77671) Cytochrome c oxidase	BAA77671	81%	9	617
  				subunit 3 (Fragment
  HBJKD16	853358	59	WUblastx.64	(Q9NXS4) CDNA FLJ20080 FIS,	Q9NXS4	91%	8	1528
  				CLONE COL03184.
  HBMBM96	561935	60	WUblastx.64	(Q9H387) PRO2550.	Q9H387	69%	661	494
  						67%	794	639
  HBMTX26	695704	63	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	46%	964	608
  				PROTEIN (FRAGMENT).		61%	272	156
  						66%	136	101
  						54%	611	507
  						58%	546	292
  HBMTY48	637521	64	WUblastx.64	(Q9H5N9) CDNA: FLJ23235 FIS,	Q9H5N9	94%	54	941
  				CLONE CAS04980.
  HBMUH74	866160	65	WUblastx.64	(Q9NVW8) CDNA FLJ10462 FIS,	Q9NVW8	100%	11	427
  				CLONE NT2RP1001494, WEAKLY
  				SIMILAR TO MAL
  HBMWE61	778066	66	WUblastx.64	(Q9BX88) MAGPHININ DELTA.	Q9BX88	100%	302	520
  						95%	869	1009
  HBNAX40	834801	67	WUblastx.64	(Q9H2K2) TANKYRASE-LIKE	Q9H2K2	100%	1	201
  				PROTEIN (TANKYRASE 2).		100%	221	481
  HBQAB79	810542	69	WUblastx.64	(Q9UQ32) AD 3 (FRAGMENT).	Q9UQ32	82%	323	204
  HBSAK32	856387	71	WUblastx.64	(Q9H1Q7) BA12M19.1.3 (NOVEL	Q9H1Q7	100%	239	412
  				PROTEIN).		100%	95	172
  HBXCM66	639039	72	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	65%	988	809
  				CLONE COL04765.		77%	836	690
  HBXCX15	637542	73	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	41%	726	827
  				PROTEIN.		52%	578	730
  HCDCY76	837972	74	WUblastx.64	frizzled protein 4 - human	pir|JC7127|JC7127	100%	1039	527
  						30%	994	785
  						79%	567	37
  HCE1G78	761204	76	HMMER	PFAM: Inositol polyphosphate	PF00783	277.3	77	775
  			2.1.1	phosphatase family, catalytic domain
  			WUblastx.64	(Q9UDT9) WUGSC: H_DJ412A9.2	Q9UDT9	72%	8	1549
  				PROTEIN (FRAGMENT).		95%	8	67
  HCE2H52	847007	77	WUblastx.64	probable transposase - human	pir|S72481|S72481	60%	564	758
  				transposon MER37		77%	430	537
  						75%	754	1251
  HCE3B04	831151	78	WUblastx.64	(O43466) HYPOTHETICAL 31.3 KDA	O43466	98%	836	1003
  				PROTEIN (FRAGMENT).		45%	217	972
  HCEDR26	771144	80	WUblastx.64	(Q9H919) CDNA FLJ13078 FIS,	Q9H919	66%	1157	1095
  				CLONE NT2RP3002002.		66%	1345	1184
  HCEEQ25	531784	82	WUblastx.64	(P78349) SODIUM CHANNEL 2.	P78349	95%	311	433
  						93%	433	480
  						100%	658	714
  HCEEU18	688041	83	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	49%	186	10
  				PRODUCT.		56%	1223	933
  HCEFZ82	831745	84	WUblastx.64	(Q9BV23) SIMILAR TO LIPASE	Q9BV23	95%	594	782
  				PROTEIN.		100%	17	604
  HCFLN88	610000	86	WUblastx.64	(Q9BQE9) SIMILAR TO B-CELL	Q9BQE9	87%	278	475
  				CLL/LYMPHOMA 7B (UNKNOWN)
  				(PROTEIN FOR MGC
  HCFLT90	788578	87	WUblastx.64	(Q9CVC2) 2210013O21RIK	Q9CVC2	53%	612	445
  				PROTEIN (FRAGMENT).		70%	850	671
  HCHAB84	834326	88	WUblastx.64	(Q9BRV3) STROMAL CELL	Q9BRV3	89%	82	744
  				PROTEIN.
  HCNSD29	862314	91	WUblastx.64	(O75400) HUNTINGTIN-	O75400	82%	628	1605
  				INTERACTING PROTEIN		78%	337	489
  				HYPA/FBP11 (FRAGMENT).
  HCRAY10	695709	96	WUblastx.64	(AAH08671) Similar to RIKEN cDNA	AAH08671	77%	72	440
  				5530601I19 gene.
  HCRBF72	828945	97	WUblastx.64	(Q9UI95) MITOTIC SPINDLE	MD22_HUMAN	94%	191	823
  				ASSEMBLY CHECKPOINT
  				PROTEIN MAD2B
  HCUCF89	637986	100	WUblastx.64	(Q9P147) PRO2822.	Q9P147	100%	421	398
  						82%	494	426
  HCUCK44	790277	101	WUblastx.64	hypothetical protein DKFZp564J157.1 -	pir|T34520|T34520	100%	29	157
  				human (fragment)		100%	377	403
  HDPDI72	897277	109	WUblastx.64	adult-specific brush border protein -	pir|C45665|C45665	64%	180	230
  				rabbit		83%	11	100
  HDPDJ58	587265	110	WUblastx.64	hypothetical protein	pir|T42691|T42691	100%	307	609
  				DKFZp434D2328.1 - human		87%	621	785
  				(fragment)		36%	101	313
  						36%	307	606
  						36%	188	316
  						42%	89	172
  						27%	23	307
  						85%	14	307
  						37%	134	307
  						35%	101	307
  						35%	89	274
  						32%	137	307
  						36%	325	594
  						34%	543	671
  						38%	307	606
  						37%	322	585
  						28%	307	606
  						29%	358	606
  						33%	337	594
  						41%	487	594
  						37%	92	307
  						36%	352	606
  						32%	89	316
  						32%	340	594
  						30%	83	316
  						35%	454	606
  						31%	654	785
  						41%	624	779
  						31%	624	794
  						40%	630	785
  						34%	624	776
  						33%	630	785
  						34%	83	307
  						35%	92	286
  						34%	1229	1402
  						36%	1265	1411
  						36%	660	785
  						32%	624	779
  						39%	645	773
  						35%	645	785
  						35%	645	785
  						32%	785	1393
  						34%	280	606
  						36%	259	594
  						32%	259	591
  						32%	319	591
  						100%	1405	1464
  						33%	322	510
  						33%	633	881
  						29%	656	1327
  						25%	902	1402
  						30%	848	1402
  						30%	1040	1420
  						36%	89	307
  						39%	827	1042
  						41%	962	1117
  						27%	803	1402
  						33%	131	307
  						25%	848	1144
  						27%	89	307
  						37%	125	307
  						30%	854	1447
  						76%	746	1450
  HDPFF10	853513	111	HMMER	PFAM: Leucine Rich Repeat	PF00560	65.1	729	800
  			2.1.1
  			WUblastx.64	garp precursor - human	pir|S42799|S42799	38%	285	965
  						42%	1153	1593
  						32%	1306	1641
  						29%	1147	1446
  						31%	1614	1898
  						26%	1159	1512
  						30%	1174	1536
  						33%	1306	1632
  						30%	1174	1494
  						31%	1162	1539
  						33%	1183	1500
  						28%	468	893
  						27%	246	965
  						37%	1629	2111
  HDPFU43	790189	112	WUblastx.64	(AAH01057) Tyrosylprotein	AAH01057	100%	360	1349
  				sulfotransferase 2.		58%	220	348
  HDPGE24	801947	114	WUblastx.64	(Q9P195) PRO1722.	Q9P195	65%	1413	1291
  						43%	1388	1278
  						77%	2528	2394
  						47%	2182	2078
  						75%	1774	1751
  						62%	2604	2557
  						68%	1301	1167
  HDPIU94	813352	115	WUblastx.64	(Q9BVF7) SIMILAR TO	Q9BVF7	99%	63	1703
  				HYPOTHETICAL PROTEIN
  				FLJ10422.
  HDPOC24	777493	116	WUblastx.64	(Q9H8K1) CDNA FLJ13518 FIS,	Q9H8K1	100%	62	208
  				CLONE PLACE1005799.
  HDPOL37	745377	117	WUblastx.64	(AAK40301) TRH4.	AAK40301	70%	502	323
  						60%	1325	483
  HDPPQ30	684292	120	WUblastx.64	(Q9H387) PRO2550.	Q9H387	51%	807	727
  						79%	1042	815
  HDPPW82	778405	121	WUblastx.64	hypothetical protein UL126 - human	pir|S09875|S09875	94%	6	116
  				cytomegalovirus (strain AD169)
  HDQHM36	852328	123	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	69%	1129	1257
  				PRODUCT.		50%	965	1153
  HDTAU35	838139	124	WUblastx.64	(Q9T9V8) NADH	Q9T9V8	87%	56	175
  				DEHYDROGENASE SUBUNIT 3.		83%	305	340
  HDTAV54	801898	125	WUblastx.64	(AAH01231) Glutathione S-transferase	AAH01231	100%	13	303
  				subunit 13 hom
  HDTGW48	827285	127	WUblastx.64	(Q9P1W8) SIRP-B2.	Q9P1W8	100%	783	1100
  						79%	1359	1757
  HE2CA60	888705	130	WUblastx.64	(O95232) OKADAIC ACID-	OA48_HUMAN	98%	1098	1265
  				INDUCIBLE PHOSPHOPROTEIN
  				OA48-18.
  HE2HC60	753265	133	WUblastx.64	(Q9NVC4) CDNA FLJ10814 FIS,	Q9NVC4	88%	125	1300
  				CLONE NT2RP4000984.
  HE6CS65	762960	136	WUblastx.64	(Q9H7C6) CDNA: FLJ21047 FIS,	Q9H7C6	98%	938	1378
  				CLONE CAS00253.
  HE6DO92	562767	137	WUblastx.64	gag polyprotein - human endogenous	pir|A46312|A46312	63%	623	895
  				virus S71		80%	19	633
  HE6EY13	847058	138	WUblastx.64	(O95476) HYPOTHETICAL 28.3 KDA	O95476	92%	5	472
  				PROTEIN.
  HE6FU11	827236	139	HMMER	PFAM: von Willebrand factor type A	PF00092	184.7	244	771
  			2.1.1	domain
  			WUblastx.64	(O95460) MATRILIN-4	MTN4_HUMAN	77%	145	789
  				PRECURSOR.		45%	782	907
  						41%	791	925
  						50%	794	907
  						38%	863	1498
  						33%	190	741
  						98%	782	1642
  HE8FC45	843781	141	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	50%	1285	1172
  				CLONE KAIA0536.		57%	1824	1663
  						75%	1672	1553
  HE8FC45	845672	142	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	50%	1285	1172
  				CLONE KAIA0536.		57%	1824	1663
  						75%	1672	1553
  HE8FD92	856544	145	WUblastx.64	(Q9UJI9) HYPOTHETICAL 105.9 KDA	Q9UJI9	84%	419	1414
  				PROTEIN.		71%	2	1060
  						76%	419	1345
  						61%	2	613
  						45%	203	1060
  						40%	449	1345
  						52%	2	328
  						33%	605	1345
  						46%	47	328
  HE8FD92	869847	146	WUblastx.64	(Q9UJI9) HYPOTHETICAL 105.9 KDA	Q9UJI9	74%	4	609
  				PROTEIN.		59%	4	540
  						50%	1	255
  						63%	346	540
  						63%	346	540
  						60%	346	540
  						49%	1	255
  						48%	1	255
  						53%	346	540
  						41%	4	255
  						33%	40	255
  HE8FD92	901142	147	WUblastx.64	(Q9UJI9) HYPOTHETICAL 105.9 KDA	Q9UJI9	76%	31	480
  				PROTEIN.		56%	31	411
  						67%	217	408
  						67%	217	408
  						63%	217	411
  						63%	217	411
  						60%	217	411
  						53%	217	411
  						59%	31	126
  						56%	31	126
  						56%	31	126
  						56%	31	126
  						39%	58	126
  HE8SG96	862016	148	WUblastx.64	(Q9P195) PRO1722.	Q9P195	58%	1997	1845
  						63%	1854	1687
  HE8TY46	899528	149	WUblastx.64	(BAB55144) CDNA FLJ14576 fis,	BAB55144	95%	318	938
  				clone NT2RM4001092, w
  HE9CY05	834826	150	WUblastx.64	(Q9CX63) 6030468B19RIK	Q9CX63	48%	434	742
  				PROTEIN.		57%	55	426
  HE9EA10	827796	151	WUblastx.64	laminin alpha-1 chain precursor -	pir|S14458|S14458	99%	761	1891
  				human		27%	878	1840
  						25%	1142	1876
  HEBFR46	847064	157	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	80%	1111	1022
  				CLONE KAIA0536.		84%	1265	1110
  HEBGE07	798096	158	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	79%	1851	1720
  				CLONE KAIA0536.
  HELAT35	693175	160	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	72%	2092	1802
  				CLONE COL04765.
  HELBU54	637624	161	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	59%	1255	1031
  				CLONE COL04765.
  HEMEY47	834491	164	WUblastx.64	(Q9H387) PRO2550.	Q9H387	68%	513	587
  						74%	578	838
  HEPBA14	855935	166	WUblastx.64	(Q9BTY9) UNKNOWN (PROTEIN	Q9BTY9	87%	423	515
  				FOR IMAGE: 2823490)		71%	15	77
  				(FRAGMENT).		92%	85	426
  HEQAH80	701984	167	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	60%	818	1045
  				PROTEIN.
  HEQBF89	786205	168	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	64%	793	638
  				CLONE COL04765.		64%	647	489
  HETCI16	844543	169	WUblastx.64	(Q9P0V3) BOG25.	Q9P0V3	99%	3	356
  HETDW58	790557	170	WUblastx.64	unidentified 27.6K protein, spliced	pir|JC7586|JC7586	95%	324	1058
  				form A - human
  HFCFE20	701985	175	WUblastx.64	(Q9CSE5) EUKARYOTIC	Q9CSE5	89%	438	581
  				TRANSLATION INITIATION		54%	1083	1187
  				FACTOR 3 (FRAGMENT).
  HFEAY59	658685	176	WUblastx.64	(Q9Z320) C29.	Q9Z320	67%	50	1153
  HFGAJ16	580824	177	WUblastx.64	CDM protein - human	pir|S44279|S44279	97%	263	403
  HFIJA29	839206	179	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	46%	889	806
  						59%	1026	880
  HFIJA68	847074	180	WUblastx.64	(Q9UHE8) SIX TRANSMEMBRANE	STEA_HUMAN	89%	13	399
  				EPITHELIAL ANTIGEN OF
  				PROSTATE.
  HFKES05	827572	181	WUblastx.64	(BAB55088) CDNA FLJ14496 fis,	BAB55088	85%	84	314
  				clone NT2RM1000035.		94%	367	1722
  HFKEU12	634006	182	WUblastx.64	hypothetical protein 3 - rat	pir|S21347|S21347	52%	695	745
  						50%	757	933
  						40%	774	1007
  						54%	387	692
  HFPDS07	821646	185	WUblastx.64	(O94925) GLUTAMINASE, KIDNEY	GLSK_HUMAN	78%	343	513
  				ISOFORM, MITOCHONDRIAL		74%	2	436
  				PRECURS
  HFVGK35	731868	189	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	65%	832	608
  				PROTEIN.
  HFVHW43	570948	190	WUblastx.64	(Q9BGX4) HYPOTHETICAL 13.8 KDA	Q9BGX4	69%	1209	1093
  				PROTEIN.
  HFXAV37	626595	191	WUblastx.64	(O60448) NEURONAL THREAD	O60448	33%	583	461
  				PROTEIN AD7C-NTP.		68%	473	333
  						60%	1240	1166
  						47%	607	539
  						45%	1454	1275
  						56%	1295	1173
  						59%	1287	1222
  						64%	607	407
  						69%	607	461
  						57%	1285	1169
  						40%	1467	1402
  						48%	1321	1232
  						37%	558	346
  						78%	402	361
  						68%	591	406
  						31%	549	355
  						60%	398	354
  						36%	1321	1232
  						41%	1364	1275
  						46%	1187	1110
  						47%	1453	1187
  HFXBT66	580831	193	WUblastx.64	(Q9H387) PRO2550.	Q9H387	73%	739	807
  						58%	809	907
  						62%	564	764
  HGBER72	826710	195	WUblastx.64	(Q9H387) PRO2550.	Q9H387	71%	1061	969
  						78%	1104	1063
  						77%	1237	1103
  HGBHP91	693011	198	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	52%	541	491
  				human		44%	537	34
  HGCAC19	851527	199	WUblastx.64	(Q9UIE9) WUGSC: H_DJ0687K01.2	Q9UIE9	34%	984	1124
  				PROTEIN.		31%	1546	1863
  						96%	361	1047
  						24%	993	1124
  						22%	984	1124
  						40%	1002	1124
  						27%	984	1124
  						27%	981	1124
  						25%	984	1088
  						31%	984	1124
  						23%	984	1124
  						29%	984	1124
  						29%	984	1124
  						32%	1023	1124
  						26%	586	801
  						95%	2017	2712
  HGCAC19	801999	200	WUblastx.64	(Q9H6A1) CDNA: FLJ22454 FIS,	Q9H6A1	34%	984	1124
  				CLONE HRC09703 (FRAGMENT).		100%	184	210
  						31%	984	1124
  						29%	984	1124
  						40%	1002	1124
  						27%	984	1124
  						29%	984	1124
  						27%	981	1124
  						21%	984	1121
  						24%	993	1124
  						23%	984	1124
  						96%	316	1056
  HGCAC19	842540	201	WUblastx.64	(Q9H6A1) CDNA: FLJ22454 FIS,	Q9H6A1	34%	982	1122
  				CLONE HRC09703 (FRAGMENT).		100%	182	208
  						31%	982	1122
  						29%	982	1122
  						40%	1000	1122
  						27%	982	1122
  						29%	982	1122
  						27%	979	1122
  						21%	982	1119
  						24%	991	1122
  						23%	982	1122
  						96%	314	1045
  HHEAK45	765278	202	WUblastx.64	(Q9NPB0) DJ202I21.1 (NOVEL	Q9NPB0	68%	1949	1458
  				PROTEIN) (CDNA FLJ11101 FIS,
  				CLONE PLACE10
  HHEOW19	886174	204	WUblastx.64	(O18973) RAB5 GDP/GTP	O18973	77%	417	623
  				EXCHANGE FACTOR, RABEX5.		91%	611	715
  						56%	166	378
  						92%	129	167
  HHFFF87	778071	205	WUblastx.64	coatomer zeta chain - bovine	pir|A49465|A49465	100%	50	145
  HHFFL34	753230	206	WUblastx.64	(BAB55306) CDNA FLJ14793 fis,	BAB55306	100%	9	710
  				clone NT2RP4001174, w
  HHFFS40	824059	207	WUblastx.64	(Q9H4A6) GOLGI PROTEIN.	Q9H4A6	100%	3	251
  HHGDT26	658692	209	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	69%	1580	1290
  				CLONE COL04765.
  HHSBI65	801910	213	WUblastx.64	(Q9H5W9) CDNA: FLJ22888 FIS,	Q9H5W9	100%	270	407
  				CLONE KAT03934.		94%	479	1300
  HHSDI53	862028	214	WUblastx.64	(Q9H387) PRO2550.	Q9H387	70%	1108	935
  						71%	1241	1107
  						75%	1276	1241
  HILCA24	782450	217	WUblastx.64	(Q9NUU6) CDNA FLJ11127 FIS,	Q9NUU6	73%	103	159
  				CLONE PLACE1006225.		100%	168	1169
  HILCA24	869856	218	WUblastx.64	(Q9NUU6) CDNA FLJ11127 FIS,	Q9NUU6	95%	104	1171
  				CLONE PLACE1006225.
  HISAT67	843549	219	WUblastx.64	(Q9UH94) PROLACTIN	Q9UH94	88%	219	797
  				REGULATORY ELEMENT-		91%	788	1447
  				BINDING PROTEIN (PROLACTIN
  				REGU
  HJBCU75	638329	220	WUblastx.64	(O45030) STRABISMUS.	O45030	44%	199	426
  						52%	464	964
  HJMAA03	824062	221	WUblastx.64	(Q9N032) UNNAMED PROTEIN	Q9N032	71%	415	528
  				PRODUCT.
  HJMAV41	862029	222	WUblastx.64	brain-specific membrane anchor	pir|JC7110|JC7110	100%	14	475
  				protein - human
  HJMAY90	793678	223	WUblastx.64	(Q9DC16) 1200007D18RIK	Q9DC16	77%	100	312
  				PROTEIN (RIKEN CDNA		98%	315	968
  				1200007D18 GENE).
  HJPBE39	801960	224	WUblastx.64	(Q9CUS4) 4833420K19RIK	Q9CUS4	33%	1	621
  				PROTEIN (FRAGMENT).		74%	213	1007
  HJPCH08	840365	226	WUblastx.64	(O95235) RABKINESIN-6 (RAB6-	RB6K_HUMAN	93%	9	596
  				INTERACTING KINESIN-LIKE
  				PROTEI
  HKABU43	838573	227	WUblastx.64	(AAH03633) Translocase of outer	AAH03633	100%	33	62
  				mitochondrial membr		92%	26	1597
  HKACI79	853361	228	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	72%	886	1104
  				PROTEIN.
  HKAFF50	790192	229	WUblastx.64	(Q9P1G7) PRO1777.	Q9P1G7	99%	1753	1424
  HKGBF25	738797	230	WUblastx.64	(Q9HBS7) HYPOTHETICAL 14.2 KDA	Q9HBS7	71%	1708	1688
  				PROTEIN.		56%	1956	1708
  HKMLK03	734213	232	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	50%	981	832
  				PRODUCT.		73%	856	731
  HLDQU79	740755	237	WUblastx.64	(O75477) KE04P.	O75477	100%	105	1142
  HLDQU79	837599	3099	blastx.2	KE04P.	sp|O75477|O75477	99%	81	1118
  HLDRT09	830544	238	WUblastx.64	(Q9HAQ7) ATP-BINDING	Q9HAQ7	86%	2	469
  				CASSETTE HALF-TRANSPORTER.
  HLHAP05	638476	239	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	55%	1553	1500
  				CLONE MAMMA1000472.		72%	1650	1585
  						77%	1807	1646
  HLIBO72	883431	241	WUblastx.64	(AAH07829) Similar to hypothetical	AAH07829	100%	65	547
  				protein AF140225
  HLICE88	840321	242	WUblastx.64	fibrinogen gamma-A chain precursor	pir|A90470|FGHUG	89%	3	584
  				[validated] - human
  HLMBW89	701996	245	WUblastx.64	(AAH07983) Unknown (protein for	AAH07983	85%	390	247
  				MGC: 16279).
  HLMGP50	647603	246	WUblastx.64	(Q9GMI7) HYPOTHETICAL 9.0 KDA	Q9GMI7	61%	765	709
  				PROTEIN.		72%	935	807
  HLQAS12	886180	249	WUblastx.64	(Q9XTA8) LECTIN-LIKE	Q9XTA8	71%	690	842
  				OXIDIZED LDL RECEPTOR.		52%	364	711
  HLQCL64	864966	250	HMMER	PFAM: Major intrinsic protein	PF00230	87.3	87	449
  			2.1.1
  			WUblastx.64	aquaporin 9 - human	pir|JC5973|JC5973	98%	18	548
  HLQCX36	584786	251	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	87%	1100	1216
  HLWDB73	838453	258	WUblastx.64	(Q9H7D7) CDNA: FLJ21016 FIS,	Q9H7D7	100%	660	872
  				CLONE CAE05735.		98%	1	657
  HLYGB19	838083	261	WUblastx.64	(Q9H0Q1) HYPOTHETICAL 12.3 KDA	Q9H0Q1	97%	204	518
  				PROTEIN.
  HLYGY91	658703	263	WUblastx.64	(Q9H8N0) CDNA FLJ13386 FIS,	Q9H8N0	94%	221	391
  				CLONE PLACE1001104, WEAKLY
  				SIMILAR TO MYO
  HMCAZ04	839783	264	WUblastx.64	(Q9Y6N5) HYPOTHETICAL 50.0 KDA	Q9Y6N5	100%	106	1455
  				PROTEIN.
  HMCAZ04	858210	265	WUblastx.64	(Q9Y6N5) HYPOTHETICAL 50.0 KDA	Q9Y6N5	100%	106	1455
  				PROTEIN.
  HMCAZ04	867910	266	WUblastx.64	(Q9Y6N5) HYPOTHETICAL 50.0 KDA	Q9Y6N5	100%	106	1455
  				PROTEIN.
  HMCAZ04	887445	267	WUblastx.64	(Q9Y6N5) HYPOTHETICAL 50.0 KDA	Q9Y6N5	100%	107	1456
  				PROTEIN.
  HMCAZ04	668249	268	WUblastx.64	(Q9UQM8) CGI-44 PROTEIN.	Q9UQM8	100%	9	1055
  HMDAB29	584789	270	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	72%	1186	890
  				CLONE KAT08285.
  HMEBB82	783077	272	WUblastx.64	(Q9NSE4) MITOCHONDRIAL	Q9NSE4	99%	2	2206
  				ISOLEUCINE TRNA SYNTHETASE
  				(FRAGMENT).
  HMEDE24	837027	273	WUblastx.64	(Q9BVH9) SIMILAR TO GLUCOSE	Q9BVH9	94%	188	1159
  				REGULATED PROTEIN, 58 KDA.		42%	101	742
  HMEDI90	840077	274	WUblastx.64	(Q9HBA3) RAB3 INTERACTING	Q9HBA3	100%	81	794
  				PROTEIN VARIANT 4
  				(FRAGMENT).
  HMELM75	587307	275	WUblastx.64	(Q9NVW5) HYPOTHETICAL 31.3 KDA	Q9NVW5	100%	137	391
  				PROTEIN.
  HMICP65	847403	279	WUblastx.64	(Q9HAU9) GUANINE	Q9HAU9	99%	8	892
  				NUCLEOTIDE BINDING PROTEIN		22%	269	943
  				BETA SUBUNIT 5L.
  HMSBE04	709672	281	WUblastx.64	(Q9H5V8) CDNA: FLJ22969 FIS,	Q9H5V8	85%	182	3
  				CLONE KAT10759.
  HMSCL38	801919	282	WUblastx.64	(Q9P195) PRO1722.	Q9P195	64%	1272	1460
  						72%	2918	2844
  						64%	2851	2759
  						76%	2769	2653
  HMSCR69	843059	283	HMMER	PFAM: Zinc finger present in	PF00569	48.2	113	250
  			2.1.1	dystrophin, CBP/p300
  			WUblastx.64	(Q9BWK2) POTASSIUM CHANNEL	Q9BWK2	78%	107	1231
  				MODULATORY FACTOR.
  HMSHC86	840402	284	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	70%	1724	1674
  				PRODUCT.		67%	1674	1420
  HMSHU20	847410	285	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	47%	1722	1453
  				CLONE COL04765.
  HMTAB77	847411	287	WUblastx.64	(P43243) MATRIN 3.	MAT3_HUMAN	95%	630	1385
  						64%	287	628
  						22%	2002	2175
  						98%	3255	3428
  						31%	2041	2190
  						22%	2047	2181
  						23%	2584	2763
  						75%	2440	2760
  						27%	2596	2709
  						35%	1705	1797
  						35%	3312	3404
  						91%	1384	2328
  HMUAE26	747403	288	WUblastx.64	(Q9P2R4) SEVEN	Q9P2R4	89%	153	575
  				TRANSMEMBRANE DOMAIN		86%	577	1272
  				ORPHAN RECEPTOR.
  HMUAN45	833072	289	WUblastx.64	(BAB55441) CDNA FLJ14993 fis,	BAB55441	70%	684	1238
  				clone Y79AA1001874, w		65%	239	955
  						100%	1247	1516
  HMVBC31	825598	290	WUblastx.64	(O60725) PROTEIN-S	ICMT_HUMAN	80%	747	938
  				ISOPRENYLCYSTEINE O-		87%	121	789
  				METHYLTRANSFERASE (E
  HMVDU15	801969	291	WUblastx.64	(Q9BTJ2) SIMILAR TO CGI-30	Q9BTJ2	100%	75	917
  				PROTEIN.
  HMWBL03	822861	292	WUblastx.64	(Q9BWT1) C-MYC TARGET JP1.	Q9BWT1	85%	137	1240
  HMWJF53	758158	293	WUblastx.64	(Q9GZU7) NUCLEAR LIM	Q9GZU7	91%	3	170
  				INTERACTOR-INTERACTING		100%	154	720
  				FACTOR.
  HNEAK81	722235	294	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	56%	770	1087
  				PRODUCT.
  HNECL22	799541	295	WUblastx.64	(Q9P0J2) MITOCHONDRIAL	Q9P0J2	94%	1771	2331
  				SOLUTE CARRIER.
  HNEDH88	815675	297	WUblastx.64	(Q9GML5) HYPOTHETICAL 8.0 KDA	Q9GML5	56%	1706	1849
  				PROTEIN.
  HNFAC50	815676	298	WUblastx.64	(Q9H286) SEROLOGICALLY	Q9H286	100%	425	282
  				DEFINED BREAST CANCER
  				ANTIGEN NY-BR-20 (FRAGME
  HNFHF34	722237	300	WUblastx.64	(Q9NZX0) HSPC068.	Q9NZX0	100%	9	431
  						34%	9	404
  						35%	3	407
  						33%	9	407
  						32%	129	422
  HNGAK51	603910	301	WUblastx.64	(O60448) NEURONAL THREAD	O60448	61%	563	601
  				PROTEIN AD7C-NTP.		67%	733	915
  						65%	702	878
  						74%	714	914
  HNGAM58	688114	302	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	71%	1020	1061
  				CLONE COL04765.		85%	1081	1143
  						53%	818	1003
  HNGGP65	597449	310	WUblastx.64	(Q9GMU5) HYPOTHETICAL 14.1 KDA	Q9GMU5	31%	69	302
  				PROTEIN.		47%	398	541
  HNGJB41	852178	313	WUblastx.64	probable oxysterol-binding protein	pir|T02435|T02435	100%	128	9
  				DJ430N08.1 - human (fragment)
  HNHFE71	834487	320	WUblastx.64	hypothetical protein	pir|T47135|T47135	67%	822	583
  				DKFZp761L0812.1 - human
  				(fragment)
  HNHGK22	597451	321	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	41%	483	37
  				human		41%	333	10
  						50%	733	485
  HNHHB10	634589	322	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	70%	658	608
  				FOR MGC: 5149).		73%	845	711
  						73%	717	661
  HNTBT17	855957	324	WUblastx.64	(Q9NZF3) BM-001.	Q9NZF3	45%	818	1342
  						61%	729	947
  						84%	556	774
  HOACG07	792928	328	WUblastx.64	(Q9GZN8) DJ1009E24.3 (A NOVEL	Q9GZN8	99%	183	704
  				PROTEIN) (CDNA FLJ14158 FIS,
  				CLONE NT2R
  HODBV05	825283	331	WUblastx.64	(Q13878) 94 KDA B-RAF PROTEIN	Q13878	100%	566	661
  				(FRAGMENT).
  HODCZ32	836069	332	WUblastx.64	(Q9NSI6) WD-REPEAT PROTEIN 9	WDR9_HUMAN	86%	8	331
  				(FRAGMENT).
  HOEBK60	789396	333	WUblastx.64	(Q9H916) CDNA FLJ13081 FIS,	Q9H916	98%	132	1916
  				CLONE NT2RP3002033.		100%	14	109
  						88%	106	159
  HOFAA78	836646	334	WUblastx.64	(Q9NXS2) CDNA FLJ20084 FIS,	Q9NXS2	90%	529	792
  				CLONE COL03526.		50%	9	80
  						88%	29	529
  HOFNB74	762821	335	WUblastx.64	(Q99JH1) HYPOTHETICAL 17.7 KDA	Q99JH1	72%	44	187
  				PROTEIN.		97%	199	471
  HORBV76	839270	339	WUblastx.64	(Q9Y2B2)	Q9Y2B2	91%	30	761
  				PHOSPHATIDYLINOSITOL
  				GLYCAN, CLASS L (EC 3.5.—.—)
  				(PIG-L PRO
  HOSDO75	862049	340	WUblastx.64	(Q9D099) 1110057L18RIK	Q9D099	89%	11	202
  				PROTEIN.		88%	259	630
  HOSEC25	688055	341	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	73%	530	631
  				PROTEIN.		65%	627	809
  						64%	1501	1451
  						56%	1440	1222
  HOSEJ94	795132	343	WUblastx.64	(Q9GZY3) HT032 (PRK1-	Q9GZY3	92%	363	986
  				ASSOCIATED PROTEIN AWP1)
  				(PROTEIN ASSOCIATED WIT
  HOUCA21	655359	344	WUblastx.64	(Q9HBS7) HYPOTHETICAL 14.2 KDA	Q9HBS7	78%	988	1110
  				PROTEIN.
  HOUDE92	580866	345	WUblastx.64	(Q9HBT2) HYPOTHETICAL 17.2 KDA	Q9HBT2	96%	21	245
  				PROTEIN.
  HOUDR07	745404	346	WUblastx.64	(Q9HBV4) ANGIOPOIETIN-LIKE	Q9HBV4	87%	170	1384
  				PROTEIN PP1158.
  HOUED72	858547	347	WUblastx.64	(Q9CRP8) RIBOSOMAL PROTEIN	Q9CRP8	84%	676	774
  				L15 (FRAGMENT).		85%	110	682
  HOUFS04	771564	348	WUblastx.64	(Q9VN45) CG12001 PROTEIN.	Q9VN45	32%	1362	1982
  						39%	915	1106
  						26%	141	380
  HOUHI25	888279	349	WUblastx.64	(O95003) WUGSC: H_DJ0593H12.2	O95003	94%	73	783
  				PROTEIN.
  HPCAL26	762822	352	WUblastx.64	(O95084) SERINE PROTEASE	O95084	98%	398	640
  				(HYPOTHETICAL 43.0 KDA		76%	135	497
  				PROTEIN) (PROTEASE, S
  HPFBA54	635539	354	WUblastx.64	(Q9HBW6) NAG13.	Q9HBW6	76%	795	733
  						73%	766	602
  						84%	602	393
  						86%	135	91
  						79%	394	128
  HPFCI36	855966	355	WUblastx.64	(Q9NX47) CDNA FLJ20445 FIS,	Q9NX47	100%	9	320
  				CLONE KAT05170.
  HPJBU43	862058	360	WUblastx.64	(Q9P1E1) PRO2221.	Q9P1E1	54%	187	44
  HPMCJ84	562779	363	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	74%	619	479
  				CLONE KAIA0536.		69%	759	613
  HPMCV30	612870	364	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	76%	384	334
  				FOR MGC: 5149).		68%	590	399
  HPQAX38	843592	366	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	74%	664	768
  				PROTEIN.		68%	543	674
  HPQAX38	845752	367	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	74%	664	768
  				PROTEIN.		68%	543	674
  HPRBH85	695752	370	WUblastx.64	(BAB55300) CDNA FLJ14784 fis,	BAB55300	62%	2	616
  				clone NT2RP4000713.		86%	534	1085
  HPRCA64	824074	371	WUblastx.64	(P55161) NCK-ASSOCIATED	NCP1_RAT	100%	1021	1926
  				PROTEIN 1 (NAP 1) (P125NAP1)		85%	387	1019
  				(MEMBR		93%	11	481
  HPRCD35	853551	372	WUblastx.64	hypothetical protein	pir|T50629|T50629	100%	320	613
  				DKFZp762L1710.1 - human		57%	2	499
  				(fragment)
  HPTRM02	812879	373	WUblastx.64	(Q9UJU6) SRC HOMOLOGY 3	Q9UJU6	92%	332	940
  				DOMAIN-CONTAINING PROTEIN		97%	2	106
  				HIP-55 (DREBRIN F).		96%	98	190
  HRAAD30	866187	376	WUblastx.64	(Q9H6V0) CDNA: FLJ21839 FIS,	Q9H6V0	89%	23	1393
  				CLONE HEP01794.
  HRADA42	827302	377	WUblastx.64	hypothetical protein C11D2.4 -	pir|T32961|T32961	48%	387	668
  				Caenorhabditis elegans		74%	668	931
  HRADF49	866481	378	WUblastx.64	(Q9H6L1) CDNA: FLJ22169 FIS,	Q9H6L1	90%	13	825
  				CLONE HRC00632.		84%	813	1379
  						75%	1291	1593
  						34%	1590	1685
  HRADN25	800628	379	WUblastx.64	(Q9HB07) MYG1 PROTEIN.	MYG1_HUMAN	96%	47	1174
  HRDAI17	560720	381	WUblastx.64	(Q9NUM6) CDNA FLJ11267 FIS,	Q9NUM6	59%	1305	1475
  				CLONE PLACE1009174.
  HRDDQ39	840405	382	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	53%	582	436
  				CLONE KAIA0536.		65%	775	578
  HRDER22	688056	383	WUblastx.64	(Q9NW07) CDNA FLJ10390 FIS,	Q9NW07	80%	9	248
  				CLONE NT2RM4000104,		100%	357	431
  				MODERATELY SIMILAR TO		39%	120	227
  						28%	15	203
  						38%	254	316
  HRDEX93	816046	384	WUblastx.64	(Q9UBV8) PEFLIN.	Q9UBV8	100%	313	864
  HRDFK37	840381	385	WUblastx.64	(Q9P195) PRO1722.	Q9P195	69%	536	652
  						40%	50	115
  HRGBD54	828436	386	WUblastx.64	(O95819) HPK/GCK-LIKE KINASE	O95819	51%	32	253
  				HGK.		74%	253	645
  						27%	6	149
  						92%	781	2019
  HSAVA08	580870	388	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	57%	949	896
  				PROTEIN.		42%	926	792
  						63%	796	764
  						66%	1059	934
  HSAWZ40	634000	391	WUblastx.64	(O00549) ORF2-LIKE PROTEIN	O00549	64%	951	610
  				(FRAGMENT).		60%	613	8
  HSDZM54	637870	393	WUblastx.64	NADH dehydrogenase (ubiquinone)	pir|A00422|DNHUN3	88%	226	360
  				(EC 1.6.5.3) chain 3 - human
  				mitochondrion
  HSHBF76	715838	394	WUblastx.64	(AAH08335) Unknown (protein for	AAH08335	86%	762	457
  				IMAGE: 3506202) (Fra		73%	882	748
  						100%	1267	836
  HSJBY32	702020	396	WUblastx.64	(Q9GZZ6) NEURONAL NICOTINIC	Q9GZZ6	81%	466	639
  				ACETYLCHOLINE ALPHA10		57%	215	514
  				SUBUNIT PRECURSOR (
  HSLHX15	777861	399	WUblastx.64	catalase (EC 1.11.1.6) -	pir|I40767|I40767	86%	162	76
  				Campylobacter jejuni
  HSNBM34	635131	402	WUblastx.64	acyl-CoA dehydrogenase (EC 1.3.99.—)	pir|S54183|S54183	84%	1548	1979
  				very-long-chain specific - human		100%	251	1546
  HSOAH16	827058	403	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	57%	682	623
  				CLONE KAIA0536.		81%	624	544
  						68%	524	384
  HSQDO85	853393	405	WUblastx.64	(Q9VCK0) CG10161 PROTEIN.	Q9VCK0	67%	485	988
  						60%	60	521
  						56%	10	57
  HSQES57	831222	406	WUblastx.64	(Q96EW4) Unknown (protein for	Q96EW4	94%	195	980
  				MGC: 19936).
  HSRBE06	871264	407	WUblastx.64	(Q9H387) PRO2550.	Q9H387	70%	1608	1327
  HSSDI26	560722	408	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	68%	1398	1264
  				FOR MGC: 5149).
  HSSEA64	853395	409	WUblastx.64	(Q9HBT2) HYPOTHETICAL 17.2 KDA	Q9HBT2	98%	7	243
  				PROTEIN.
  HSSEF77	658725	410	WUblastx.64	(O95637) WW DOMAIN BINDING	O95637	42%	10	246
  				PROTEIN-1.		83%	296	829
  HSSFE38	742512	411	HMMER	PFAM: Ribonuclease HII	PF01351	76.3	184	−142
  			2.1.1
  			WUblastx.64	(O75792) RIBONUCLEASE HI	RNHL_HUMAN	91%	156	635
  				LARGE SUBUNIT (EC 3.1.26.—)		99%	587	1051
  				(RNASE
  HSWBE76	751308	413	WUblastx.64	(Q9NW15) CDNA FLJ10375 FIS,	Q9NW15	100%	126	266
  				CLONE NT2RM2001950.
  HSXCP38	895392	414	WUblastx.64	hydroxymethylglutaryl-CoA lyase (EC	pir|B45470|B45470	70%	17	895
  				4.1.3.4) - chicken
  HSYBI06	740766	415	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	69%	916	954
  				PROTEIN.		78%	821	913
  HT5GR59	801930	420	WUblastx.64	(O60496) DOCKING PROTEIN.	O60496	72%	70	1284
  HTAEI78	637684	421	WUblastx.64	(Q9UKQ2) ADAM 28 PRECURSOR	AD28_HUMAN	90%	85	174
  				(EC 3.4.24.—) (A DISINTEGRIN AND
  HTDAA78	566861	422	WUblastx.64	(Q9D8E7) 5830443F10RIK	Q9D8E7	58%	84	302
  				PROTEIN.
  HTEAG62	812332	423	WUblastx.64	(Q9Y5Z7) HOST CELL FACTOR 2.	Q9Y5Z7	60%	1	57
  						93%	14	2011
  						30%	107	631
  HTECB02	806305	424	WUblastx.64	(AAK39520) BTB domain protein	AAK39520	95%	33	1211
  				(Fragment).
  HTECC15	866488	425	WUblastx.64	(Q92558) WISKOTT-ALDRICH	WAS1_HUMAN	95%	321	1100
  				SYNDROME PROTEIN FAMILY		70%	1525	1998
  				MEMBER 1 (		89%	1105	1281
  HTEDS12	838621	428	WUblastx.64	(Q9H0K0) HYPOTHETICAL 81.8 KDA	Q9H0K0	97%	1029	1391
  				PROTEIN.		42%	1269	1490
  						100%	16	1011
  HTEEF26	789606	431	WUblastx.64	(Q9H7X7) CDNA FLJ14117 FIS,	Q9H7X7	81%	80	634
  				CLONE MAMMA1001785.
  HTEEF26	879704	432	WUblastx.64	(Q9H7X7) CDNA FLJ14117 FIS,	Q9H7X7	81%	80	634
  				CLONE MAMMA1001785.
  HTEEW69	764835	433	WUblastx.64	(Q9Z1H7) GSG1.	Q9Z1H7	65%	850	927
  						85%	707	769
  						50%	519	662
  						66%	908	943
  						65%	182	544
  HTEGS07	827700	434	WUblastx.64	(Q9D143) 1110030K22RIK	Q9D143	96%	183	593
  				PROTEIN.
  HTEHA56	806461	436	WUblastx.64	(Q9H9A0) CDNA FLJ12895 FIS,	Q9H9A0	94%	2	217
  				CLONE NT2RP2004187, WEAKLY		65%	70	468
  				SIMILAR TO ZIN
  HTEJD29	695798	438	WUblastx.64	(Q60713) REVERSE	Q60713	42%	1115	1285
  				TRANSCRIPTASE.		47%	874	1089
  HTEMQ17	840387	440	WUblastx.64	(Q9D4P8) 4930579G24RIK	Q9D4P8	90%	120	359
  				PROTEIN.
  HTENR63	877952	441	WUblastx.64	(Q9HD71) HYPOTHETICAL	Q9HD71	33%	1278	1358
  				NUCLEAR FACTOR SBBI22.		78%	26	1168
  HTGGM44	842856	442	WUblastx.64	probable phosphodiesterase I (EC	pir|T43461|T43461	100%	1400	1924
  				3.1.4.1) - human (fragment)		83%	1925	2488
  HTLBT80	840045	445	WUblastx.64	(Q9NQQ7) BA394O2.1 (CGI-15	Q9NQQ7	76%	1214	1405
  				PROTEIN).		74%	804	1223
  						47%	780	845
  						78%	313	825
  HTLDA84	686397	446	WUblastx.64	(Q9H387) PRO2550.	Q9H387	79%	1265	1134
  						60%	1442	1398
  						65%	1398	1243
  HTLDN29	790195	447	WUblastx.64	(Q9CWL8) 5730471K09RIK	Q9CWL8	96%	15	1226
  				PROTEIN.
  HTLEC82	811992	449	WUblastx.64	(Q99MI0) CELL GROWTH	Q99MI0	98%	111	455
  				REGULATOR FALKOR.
  HTLEM16	779133	450	WUblastx.64	(O95638) WW DOMAIN BINDING	O95638	92%	50	541
  				PROTEIN-2.		28%	987	1142
  						48%	617	841
  HTLEV48	723799	451	WUblastx.64	(BAB55550) Bk125H2.1 protein.	BAB55550	94%	10	825
  HTLFA13	535937	452	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	57%	1118	873
  HTLGI89	835069	454	WUblastx.64	(Q9BXS5) CLATHRIN-	Q9BXS5	98%	104	682
  				ASSOCIATED PROTEIN AP47.		99%	675	1370
  HTLIF11	843506	455	WUblastx.64	(Q9I8S4) ORNITHINE	Q9I8S4	68%	309	356
  				DECARBOXYLASE-2.		59%	353	1687
  HTLIF12	834946	456	WUblastx.64	(Q9DAR1) 1700001K04RIK	Q9DAR1	47%	291	752
  				PROTEIN.
  HTLIF12	842691	457	WUblastx.64	(Q9DAR1) 1700001K04RIK	Q9DAR1	47%	293	754
  				PROTEIN.
  HTLIF12	870167	458	WUblastx.64	(Q9DAR1) 1700001K04RIK	Q9DAR1	47%	293	754
  				PROTEIN.
  HTLIF12	886780	459	WUblastx.64	(Q9DAR1) 1700001K04RIK	Q9DAR1	47%	293	754
  				PROTEIN.
  HTLIF12	891533	460	WUblastx.64	(Q9DAR1) 1700001K04RIK	Q9DAR1	47%	293	754
  				PROTEIN.
  HTLIF12	901225	461	WUblastx.64	(Q9DAR1) 1700001K04RIK	Q9DAR1	47%	293	754
  				PROTEIN.
  HTNBK13	831967	463	WUblastx.64	(Q9Y3M2) HYPOTHETICAL 14.5 KDA	Q9Y3M2	81%	123	500
  				PROTEIN.
  HTOAM11	664508	465	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	77%	428	363
  				CLONE LNG09295.		75%	586	425
  HTOEV16	853616	468	WUblastx.64	(Q9NRZ5) 1-ACYL-SN-	PLCD_HUMAN	98%	201	383
  				GLYCEROL-3-PHOSPHATE		95%	379	1164
  				ACYLTRANSFERASE DEL
  HTOHO21	732808	470	WUblastx.64	P47 LBC oncogene - human	pir|I38434|I38434	97%	581	438
  HTOHQ05	853621	471	WUblastx.64	(Q9UII4) CYCLIN-E BINDING	Q9UII4	100%	669	791
  				PROTEIN 1.
  HTOJL95	806212	472	WUblastx.64	(Q15605) ORF1 CODES FOR A 40 KDA	Q15605	86%	192	61
  				PRODUCT.		57%	876	730
  						57%	751	161
  HTOJL95	762851	473	WUblastx.64	(Q15401) LINE-1 REPEAT MRNA	Q15401	36%	683	609
  				WITH 2 OPEN READING FRAMES.		59%	966	820
  						71%	607	248
  HTPDU17	840596	474	WUblastx.64	(Q9NW00) CDNA FLJ10404 FIS,	Q9NW00	80%	553	1308
  				CLONE NT2RM4000486.		64%	1143	1664
  HTSFJ32	637720	475	WUblastx.64	(Q9WUW2) VESICLE	Q9WUW2	64%	747	788
  				ASSOCIATED MEMBRANE		94%	448	609
  				PROTEIN 2B.
  HTTCB60	853401	476	WUblastx.64	(Q9HAW0) RNA POLYMERASE III	Q9HAW0	90%	6	881
  				TRANSCRIPTION INITIATION
  				FACTOR BRFU.
  HTTEE41	840950	477	WUblastx.64	(P78371) T-COMPLEX PROTEIN 1,	TCPB_HUMAN	98%	92	1696
  				BETA SUBUNIT (TCP-1-BETA) (CC
  HTTEZ02	702027	478	WUblastx.64	(Q9UEZ7) MAKORIN 1.	Q9UEZ7	56%	278	346
  						98%	6	272
  HTWEH94	561680	479	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	60%	1150	929
  				PROTEIN.
  HTXDC38	801935	482	WUblastx.64	(Q9BTX3) SIMILAR TO HSPC171	Q9BTX3	99%	100	573
  				PROTEIN.
  HTXDC77	844258	483	HMMER	PFAM: Class I Histocompatibility	PF00129	103.3	137	259
  			2.1.1	antigen, domains alpha 1 and 2
  			WUblastx.64	(P03989) HLA CLASS I	1B14_HUMAN	63%	880	945
  				HISTOCOMPATIBILITY ANTIGEN,		71%	65	256
  				B-27 ALPHA		80%	282	863
  HTXFA72	853410	487	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	59%	1688	1557
  				PRODUCT.		66%	1839	1681
  HTXKF95	834438	489	WUblastx.64	(AAH08360) Similar to hypothetical	AAH08360	85%	233	553
  				protein FLJ22376		100%	2	112
  HTXMZ07	834881	490	WUblastx.64	(Q9BRF3) SIMILAR TO RIKEN	Q9BRF3	90%	3	1469
  				CDNA 2810468K17 GENE.
  HUFCL31	801938	491	WUblastx.64	(Q9D311) 9030623N16RIK	Q9D311	60%	280	1224
  				PROTEIN.
  HUKBT67	844446	492	WUblastx.64	(BAB55428) CDNA FLJ14975 fis,	BAB55428	100%	1040	1216
  				clone THYRO1001405, w		100%	8	61
  						30%	80	241
  HUKDY82	570896	494	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	59%	1405	1145
  				CLONE MAMMA1000472.
  HUSCJ14	894699	495	WUblastx.64	tex261 protein - mouse	pir|S47481|S47481	99%	74	661
  HUSGL67	792637	496	WUblastx.64	(Q9Y2G2) CARD DOMAIN	CRD8_HUMAN	100%	347	421
  				PROTEIN 8 (APOPTOTIC PROTEIN		65%	947	1006
  				NDPP1) (D		97%	469	954
  HUSGU40	684975	497	WUblastx.64	(Q9BX98) UBIQUITIN A-52	Q9BX98	75%	840	433
  				RESIDUE RIBOSOMAL PROTEIN
  				FUSION PRODUCT 1 (F
  HUVDJ48	564853	499	WUblastx.64	SHORT ISOFORM OF Q9P2N4	sp_vs|Q9P2N4-	92%	1510	1668
  					01|Q9P2N4
  HWAAI12	830432	500	WUblastx.64	(Q9BWW4) SINGLE STRANDED	Q9BWW4	82%	512	829
  				DNA BINDING PROTEIN-1.		87%	92	394
  						69%	941	1252
  						36%	521	685
  						37%	752	826
  HWBCN36	722259	502	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	69%	1007	900
  				PROTEIN.		57%	887	846
  HWBDJ08	762860	503	WUblastx.64	probable pol polyprotein-related	pir|S21348|S21348	47%	901	833
  				protein 4 - rat		43%	1262	1131
  						53%	1134	904
  HWDAC26	821335	505	WUblastx.64	(Q14287) HYPOTHETICAL	Q14287	51%	1316	1471
  				PROTEIN (FRAGMENT).		57%	1093	1323
  HWDAG96	796743	506	WUblastx.64	(AAH01119) Integrin beta 4 binding	AAH01119	100%	108	842
  				protein.
  HWHPB78	740778	508	WUblastx.64	(Q9BUK4) SIMILAR TO	Q9BUK4	61%	360	614
  				HYPOTHETICAL PROTEIN		100%	677	817
  				FLJ10709.
  HYABC84	789854	509	WUblastx.64	(Q99L03) SIMILAR TO TRP4-	Q99L03	89%	209	553
  				ASSOCIATED PROTEIN TAP1
  				(FRAGMENT).
  HYABC84	865064	510	WUblastx.64	(Q9H429) DJ756N5.2 (A NOVEL	Q9H429	92%	163	618
  				PROTEIN (DKFZP727M231)
  				SIMILAR TO TRP4-AS
  H2CBD20	570796	511	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	42%	758	988
  				PROTEIN (FRAGMENT).		51%	987	1223
  H2CBH91	826669	512	WUblastx.64	(Q9NXA3) CDNA FLJ20357 FIS,	Q9NXA3	63%	133	222
  				CLONE HEP16545.		53%	3	125
  						75%	603	737
  						68%	263	643
  H2LBA54	684290	513	WUblastx.64	(O60875) APOPTOSIS SPECIFIC	O60875	97%	172	996
  				PROTEIN (DJ134E15.2)
  				(APOPTOSIS SPECIFIC
  H2LBB09	658667	514	WUblastx.64	(Q9P0R6) HSPC210.	Q9P0R6	95%	465	536
  						97%	73	477
  H2LBB09	830636	515	WUblastx.64	(Q9P0R6) HSPC210.	Q9P0R6	100%	504	575
  						97%	112	516
  H2MBF60	695714	518	WUblastx.64	transcription factor TFIID 32K chain	pir|I39141|I39141	100%	163	909
  				TAFII32 - human
  H6EEA48	847111	520	WUblastx.64	(AAH07644) Similar to RIKEN cDNA	AAH07644	90%	478	933
  				9430029K10 gene (F		96%	27	482
  H6EEN71	829201	521	WUblastx.64	(Q9BW90) SIMILAR TO	Q9BW90	100%	1943	1779
  				PEROXISOME BIOGENESIS
  				FACTOR 10.
  H6EEO05	865424	522	HMMER	PFAM: EF hand	PF00036	25	328	414
  			2.1.1
  			WUblastx.64	hypothetical protein	pir|T17225|T17225	97%	265	522
  				DKFZp564C246.1 - human
  HACBJ11	797625	528	WUblastx.64	band-6-protein - human	pir|S60712|S60712	100%	8	139
  HACBS86	603946	529	WUblastx.64	(Q9BQB1) HYPOTHETICAL 22.5 KDA	Q9BQB1	66%	89	682
  				PROTEIN.
  HADCL19	599065	535	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	57%	1070	780
  				CLONE COL04765.
  HADDC04	601695	537	WUblastx.64	(Q9GML5) HYPOTHETICAL 8.0 KDA	Q9GML5	57%	2522	2379
  				PROTEIN.
  HADDP51	853356	539	HMMER	PFAM: TBC domain	PF00566	52.3	357	509
  			2.1.1
  			WUblastx.64	(Q9H695) CDNA: FLJ22474 FIS,	Q9H695	99%	12	701
  				CLONE HRC10568.
  HADET62	607615	541	WUblastx.64	retrovirus-related hypothetical protein	pir|S23650|S23650	33%	506	330
  				II - human 1		27%	325	77
  						50%	657	592
  						44%	808	668
  HADEY08	799507	542	WUblastx.64	(Q9NX94) CDNA FLJ20367 FIS,	Q9NX94	98%	663	1076
  				CLONE HEP18101.
  HADEY22	861628	544	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	77%	590	459
  				CLONE COL04765.
  HADFB84	668229	545	WUblastx.64	(Q9H387) PRO2550.	Q9H387	67%	758	1009
  HADFD10	843934	547	WUblastx.64	(Q9P1C6) PRO2738.	Q9P1C6	52%	1254	1054
  HADFW20	599066	550	WUblastx.64	(Q9H9H0) CDNA FLJ12759 FIS,	Q9H9H0	83%	1065	823
  				CLONE NT2RP2001347.
  HADFX10	741054	551	WUblastx.64	(O60448) NEURONAL THREAD	O60448	45%	1225	1040
  				PROTEIN AD7C-NTP.		62%	726	646
  						35%	1221	808
  						39%	1194	850
  						56%	917	729
  						33%	965	729
  						57%	1022	939
  						45%	726	628
  						51%	972	772
  						59%	972	715
  						57%	1225	1169
  						34%	1188	940
  HADFY80	654831	552	WUblastx.64	(Q9H8X9) CDNA FLJ13153 FIS,	Q9H8X9	94%	2	169
  				CLONE NT2RP3003409, WEAKLY
  				SIMILAR TO HUM
  HADXA10	772423	555	WUblastx.64	(Q9NZ51) NEUROENDOCRINE	Q9NZ51	78%	132	311
  				DIFFERENTIATION FACTOR.		79%	295	798
  HADXA10	859777	556	WUblastx.64	(Q9D792) 9130011K15RIK	Q9D792	49%	173	472
  				PROTEIN.
  HAFBB15	608180	557	HMMER	PFAM: RNA 3′-terminal phosphate	PF01137	159.5	186	440
  			2.1.1	cyclase
  			WUblastx.64	RNA-3′-phosphate cyclase (EC	pir|T48844|T48844	93%	6	446
  				6.5.1.4) 1 [validated] - human
  HAGAB62	588471	559	WUblastx.64	(Q9H2I4) DC42.	Q9H2I4	77%	8	232
  HAGAB83	823044	560	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	47%	1597	1472
  				CLONE KAT08285.		76%	1817	1599
  HAGAF75	561933	562	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	77%	1203	1310
  HAGAK40	731929	563	WUblastx.64	(Q9H387) PRO2550.	Q9H387	80%	506	658
  						70%	707	838
  HAGAZ36	564230	565	WUblastx.64	pro-pol-dUTPase polyprotein - murine	pir|T29097|T29097	66%	613	578
  				endogenous retrovirus ERV-L		38%	897	655
  				(fragment)		34%	572	27
  HAGBL31	679582	567	WUblastx.64	(Q13416) ORIGIN RECOGNITION	ORC2_HUMAN	97%	509	610
  				COMPLEX SUBUNIT 2.
  HAGBO09	853357	568	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	78%	1533	1249
  				CLONE KAT08285.
  HAGBO12	601431	569	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	63%	348	241
  				PROTEIN.		76%	532	353
  HAGBS89	846292	571	WUblastx.64	(Q9P195) PRO1722.	Q9P195	56%	1125	841
  HAGBV06	701966	572	WUblastx.64	(Q9UHC7) MAKORIN 1.	Q9UHC7	66%	28	195
  HAGBV25	838174	573	WUblastx.64	(Q9H7C8) CDNA: FLJ21040 FIS,	Q9H7C8	99%	1	1644
  				CLONE CAE10642.
  HAGBV29	837203	574	WUblastx.64	(Q9H387) PRO2550.	Q9H387	70%	2054	1761
  						71%	439	585
  						60%	309	455
  						56%	749	955
  						48%	732	836
  						73%	687	731
  HAGCC87	638587	575	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	65%	992	1105
  				PROTEIN.		36%	54	116
  						57%	801	980
  HAGCI69	560596	577	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	51%	1015	1185
  HAGCZ70	747697	579	WUblastx.64	(Q9H397) PRO2852.	Q9H397	41%	1672	1544
  						75%	1490	1359
  						77%	1366	1235
  HAGDC73	724860	580	WUblastx.64	(Q9V662) CG12367 PROTEIN.	Q9V662	37%	536	1006
  HAGDJ53	821315	584	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	70%	1873	1781
  				PROTEIN.		46%	1796	1713
  						41%	1782	1582
  HAGDL51	637488	586	WUblastx.64	(O60448) NEURONAL THREAD	O60448	61%	1639	1532
  				PROTEIN AD7C-NTP.		70%	110	3
  HAGDO70	812393	587	WUblastx.64	platelet-activating factor	pir|JC4246|JC4246	94%	185	877
  				acetylhydrolase (EC 3.1.1.—) gamma
  				chain - human
  HAGDT30	589514	588	WUblastx.64	(O75592) PROTEIN ASSOCIATED	O75592	100%	11	280
  				WITH MYC.		24%	11	208
  						100%	1125	1946
  						25%	295	933
  						90%	511	1131
  						45%	1445	1504
  HAGDW68	835631	589	WUblastx.64	(AAC14670)	AAC14670	100%	390	470
  				WUGSC: H_DJ0651K02.1 protein		99%	2	385
  				(Fragment).
  HAGEK86	748222	592	WUblastx.64	(O43237) DYNEIN LIGHT	DYJ2_HUMAN	88%	1066	1617
  				INTERMEDIATE CHAIN 2,
  				CYTOSOLIC (LIC5
  HAGEP30	604478	593	WUblastx.64	(Q9H387) PRO2550.	Q9H387	84%	594	517
  						67%	788	606
  HAGEQ67	838445	595	WUblastx.64	(Q94532) PUTATIVE TYPE III	Q94532	55%	22	123
  				ALCOHOL DEHYDROGENASE.		59%	108	722
  HAGEU26	608183	596	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	59%	356	481
  				PROTEIN.
  HAGFM58	604536	603	WUblastx.64	(Q9H3C0) PRO0898.	Q9H3C0	62%	10	144
  HAGFT48	780112	604	WUblastx.64	(Q9Y2Y7) FOOCEN-M (NOGO-B	Q9Y2Y7	80%	761	1090
  				PROTEIN) (RTN-XS) (RETICULON		100%	1156	1281
  				4B).		48%	233	559
  HAGFU31	751713	605	WUblastx.64	(O60448) NEURONAL THREAD	O60448	65%	1214	1083
  				PROTEIN AD7C-NTP.		68%	1245	1084
  						45%	1093	1028
  						52%	1015	953
  						68%	1094	1029
  						71%	1230	967
  HAGFW13	634611	606	WUblastx.64	(O00549) ORF2-LIKE PROTEIN	O00549	65%	398	321
  				(FRAGMENT).		65%	453	385
  						70%	307	164
  HAGHE85	838059	607	WUblastx.64	(Q9P195) PRO1722.	Q9P195	58%	1665	1498
  						69%	1511	1365
  HAHSD51	847014	612	WUblastx.64	(Q9D8B4) 2010012C24RIK	Q9D8B4	50%	99	176
  				PROTEIN.		61%	182	505
  HAIBV91	852223	615	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	59%	672	899
  				PRODUCT.		40%	1911	1955
  HAICE62	834523	616	WUblastx.64	(Q9H3T5) MOB1 PROTEIN	Q9H3T5	100%	203	850
  				(HYPOTHETICAL 25.1 KDA
  				PROTEIN).
  HAICL90	637491	617	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	63%	716	438
  				PRODUCT.
  HAIDP45	847015	619	WUblastx.64	(AAH08590) Hypothetical 47.9 kDa	AAH08590	100%	986	1096
  				protein.
  HAJAB88	780114	620	HMMER	PFAM: Ribosomal protein L34	PF00468	32.3	276	401
  			2.1.1
  			WUblastx.64	(Q9BQ48) MITOCHONDRIAL	Q9BQ48	98%	126	374
  				RIBOSOMAL PROTEIN L34
  				(L34MT) (UNKNOWN) (PROTE
  HAMGG01	783864	624	WUblastx.64	(Q9BY78) RING FINGER PROTEIN	Q9BY78	86%	7	342
  				WITH LEUCINE ZIPPER RNF26.
  HANKC93	847018	626	WUblastx.64	(Q9H387) PRO2550.	Q9H387	67%	648	565
  						52%	876	820
  						61%	748	656
  HAPAD35	840584	627	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	76%	1346	1245
  				CLONE KAIA0536.		63%	1237	1064
  HAPBR13	609976	628	WUblastx.64	(Q9H6T0) CDNA: FLJ21918 FIS,	Q9H6T0	26%	273	452
  				CLONE HEP04006.		51%	95	274
  						40%	424	762
  HAPBU09	762803	629	WUblastx.64	(O60859) NEUROPATHY TARGET	O60859	100%	3	545
  				ESTERASE.
  HAPNJ33	835554	632	WUblastx.64	(BAA85159) Sec61.	BAA85159	100%	1200	1403
  						98%	163	1230
  HAPNL62	790340	633	WUblastx.64	(BAB55063) CDNA FLJ14456 fis,	BAB55063	93%	1	1977
  				clone HEMBB1001915, m
  HAPNO50	834384	634	WUblastx.64	hypothetical protein	pir|T08701|T08701	98%	27	332
  				DKFZp564N123.1 - human (fragment)		88%	3	29
  						86%	323	883
  HAPPW83	847020	636	WUblastx.64	(Q9H387) PRO2550.	Q9H387	77%	626	757
  						84%	475	627
  HAPRK55	735887	643	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	63%	1379	1068
  				CLONE KAT08285.
  HAPSH37	847021	644	WUblastx.64	(Q9H387) PRO2550.	Q9H387	75%	63	28
  						84%	199	68
  HAQBY85	832384	646	WUblastx.64	(Q9D4H9) 4932409F11RIK	Q9D4H9	100%	17	358
  				PROTEIN.		55%	1663	1716
  						28%	1066	1212
  						34%	1459	1728
  						93%	361	1539
  HAQBZ15	801966	647	WUblastx.64	(AAH07201) Unknown (protein for	AAH07201	100%	6	86
  				IMAGE: 2961284) (Fra		30%	1717	1824
  						93%	175	711
  						37%	1019	1564
  						29%	1277	1510
  						35%	1711	1836
  						97%	1705	1956
  						35%	843	953
  						31%	205	357
  						27%	187	408
  						67%	641	1708
  						52%	1711	1836
  						31%	849	962
  						31%	1078	1296
  						26%	1241	1696
  						36%	1123	1266
  						30%	1292	1564
  						27%	1081	1146
  						37%	882	953
  						30%	205	498
  						29%	1684	1938
  						29%	196	357
  						27%	1054	1317
  						90%	83	181
  HARAE26	560598	650	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	65%	1204	1076
  				CLONE COL03536.		75%	1068	925
  HARAT69	769389	651	WUblastx.64	(Q9VZ55) CG1582 PROTEIN.	Q9VZ55	48%	2	1360
  HARAZ81	832380	652	WUblastx.64	(Q9BQ36) MITOCHONDRIAL	Q9BQ36	97%	7	327
  				RIBOSOMAL PROTEIN BMRP64
  				(HYPOTHETICAL 15.1 KD
  HASAU26	845848	653	WUblastx.64	(Q9H964) CDNA FLJ12984 FIS,	Q9H964	94%	3	107
  				CLONE NT2RP3000047, WEAKLY		95%	132	194
  				SIMILAR TO NPL		98%	820	1425
  						40%	2405	2479
  						98%	188	829
  						100%	98	133
  HASAY07	834511	655	WUblastx.64	catalase (EC 1.11.1.6) -	pir|I40767|I40767	96%	263	177
  				Campylobacter jejuni
  HATAE01	654834	656	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	71%	569	507
  				CLONE KAT08285.		95%	636	574
  						72%	790	647
  HATAL05	847023	658	WUblastx.64	(Q9ER61) MDM2 BINDING	Q9ER61	68%	10	876
  				PROTEIN.
  HATCF80	780460	661	WUblastx.64	(Q99LV8) UNKNOWN (PROTEIN	Q99LV8	78%	9	479
  				FOR IMAGE: 3489486)
  				(FRAGMENT).
  HATCI67	847024	662	WUblastx.64	(Q9UHU3) PRO1659.	Q9UHU3	85%	214	528
  HATDH23	603959	668	WUblastx.64	(O43439) MTG8-LIKE PROTEIN	O43439	98%	1	348
  				(MTG8 RELATED PROTEIN) (EHT)
  				(EHT PROTEIN)
  HATDO84	609850	670	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	58%	798	646
  				CLONE KAIA0536.		77%	919	812
  HATDU01	847028	671	WUblastx.64	(Q9H387) PRO2550.	Q9H387	81%	1257	1225
  						58%	1198	974
  HAUCC84	830672	677	WUblastx.64	(O75353) ANTI-DEATH PROTEIN.	O75353	72%	44	259
  						98%	250	498
  HAWAS41	877621	678	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	66%	754	930
  				PROTEIN.
  HAWBA65	542056	679	WUblastx.64	(Q24333) ELASTIN LIKE PROTEIN	Q24333	100%	41	112
  				(FRAGMENT).
  HBAGH64	801884	680	WUblastx.64	(Q14287) HYPOTHETICAL	Q14287	37%	810	568
  				PROTEIN (FRAGMENT).
  HBBBA42	841010	684	WUblastx.64	(Q9UIL1) HRIHFB2072 PROTEIN	Q9UIL1	58%	3	263
  				(FRAGMENT).		57%	6	269
  HBCAQ48	525002	690	WUblastx.64	(O75528) ADA3-LIKE PROTEIN.	O75528	84%	73	345
  HBGBE75	897455	694	WUblastx.64	(O60830) MITOCHONDRIAL	I17B_HUMAN	97%	98	307
  				IMPORT INNER MEMBRANE
  				TRANSLOCASE SU
  HBHAA53	603183	699	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	65%	439	326
  				PRODUCT.		68%	594	415
  HBIAU43	840354	700	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	76%	1109	1225
  HBIAW58	596805	701	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	66%	1268	1188
  				CLONE COL04765.		77%	1335	1270
  						68%	1486	1337
  HBIBF26	845743	703	WUblastx.64	(Q9NQQ7) BA394O2.1 (CGI-15	Q9NQQ7	76%	1214	1405
  				PROTEIN).		74%	804	1223
  						47%	780	845
  						78%	313	825
  HBIBQ69	580807	706	WUblastx.64	(BAB55217) CDNA FLJ14686 fis,	BAB55217	94%	671	724
  				clone NT2RP2004961, m		100%	721	810
  HBIBR38	612783	707	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	73%	802	503
  				CLONE KAT08285.
  HBIBS33	590280	709	WUblastx.64	(Q9H960) CDNA FLJ12988 FIS,	Q9H960	62%	595	452
  				CLONE NT2RP3000080.
  HBIBZ20	688861	711	WUblastx.64	(O75717) ACIDIC	O75717	99%	229	603
  				NUCLEOPLASMIC DNA-BINDING
  				PROTEIN 1 (AND-1).
  HBICB80	637516	712	WUblastx.64	(Q9BV17) SIMILAR TO CG9172	Q9BV17	90%	545	637
  				GENE PRODUCT.		91%	18	557
  HBJAC40	841235	713	WUblastx.64	(Q9P112) CHROMOSOME 16 OPEN	Q9P112	100%	8	73
  				READING FRAME 5.		36%	5	70
  						57%	11	52
  						53%	85	180
  						100%	192	632
  HBJAV56	603529	714	WUblastx.64	(Q9H387) PRO2550.	Q9H387	58%	642	478
  						67%	715	623
  						75%	780	745
  HBJBR40	581104	717	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	54%	931	860
  				PROTEIN.		66%	861	745
  HBJCH46	609859	718	WUblastx.64	conserved hypothetical protein	pir|D83014|D83014	53%	1070	900
  				PA5065 [imported] - Pseudomonas		78%	900	1
  				aeruginosa (strain PAO1)
  HBJCS26	821682	720	WUblastx.64	(Q9H0D4) HYPOTHETICAL 54.1 KDA	Q9H0D4	92%	227	1576
  				PROTEIN.
  HBJDR18	604907	723	WUblastx.64	(Q9H6G8) CDNA: FLJ22294 FIS,	Q9H6G8	81%	940	875
  				CLONE HRC04426.		77%	1092	934
  HBJDR83	600395	724	WUblastx.64	(Q14273) POL/ENV ORF.	Q14273	71%	559	621
  						60%	813	902
  						68%	614	718
  						41%	1223	1291
  						62%	14	556
  HBJEL21	866158	726	WUblastx.64	(Q9UQR1) ZINC FINGER PROTEIN	Z148_HUMAN	97%	807	2063
  				148 (ZINC FINGER DNA BINDING P
  HBJFH84	836997	727	WUblastx.64	(Q9LM25) T10O22.22.	Q9LM25	30%	118	921
  HBJFJ26	873844	3102	WUblastx.64	(Q9BWK5) UNKNOWN (PROTEIN	Q9BWK5	94%	657	758
  				FOR MGC: 5242).		100%	336	389
  						100%	427	657
  HBJHO83	610259	736	HMMER	PFAM: ENV polyprotein (coat	PF00429	40.5	207	389
  			2.1.1	polyprotein)
  			WUblastx.64	(Q85641) 3′ END OF THE GENOME	Q85641	85%	2	61
  				OF MOLONEY MURINE		28%	177	365
  				LEUKEMIA VIRUS (CODES		46%	64	180
  HBJIR14	793391	741	WUblastx.64	(Q9H832) CDNA FLJ13968 FIS,	Q9H832	91%	134	871
  				CLONE Y79AA1001493, WEAKLY
  				SIMILAR TO UBI
  HBJJA26	743181	742	WUblastx.64	(Q9BRM8) UNKNOWN (PROTEIN	Q9BRM8	47%	647	423
  				FOR MGC: 13219).
  HBJND04	837242	745	HMMER	PFAM: Bacterial mutT protein	PF00293	26.2	−360	−479
  			2.1.1
  			WUblastx.64	(Q9BW91) UNKNOWN (PROTEIN	Q9BW91	91%	309	1352
  				FOR MGC: 3037).
  HBJND57	612785	746	WUblastx.64	(Q9VCB4) CG17741 PROTEIN.	Q9VCB4	52%	1	318
  HBKEE60	793788	749	WUblastx.64	pro-pol-dUTPase polyprotein - murine	pir|T29097|T29097	64%	97	204
  				endogenous retrovirus ERV-L		39%	3	128
  				(fragment)		62%	273	620
  HBKEI41	827278	750	WUblastx.64	(BAB47402) CG10671-like.	BAB47402	100%	436	465
  						90%	596	757
  						59%	498	623
  						77%	5	436
  HBMBD51	842176	751	WUblastx.64	hypothetical protein	pir|T50632|T50632	98%	2197	2709
  				DKFZp762E1511.1 - human
  				(fragment)
  HBMBD73	839564	752	WUblastx.64	MAP kinase 1 (EC 2.7.1.—) - human	pir|JQ1400|JQ1400	91%	93	161
  						92%	127	1137
  HBMBM17	637518	754	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	75%	950	840
  				CLONE KAIA0536.		75%	1083	949
  HBMCL59	608668	755	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	45%	630	472
  				CLONE COL04765.		60%	796	608
  HBMCM96	821318	756	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	57%	2253	2155
  				CLONE COL04765.		63%	2177	1971
  HBMCQ74	856461	757	WUblastx.64	(Q9JKP5) MUSCLEBLIND.	Q9JKP5	100%	561	590
  						52%	13	213
  						83%	1	465
  HBMCQ74	864382	758	WUblastx.64	(Q9JKP5) MUSCLEBLIND.	Q9JKP5	100%	561	590
  						52%	13	213
  						83%	1	465
  HBMDM08	837927	760	WUblastx.64	(Q9P0I2) 30 KDA PROTEIN.	Q9P0I2	94%	190	972
  HBMSO30	843389	762	WUblastx.64	(Q9D4B1) 4933405A16RIK	Q9D4B1	78%	52	174
  				PROTEIN.
  HBMTM50	609988	763	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	76%	763	689
  				CLONE KAIA0536.		55%	962	765
  HBMUD59	701970	764	WUblastx.64	(Q9BR03) C367G8.2 (NOVEL	Q9BR03	91%	9	647
  				PROTEIN) (FRAGMENT).
  HBMUR39	647594	767	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	55%	883	803
  				PRODUCT.		39%	810	577
  HBMWC39	523713	770	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	71%	1055	1117
  				CLONE COL04765.		67%	820	1053
  HBMWS52	872553	772	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	62%	1341	997
  				CLONE COL04765.
  HBMXG01	689522	774	WUblastx.64	(Q9BGX7) HYPOTHETICAL 13.0 KDA	Q9BGX7	58%	838	885
  				PROTEIN.		71%	900	1055
  HBMXG76	580812	775	WUblastx.64	probable phosphodiesterase I (EC	pir|T43461|T43461	98%	37	222
  				3.1.4.1) - human (fragment)
  HBMXW83	725335	777	WUblastx.64	glutamate-cysteine ligase (EC 6.3.2.2)	pir|JH0611|JH0611	100%	492	584
  				heavy chain - human		90%	373	498
  HBNAE74	637524	778	WUblastx.64	(Q9NU78) DJ622L5.7.1 (NOVEL	Q9NU78	100%	6	146
  				PROTEIN (ISOFORM 1)).
  HBNAX16	843727	779	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	92%	360	2639
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HBODK40	852382	781	WUblastx.64	(Q9Y6B7) ADAPTER-RELATED	A4B1_HUMAN	89%	55	696
  				PROTEIN COMPLEX 4 BETA 1		99%	674	1741
  				SUBUNIT (
  HBODV76	866420	782	WUblastx.64	(O60662) KELCH-RELATED	KRP1_HUMAN	83%	474	902
  				PROTEIN 1 (KEL-LIKE PROTEIN		100%	71	475
  				23) (SAR		96%	808	1905
  HBPAF39	850786	784	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	51%	1217	849
  				CLONE KAIA0536.
  HBQAC72	799512	786	WUblastx.64	transcription factor znf6 - human	pir|S25409|S25409	90%	2072	1110
  						33%	1844	1227
  						30%	2024	1215
  						30%	1757	1194
  						31%	1844	1401
  HBSAJ63	848683	788	WUblastx.64	(BAB55144) CDNA FLJ14576 fis,	BAB55144	92%	11	406
  				clone NT2RM4001092, w
  HBSDD24	839802	790	WUblastx.64	(Q9UJ70) N-	Q9UJ70	100%	886	1251
  				ACETYLGLUCOSAMINE KINASE
  				PROTEIN (EC 2.7.1.59).
  HBWBD25	800765	791	WUblastx.64	(O08872) PUTATIVE RNA	O08872	49%	850	698
  				BINDING PROTEIN 1		30%	1473	853
  				(FRAGMENT).
  HBXAS93	836513	792	WUblastx.64	(Q14940) SODIUM/HYDROGEN	NAH5_HUMAN	85%	532	1023
  				EXCHANGER 5 (NA(+)/H(+)		78%	2	238
  				EXCHANGER		90%	256	321
  HBXAW57	815650	794	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	56%	1223	1071
  				PRODUCT.
  HBXBM78	812527	797	WUblastx.64	(Q9Y613) FH1/FH2 DOMAINS-	FHOS_HUMAN	84%	846	259
  				CONTAINING PROTEIN (FORMIN		28%	2503	2138
  				HOMOLOG		100%	1004	852
  						97%	1903	1046
  						100%	2311	2042
  						42%	2184	2122
  						28%	1777	1334
  						55%	2520	2248
  HBXCD59	860439	798	WUblastx.64	(Q9C026) TRIPARTITE MOTIF	Q9C026	99%	59	823
  				PROTEIN TRIM9 ISOFORM BETA.
  HBXCG08	628501	800	WUblastx.64	(Q9BSG3) UNKNOWN (PROTEIN	Q9BSG3	98%	758	1024
  				FOR MGC: 12974).
  HBXCM52	799513	801	WUblastx.64	(Q9BH03) HYPOTHETICAL 12.0 KDA	Q9BH03	53%	683	850
  				PROTEIN.		73%	784	942
  						80%	903	1028
  HBXCQ03	589516	802	WUblastx.64	(Q9Y296) PTD009 (HSPC172).	Q9Y296	100%	382	672
  						100%	17	382
  HBXDN08	566765	806	WUblastx.64	(O43597) SPROUTY HOMOLOG 2	SPY2_HUMAN	100%	8	256
  				(SPRY-2).
  HBXDN65	840021	807	WUblastx.64	(Q9H387) PRO2550.	Q9H387	75%	1925	1641
  HBXFA04	842901	808	WUblastx.64	(AAH08467) Similar to RIKEN cDNA	AAH08467	95%	287	499
  				1110001J03 gene.
  HBXFE64	838824	809	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	57%	1646	1320
  				CLONE COL04765.
  HBXFP72	688040	811	WUblastx.64	(Q9H754) CDNA: FLJ21308 FIS,	Q9H754	93%	7	1155
  				CLONE COL02131.
  HBXFS31	815651	812	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	65%	469	678
  				CLONE KAT08285.		70%	1551	1261
  HBXFW01	847001	813	HMMER	PFAM: Stathmin family	PF00836	185.7	266	523
  			2.1.1
  			WUblastx.64	(Q9H169) RB3 PROTEIN	Q9H169	100%	523	690
  				(HYPOTHETICAL 22.1 KDA		100%	125	523
  				PROTEIN).
  HBXGE12	1310891	814	WUblastx.64	(O95902) UNKNOWN	O95902	100%	31	411
  				(FRAGMENT).
  HBXGE12	745398	3103	WUblastx.64	(AF131851) Unknown [Homo sapiens]	gb|AAD20061.1|	100%	10	390
  HBXGL91	901845	815	WUblastx.64	(Q9H2J7) ORPHAN	Q9H2J7	100%	949	1470
  				NEUROTRANSMITTER		100%	10	99
  				TRANSPORTER V7-3.		95%	389	979
  HBXGM24	821320	816	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	77%	712	647
  				human		46%	740	660
  						48%	639	475
  						57%	85	23
  						64%	530	84
  HCBAB34	847002	821	WUblastx.64	(Q9BWF8) UNKNOWN (PROTEIN	Q9BWF8	95%	1009	1308
  				FOR IMAGE: 3355813)		98%	814	963
  				(FRAGMENT).		95%	680	823
  						60%	29	583
  HCDAH02	653066	825	WUblastx.64	(BAB55208) CDNA FLJ14668 fis,	BAB55208	81%	655	491
  				clone NT2RP2003194.		83%	492	241
  HCDAP33	566794	826	WUblastx.64	(O75964) ATP SYNTHASE G	ATPN_HUMAN	73%	407	664
  				CHAIN, MITOCHONDRIAL (EC
  				3.6.1.34)
  HCDAR40	654821	827	WUblastx.64	(Q9P195) PRO1722.	Q9P195	63%	837	514
  HCDAS02	896667	828	WUblastx.64	(O75153) PUTATIVE EUKARYOTIC	IF3X_HUMAN	89%	79	867
  				TRANSLATION INITIATION
  				FACTOR
  HCDBO32	831942	830	WUblastx.64	(AAH17472) Hypothetical 21.3 kDa	AAH17472	69%	643	801
  				protein.		100%	239	583
  HCDBW67	733860	831	WUblastx.64	(Q9H410) DJ469A13.2 (NOVEL	Q9H410	97%	43	255
  				PROTEIN) (FRAGMENT).
  HCDCB03	571037	833	HMMER	PFAM: Zinc carboxypeptidase	PF00246	103.3	35	337
  			2.1.1
  			WUblastx.64	carboxypeptidase H (EC 3.4.17.10)	pir|S09489|S09489	91%	657	728
  				precursor - human		66%	11	664
  HCDCE51	813504	834	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	55%	1016	837
  				PRODUCT.
  HCDCI42	847004	835	WUblastx.64	(Q9UHD2) TANK BINDING	Q9UHD2	100%	678	875
  				KINASE TBK1 (NF-KB-
  				ACTIVATING KINASE NAK).
  HCDDB15	841041	836	WUblastx.64	(Q9BW53) UNKNOWN (PROTEIN	Q9BW53	100%	98	703
  				FOR IMAGE: 3343149)
  				(FRAGMENT).
  HCDDY28	892137	838	WUblastx.64	(Q14287) HYPOTHETICAL	Q14287	55%	1458	1312
  				PROTEIN (FRAGMENT).		60%	1312	1013
  HCDEB19	587264	839	WUblastx.64	nuclear matrix protein NMP 238 -	pir|JE0334|JE0334	100%	1218	1436
  				human		93%	426	470
  						99%	473	829
  						98%	76	438
  						31%	605	700
  						31%	1335	1430
  						91%	838	1218
  HCDES69	609997	841	WUblastx.64	(Q9H8N2) CDNA FLJ13381 FIS,	Q9H8N2	61%	524	423
  				CLONE PLACE1001010.		62%	687	553
  HCE1D45	664481	842	WUblastx.64	(Q9UDU0) WUGSC: H_DJ400N23.1	Q9UDU0	84%	10	279
  				PROTEIN (ZINC FINGER
  				SARCOMA GENE LONG A
  HCE1T53	843680	844	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	71%	725	663
  				CLONE KAT08285.		65%	843	739
  						92%	1450	1412
  						67%	1409	1140
  HCE1Y27	637529	845	WUblastx.64	(O95741) COPINE VI (NEURONAL-	CNE6_HUMAN	80%	887	946
  				COPINE) (N-COPINE).		98%	519	896
  						95%	901	1353
  						93%	16	561
  HCE1Y34	809084	846	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	61%	1718	1680
  				CLONE KAIA0536.		51%	1680	1411
  HCE2E47	886155	848	WUblastx.64	(Q9H387) PRO2550.	Q9H387	72%	1254	1448
  						67%	1450	1533
  HCE2P90	737935	850	WUblastx.64	(AAH07558) Unknown (protein for	AAH07558	47%	792	896
  				MGC: 15483).		42%	640	795
  HCE3C46	737889	852	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	41%	186	260
  				CLONE KAT08285.		62%	751	1032
  HCE3D58	873227	853	WUblastx.64	(Q9H026) HYPOTHETICAL 16.0 KDA	Q9H026	70%	421	879
  				PROTEIN (FRAGMENT).
  HCE3N23	810211	857	WUblastx.64	(Q9D2L0) 4833417L20RIK	Q9D2L0	42%	1150	1539
  				PROTEIN.		38%	1147	1536
  						71%	423	590
  						70%	913	1044
  						55%	584	925
  						54%	916	1047
  						44%	370	423
  						58%	863	913
  						72%	77	358
  HCE3R01	834836	858	WUblastx.64	(O95251) HISTONE	O95251	76%	6	320
  				ACETYLTRANSFERASE.		84%	68	1111
  HCE3R01	841472	859	WUblastx.64	(O95251) HISTONE	O95251	76%	6	320
  				ACETYLTRANSFERASE.		84%	68	1111
  HCE3R01	844574	860	WUblastx.64	(O95251) HISTONE	O95251	76%	6	320
  				ACETYLTRANSFERASE.		84%	68	1111
  HCE3R46	844450	861	WUblastx.64	(O95894) UNKNOWN (DERP2).	O95894	91%	131	1165
  HCE4H32	843941	862	WUblastx.64	(Q9NVF5) CDNA FLJ10769 FIS,	Q9NVF5	92%	926	964
  				CLONE NT2RP4000151.		65%	964	1104
  						64%	73	402
  						94%	333	893
  HCE4H32	874256	863	WUblastx.64	(Q9P0P1) HSPC237.	Q9P0P1	100%	1260	1322
  						100%	1325	1765
  HCE4W88	792953	865	WUblastx.64	(Q9Y5W4) MLL SEPTIN-LIKE	Q9Y5W4	100%	3676	2003
  				FUSION PROTEIN (CELL
  				DIVISION CONTROL PROTEI
  HCE5B62	566864	866	WUblastx.64	(O60448) NEURONAL THREAD	O60448	70%	1011	850
  				PROTEIN AD7C-NTP.		47%	1056	757
  						64%	1653	1486
  						58%	1563	1441
  						47%	953	810
  						70%	1470	1420
  						34%	820	725
  						40%	1023	943
  						62%	956	855
  						58%	778	728
  						58%	1553	1425
  						63%	1653	1546
  						61%	1632	1579
  						38%	1631	1500
  						36%	981	748
  						55%	1696	1637
  						56%	1505	1410
  						50%	1496	1431
  						45%	823	725
  						59%	846	781
  						61%	995	834
  						43%	1557	1420
  						84%	1683	1645
  						62%	1673	1425
  HCE5H86	847032	867	WUblastx.64	(O95637) WW DOMAIN BINDING	O95637	83%	1574	2107
  				PROTEIN-1.		66%	847	1248
  						66%	1300	1539
  HCE5J64	688883	868	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	64%	2021	2122
  				PROTEIN.		56%	1903	2031
  HCEBF54	847033	869	WUblastx.64	(Q9NQ43) HYPOTHETICAL 18.4 KDA	Q9NQ43	100%	338	583
  				PROTEIN (FRAGMENT).
  HCEDN07	847034	874	WUblastx.64	(Q9H387) PRO2550.	Q9H387	93%	1256	1212
  						86%	1186	1121
  						71%	1410	1255
  HCEEG48	896688	876	WUblastx.64	(AAK55521) PRO0764.	AAK55521	70%	919	680
  HCEEM33	821322	877	WUblastx.64	hypothetical protein	pir|T46310|T46310	100%	3	746
  				DKFZp434G0511.1 - human
  HCEFA94	822850	882	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	56%	1066	1224
  				PROTEIN.
  HCEFG93	745400	884	WUblastx.64	(Q9H387) PRO2550.	Q9H387	66%	1178	1134
  						80%	1114	1052
  						76%	1323	1186
  HCEFH31	801890	885	WUblastx.64	(Q9ULW1) SPONDIN 2.	Q9ULW1	100%	1503	1685
  HCEFK56	872554	886	WUblastx.64	(Q9UIU2) DYNACTIN 1 P150	Q9UIU2	92%	7	2088
  				ISOFORM.
  HCEGK81	844452	890	WUblastx.64	(Q9H387) PRO2550.	Q9H387	88%	524	649
  						68%	390	455
  						76%	487	525
  HCEGS49	846298	891	WUblastx.64	(O60448) NEURONAL THREAD	O60448	42%	711	529
  				PROTEIN AD7C-NTP.		92%	651	610
  						61%	856	710
  						72%	870	838
  						66%	721	659
  						61%	872	711
  						68%	857	579
  HCEGY33	753258	893	WUblastx.64	(O75843) ADAPTER-RELATED	A1G2_HUMAN	97%	1131	988
  				PROTEIN COMPLEX 1 GAMMA 2		89%	873	760
  				SUBUNIT		100%	1396	1274
  						68%	393	256
  						96%	603	523
  						98%	2024	1818
  HCEHW24	560610	894	WUblastx.64	(Q9BGX7) HYPOTHETICAL 13.0 KDA	Q9BGX7	55%	1001	1297
  				PROTEIN.
  HCEJL08	722208	895	WUblastx.64	(AAH08203) Chromosome X open	AAH08203	59%	36	170
  				reading frame 12.		96%	581	775
  						58%	2	103
  						79%	130	570
  HCELB04	847375	897	WUblastx.64	(Q9P290) POTENT BRAIN TYPE	Q9P290	96%	994	1083
  				ORGANIC ION TRANSPORTER.		94%	156	263
  						71%	677	1024
  						100%	317	544
  						62%	1106	1177
  						73%	500	544
  						75%	1064	1579
  HCEMA08	846468	898	WUblastx.64	ATPase inhibitor precursor,	pir|JC7175|JC7175	65%	1599	1739
  				mitochondrial - human		100%	67	216
  HCENQ22	740746	900	WUblastx.64	(Q9H926) CDNA FLJ13059 FIS,	Q9H926	80%	1086	934
  				CLONE NT2RP3001589.
  HCEOF01	564906	901	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	50%	1669	1586
  				CLONE COL04765.		75%	1551	1366
  HCEOF01	850521	902	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	50%	1668	1585
  				CLONE COL04765.		75%	1550	1365
  HCEOV48	850681	905	WUblastx.64	(Q9DA75) 1700018O18RIK	Q9DA75	83%	1344	1739
  				PROTEIN.		76%	193	1311
  HCEPO08	637543	907	WUblastx.64	ribosomal protein L29, cytosolic -	pir|S65784|S65784	100%	614	504
  				human		64%	516	145
  HCESB03	812940	908	WUblastx.64	(Q9HD86) NAG18.	Q9HD86	76%	729	857
  HCETL19	702090	911	WUblastx.64	retrovirus-related env polyprotein	pir|E24483|VCHUER	79%	580	464
  				pseudogene - human
  HCFAT42	815652	915	WUblastx.64	(Q9G6T1) CYTOCHROME	Q9G6T1	79%	699	884
  				OXIDASE SUBUNIT 3.		47%	369	431
  HCFAT66	821337	916	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	64%	1754	1662
  				CLONE HEMBA1006036.		69%	1641	1453
  HCFBA30	608166	917	WUblastx.64	(Q9BU56) UNKNOWN (PROTEIN	Q9BU56	75%	357	692
  				FOR IMAGE: 3940029)		45%	192	245
  				(FRAGMENT).
  HCFBM77	604576	918	WUblastx.64	(O60448) NEURONAL THREAD	O60448	59%	729	664
  				PROTEIN AD7C-NTP.		75%	880	719
  						77%	865	605
  HCFCB72	824165	920	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	63%	1537	1370
  				CLONE KAIA0536.		59%	1707	1555
  HCFCG91	897509	921	WUblastx.64	(Q9V677) CG8858 PROTEIN.	Q9V677	47%	53	511
  						32%	786	2780
  						43%	492	785
  						24%	480	950
  						23%	1269	1565
  						21%	1275	1643
  						26%	1800	1922
  HCFCM81	847378	922	WUblastx.64	(O00398) PUTATIVE PURINERGIC	O00398	96%	281	1297
  				RECEPTOR P2Y10.
  HCFLJ52	1307037	928	WUblastx.64	(O00466) K12 PROTEIN	O00466	95%	362	418
  				PRECURSOR.		100%	415	561
  						100%	121	372
  HCFLJ52	753260	3104	WUblastx.64	(AX055560) unnamed protein product	emb|CAC22100.1|	100%	91	126
  				[Homo sapiens]		56%	14	94
  						98%	123	287
  HCFLY20	786452	932	WUblastx.64	hypothetical protein	pir|T46299|T46299	85%	57	380
  				DKFZp434J0310.1 - human		93%	437	784
  						65%	936	1022
  						40%	167	226
  						76%	757	936
  HCFLY20	858875	933	WUblastx.64	hypothetical protein	pir|T46299|T46299	85%	68	391
  				DKFZp434J0310.1 - human		72%	959	1033
  						40%	178	237
  						89%	448	951
  HCFMX16	581042	938	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	54%	304	29
  				PROTEIN.
  HCFMX88	825989	939	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	71%	1068	1130
  				CLONE HEMBA1006036.		77%	853	945
  						69%	959	1066
  HCFNM40	746864	940	WUblastx.64	catalase (EC 1.11.1.6) -	pir|I40767|I40767	84%	246	130
  				Campylobacter jejuni
  HCFNM50	732010	941	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	75%	1258	1103
  				CLONE KAT08285.
  HCFNN75	762959	943	WUblastx.64	(Q9BGZ4) HYPOTHETICAL 11.6 KDA	Q9BGZ4	56%	821	696
  				PROTEIN.
  HCGBA15	604603	946	WUblastx.64	(AAK55521) PRO0764.	AAK55521	81%	791	726
  						80%	925	881
  						80%	724	662
  						65%	903	790
  HCHAC68	610370	947	HMMER	PFAM: Ank repeat	PF00023	58.1	462	560
  			2.1.1
  			WUblastx.64	(Q9Y290) RELA ASSOCIATED	Q9Y290	90%	201	770
  				INHIBITOR.		93%	52	96
  						100%	770	871
  						70%	6	50
  HCHBP49	892141	948	WUblastx.64	hypothetical protein	pir|T47147|T47147	91%	8	457
  				DKFZp761H229.1 - human (fragment)
  HCHCG33	862534	950	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	66%	2068	1988
  				CLONE COL04765.		75%	2285	2073
  HCHMY57	833049	951	WUblastx.64	(Q9BV10) UNKNOWN (PROTEIN	Q9BV10	84%	12	1202
  				FOR MGC: 3136).
  HCHOC06	688042	952	WUblastx.64	(Q9BZH7) GASTRIC CANCER-	Q9BZH7	75%	131	424
  				RELATED PROTEIN VRG107.
  HCHOY52	837297	953	WUblastx.64	(Q9BT25) UNKNOWN (PROTEIN	Q9BT25	89%	27	1223
  				FOR IMAGE: 3636299)
  				(FRAGMENT).
  HCHQB93	793648	954	WUblastx.64	GTP-binding protein 2 - human	pir|PC7084|PC7084	89%	767	985
  				(fragment)		92%	258	788
  						86%	2	46
  						37%	36	143
  						100%	61	258
  HCHQB93	875853	955	WUblastx.64	GTP-binding protein 2 - human	pir|PC7084|PC7084	89%	767	985
  				(fragment)		92%	258	788
  						86%	2	46
  						37%	36	143
  						100%	61	258
  HCLCU75	862406	957	WUblastx.64	(Q24333) ELASTIN LIKE PROTEIN	Q24333	95%	51	122
  				(FRAGMENT).
  HCMSA37	598712	958	WUblastx.64	(Q9UHS7) PRO1992.	Q9UHS7	63%	902	795
  HCMSR07	821338	959	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	91%	1388	1489
  HCNSF01	901060	964	WUblastx.64	(Q9Y6N5) HYPOTHETICAL 50.0 KDA	Q9Y6N5	100%	107	1456
  				PROTEIN.
  HCPAE41	799546	967	WUblastx.64	(Q9P0P0) HSPC238	Q9P0P0	81%	490	443
  				(HYPOTHETICAL 17.9 KDA		73%	444	31
  				PROTEIN).
  HCQAS72	828082	970	WUblastx.64	(AAK58423) PC2-glutamine-rich-	AAK58423	91%	22	57
  				associated protein.		93%	1010	1054
  						100%	57	119
  						71%	122	463
  HCQBM95	841011	971	WUblastx.64	ribosomal protein L39, cytosolic	pir|JC4229|R6RT39	84%	409	447
  				[validated] - rat		88%	282	389
  HCRAI29	844084	979	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	34%	329	132
  				human		56%	48	1
  						39%	493	305
  						61%	89	12
  HCRBL20	709660	981	WUblastx.64	(Q9NWM9) CDNA FLJ20730 FIS,	Q9NWM9	94%	1	678
  				CLONE HEP10359.
  HCRBX84	1007105	982	WUblastx.64	(Q9H1F6) DJ453C12.6.1	Q9H1F6	91%	37	177
  				(UNCHARACTERIZED		63%	51	617
  				HYPOTHALAMUS PROTEIN
  				(ISOFORM
  HCRMA24	897005	983	WUblastx.64	hypothetical protein	pir|T08683|T08683	100%	388	591
  				DKFZp564J2123.1 - human
  				(fragment)
  HCRMR35	849071	984	WUblastx.64	(Q9H175) HYPOTHETICAL 59.6 KDA	Q9H175	48%	43	858
  				PROTEIN.
  HCRMR35	874743	985	WUblastx.64	(Q9H175) HYPOTHETICAL 59.6 KDA	Q9H175	48%	43	858
  				PROTEIN.
  HCRMR35	897434	986	WUblastx.64	(Q9H175) HYPOTHETICAL 59.6 KDA	Q9H175	49%	43	858
  				PROTEIN.
  HCROC18	884144	987	WUblastx.64	(Q9H8A5) CDNA FLJ13824 FIS,	Q9H8A5	95%	7	1695
  				CLONE THYRO1000505.
  HCUAE53	665716	988	WUblastx.64	hypothetical protein [imported] -	pir|E86188|E86188	58%	504	304
  				Arabidopsis thaliana		80%	225	76
  						56%	895	653
  						28%	542	459
  						62%	1299	1156
  HCUAT74	834611	990	WUblastx.64	hypothetical protein UL126 - human	pir|S09875|S09875	68%	3	191
  				cytomegalovirus (strain AD169)
  HCUBN69	826610	994	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	78%	831	763
  				FOR MGC: 5149).		72%	626	573
  						80%	760	626
  HCUBY47	842787	995	WUblastx.64	(Q9EPL2) CALSYNTENIN-1	Q9EPL2	97%	444	313
  				PROTEIN PRECURSOR.		97%	1003	869
  HCUCV66	806577	996	WUblastx.64	(BAB22833) 18 days embryo cDNA,	BAB22833	100%	36	362
  				RIKEN full-length e
  HCUFC77	745374	1001	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	77%	1526	1461
  				CLONE KAIA0536.		61%	1449	1234
  HCUFD17	847039	1002	WUblastx.64	(Q9HBZ6) HT005 PROTEIN.	Q9HBZ6	100%	10	195
  HCUFD46	757481	1003	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	43%	580	386
  				CLONE COL04765.
  HCUFX08	612843	1008	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	55%	1214	1128
  				PROTEIN.		65%	1329	1216
  HCUHE27	562766	1014	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	61%	764	639
  				PRODUCT.
  HCWAK88	839083	1017	WUblastx.64	probable excinuclease ABC chain A -	pir|T33732|T33732	86%	435	370
  				Zymomonas mobilis		31%	815	321
  						32%	1969	1814
  						64%	956	501
  						67%	362	42
  						57%	1237	1181
  						69%	1306	998
  						85%	1993	1538
  HCWAL10	639023	1018	WUblastx.64	(Q9Y2N3) NUCLEAR ENVELOPE	N121_HUMAN	100%	754	680
  				PORE MEMBRANE PROTEIN POM		100%	240	118
  				121 (PO
  HCWDM69	684527	1022	WUblastx.64	(Q9P195) PRO1722.	Q9P195	66%	819	748
  						78%	886	818
  						70%	1001	900
  HCWEB72	639041	1024	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	56%	872	798
  				CLONE KAIA0536.		48%	588	508
  						65%	757	617
  HCWEI82	799516	1026	WUblastx.64	(Q9P1N7) PRO0974.	Q9P1N7	47%	384	656
  HCWEM96	812735	1027	WUblastx.64	probable ATP-binding component of	pir|G83165|G83165	74%	1901	1146
  				ABC transporter PA3838 [imported] -
  				Pseudomonas aeruginosa (strain
  				PAO1)
  HCWHD30	834724	1031	WUblastx.64	(AAH07609) Similar to hypothetical	AAH07609	86%	634	590
  				protein PRO1722.		80%	782	633
  HCWHT34	692400	1032	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	68%	556	777
  HCWHT52	834618	1033	WUblastx.64	hypothetical protein UL126 - human	pir|S09875|S09875	75%	20	274
  				cytomegalovirus (strain AD169)
  HCWKO32	610619	1034	WUblastx.64	(Q9GMU5) HYPOTHETICAL 14.1 KDA	Q9GMU5	82%	496	600
  				PROTEIN.
  HCWUW24	799517	1037	WUblastx.64	(Q9H387) PRO2550.	Q9H387	45%	541	696
  						33%	60	245
  						77%	686	817
  HCYBA32	847042	1038	WUblastx.64	(Q9H3F8) MSTP016.	Q9H3F8	100%	329	718
  						79%	925	1800
  HDABR74	861789	1040	WUblastx.64	(O60717) 7-	O60717	81%	5	37
  				DEHYDROCHOLESTEROL		95%	69	908
  				REDUCTASE (EC 1.3.1.21)
  				(FRAGMENT).
  HDFIB37	821339	1045	WUblastx.64	(Q9D8F2) 2010004A03RIK	Q9D8F2	74%	792	884
  				PROTEIN.		64%	357	431
  						65%	388	816
  HDHEA33	847044	1048	WUblastx.64	(Q9BU66) UNKNOWN (PROTEIN	Q9BU66	40%	482	619
  				FOR MGC: 10433).		45%	159	638
  						82%	1054	1494
  HDHEB12	610260	1049	WUblastx.64	(Q9NZ96) NEUROLIGIN 3	Q9NZ96	94%	2	697
  				ISOFORM HNL3S (FRAGMENT).
  HDLAL94	843583	1054	HMMER	PFAM: ADP-ribosylation factor	PF00025	309.2	195	728
  			2.1.1	family
  			WUblastx.64	(Q9D4P0) 4930587A11RIK	Q9D4P0	100%	192	728
  				PROTEIN.
  HDPAB86	901851	1055	WUblastx.64	(Q9NYH9) HEPATOCELLULAR	Q9NYH9	56%	74	538
  				CARCINOMA-ASSOCIATED		97%	310	1863
  				ANTIGEN 66.
  HDPAE80	778068	1056	WUblastx.64	(Q9BR63) PHENYLALANYL-TRNA	Q9BR63	93%	1435	1662
  				SYNTHETASE BETA-SUBUNIT		78%	1604	1744
  				(FRAGMENT).		54%	383	661
  						32%	417	761
  						98%	905	1438
  HDPAQ86	612855	1057	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	48%	1419	1246
  				CLONE COL03536.		76%	1250	1122
  HDPBN48	838593	1059	WUblastx.64	(Q9H013) MELTRIN-BETA/ADAM	Q9H013	84%	525	773
  				19 HOMOLOGUE.
  HDPCG79	691350	1060	WUblastx.64	(Q9H9H4) CDNA FLJ12750 FIS,	Q9H9H4	98%	415	588
  				CLONE NT2RP2001168, WEAKLY
  				SIMILAR TO VER
  HDPCV29	567228	1061	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	40%	546	1196
  				human
  HDPDA36	827283	1062	WUblastx.64	(Q9CZU3) 2610528A15RIK	Q9CZU3	96%	3443	3604
  				PROTEIN.
  HDPDC59	872451	1063	WUblastx.64	(Q9H251) CADHERIN RELATED	Q9H251	99%	142	1737
  				23.		32%	145	1761
  						33%	130	1737
  						32%	130	1734
  						32%	112	1740
  						34%	145	1743
  						31%	124	1731
  						30%	145	1743
  						31%	256	1725
  						31%	145	1755
  						31%	145	1698
  						31%	139	1491
  						30%	658	1737
  						29%	154	1731
  HDPFG13	637580	1064	WUblastx.64	(Q9H8S0) CDNA FLJ13287 FIS,	Q9H8S0	100%	55	441
  				CLONE OVARC1001161.
  HDPFZ05	824390	1066	WUblastx.64	(Q9CZC8) 2810019K23RIK	Q9CZC8	84%	500	658
  				PROTEIN.
  HDPGR80	844812	1068	WUblastx.64	(Q9BRR6) SIMILAR TO RIKEN	Q9BRR6	87%	2434	1067
  				CDNA 2610017G09 GENE.		83%	2556	2377
  HDPGU14	832543	1069	WUblastx.64	mda-9 protein - human	pir|JC6537|JC6537	100%	292	1185
  HDPGX09	580818	1070	WUblastx.64	(Q9D0M0) 2610002K22RIK	Q9D0M0	94%	832	999
  				PROTEIN.		33%	995	1093
  						91%	1020	1220
  HDPIE44	899328	1071	WUblastx.64	(Q9D666) 4632417G13RIK	Q9D666	62%	102	2453
  				PROTEIN.
  HDPIE73	886158	1072	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	58%	1852	2145
  				CLONE COL04765.
  HDPIF35	826781	1073	WUblastx.64	(BAB55422) CDNA FLJ14966 fis,	BAB55422	100%	1000	887
  				clone THYRO1000034, w
  HDPIF65	847047	1074	WUblastx.64	(Q9H7Z0) CDNA FLJ14058 FIS,	Q9H7Z0	60%	236	12
  				CLONE HEMBB1000554.
  HDPIH25	740749	1075	WUblastx.64	(Q9H7Q7) FLJ00010 PROTEIN	Q9H7Q7	78%	99	539
  				(FRAGMENT).		100%	876	968
  						76%	971	2266
  						98%	536	796
  						50%	2190	2300
  						75%	796	876
  HDPIY31	886159	1076	WUblastx.64	hypothetical protein	pir|T46448|T46448	72%	1714	1899
  				DKFZp434N1429.1 - human
  				(fragment)
  HDPJH72	847048	1077	WUblastx.64	(O15427) MONOCARBOXYLATE	MOT4_HUMAN	96%	294	392
  				TRANSPORTER 4 (MCT 4) (MCT 3).		81%	1204	1473
  						100%	77	295
  						89%	854	1192
  						37%	1427	1498
  						71%	394	855
  HDPKC55	822869	1080	HMMER	PFAM: Tubulin/FtsZ family	PF00091	177.8	108	797
  			2.1.1
  			WUblastx.64	(Q9UJT1) TUBULIN DELTA CHAIN	TBD_HUMAN	100%	102	1460
  				(DELTA TUBULIN).
  HDPKD16	897304	1081	WUblastx.64	(AAG10506) 2P domain K+ channel	AAG10506	100%	246	857
  				TWIK-2.
  HDPMC52	847049	1082	WUblastx.64	(Q9Y275) TUMOR NECROSIS	T13B_HUMAN	98%	67	447
  				FACTOR LIGAND SUPERFAMILY		100%	7	72
  				MEMBER 13
  HDPML04	822851	1083	WUblastx.64	X-linked retinopathy protein (C-	pir|A46010|A46010	73%	1403	1326
  				terminal, clone XEH.8c) - human		68%	1327	1223
  				(fragment)
  HDPNC21	839263	1085	WUblastx.64	(AAK55521) PRO0764.	AAK55521	81%	1290	964
  HDPNJ26	852338	1086	WUblastx.64	(Q9CQ35) 4921508O11RIK	Q9CQ35	67%	115	651
  				PROTEIN.
  HDPOD73	876202	1087	WUblastx.64	(Q9P195) PRO1722.	Q9P195	84%	173	211
  						60%	16	90
  						61%	78	185
  HDPOT33	892319	1088	WUblastx.64	(Q92482) AQUAPORIN 3.	AQP3_HUMAN	100%	53	928
  HDPPB70	874027	1089	WUblastx.64	(Q96DV8) Unknown (protein for	Q96DV8	68%	257	1186
  				MGC: 3551).
  HDPPE05	809109	1091	WUblastx.64	(Q9P0G1) HSPC075 (FRAGMENT).	Q9P0G1	100%	355	459
  						100%	64	153
  HDPSA70	722216	1092	WUblastx.64	(Q9UL01) SQUAMOUS CELL	Q9UL01	100%	13	261
  				CARCINOMA ANTIGEN
  				RECOGNIZED BY T CELL.
  HDPTI49	847052	1097	WUblastx.64	(Q14185) DOCK180 PROTEIN.	Q14185	79%	927	1190
  HDPYE25	636058	1099	WUblastx.64	(O42346) NEURULA-SPECIFIC	O42346	73%	3	722
  				FERRODOXIN REDUCTASE-LIKE
  				PROTEIN.
  HDQGD06	838076	1100	WUblastx.64	(Q9H1C4) UNC-93 RELATED	Q9H1C4	80%	969	1535
  				PROTEIN.		90%	59	463
  						87%	454	996
  HDQGD06	852673	1101	WUblastx.64	(Q9H1C4) UNC-93 RELATED	Q9H1C4	81%	969	1559
  				PROTEIN.		90%	59	463
  						87%	454	996
  HDQGD06	881280	1102	WUblastx.64	(Q9H1C4) UNC-93 RELATED	Q9H1C4	86%	59	1537
  				PROTEIN.
  HDQGN08	840588	1103	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	56%	782	510
  				CLONE HEMBA1006036.
  HDQGO62	837353	1104	WUblastx.64	(Q9D6E9) 2900070E19RIK	Q9D6E9	96%	222	515
  				PROTEIN.
  HDQPM16	886161	1105	WUblastx.64	(Q9DCP9) PROLINE RICH	Q9DCP9	76%	145	648
  				PROTEIN EXPRESSED IN BRAIN.
  HDSAH37	603518	1109	WUblastx.64	(Q9H9U3) CDNA FLJ12547 FIS,	Q9H9U3	41%	341	156
  				CLONE NT2RM4000634.		36%	449	147
  						35%	344	105
  HDSAM57	834822	1110	WUblastx.64	(Q9P195) PRO1722.	Q9P195	76%	752	690
  						80%	685	623
  						61%	897	742
  HDSAO14	847054	1111	WUblastx.64	(Q9H0I1) HYPOTHETICAL 83.8 KDA	Q9H0I1	100%	241	348
  				PROTEIN.
  HDSAP15	772712	1113	WUblastx.64	(Q9H059) HYPOTHETICAL 99.0 KDA	Q9H059	46%	160	378
  				PROTEIN.
  HDTAR39	847055	1114	WUblastx.64	(Q9NVH2) HYPOTHETICAL 106.8 KDA	Q9NVH2	100%	13	273
  				PROTEIN.		85%	242	772
  HDTDA48	809110	1118	WUblastx.64	(O15254) PRISTANOYL-COA	O15254	98%	149	601
  				OXIDASE.
  HDTDE66	610074	1119	WUblastx.64	(Q9Y609) LR8.	Q9Y609	88%	1	447
  HDTDG75	799892	1120	WUblastx.64	(Q9H2I8) CDA017.	Q9H2I8	100%	434	514
  HDTGW76	862012	1123	WUblastx.64	(Q9NY74) ETAA16 PROTEIN.	Q9NY74	73%	1290	1835
  						27%	1422	1616
  						23%	296	829
  						82%	20	1234
  HDTGZ56	800756	1124	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	54%	27	257
  				PROTEIN.
  HDTIM39	886164	1126	WUblastx.64	(Q9P0Q9) HSPC217.	Q9P0Q9	80%	254	523
  						35%	302	403
  						90%	504	788
  HDTKJ29	840023	1127	HMMER	PFAM: Uncharacterized protein family	PF01027	137.6	184	726
  			2.1.1
  			WUblastx.64	(Q9Y3C2) CGI-119 PROTEIN.	Q9Y3C2	71%	19	732
  HE2AC74	901343	1131	WUblastx.64	(AAK53385) G protein gamma 12	AAK53385	100%	207	422
  				subunit.
  HE2AX36	812337	1135	WUblastx.64	(Q9Y5B5) UBIQUITIN-SPECIFIC	Q9Y5B5	85%	1292	300
  				PROTEASE HOMOLOG.
  HE2AY96	796800	1136	WUblastx.64	(BAB22850) 18 days embryo cDNA,	BAB22850	87%	56	751
  				RIKEN full-length e
  HE2BD72	600349	1137	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	66%	496	371
  				CLONE KAIA0536.		77%	650	522
  HE2BH50	847056	1138	WUblastx.64	(Q9GZW0) DJ604K5.1 (15 KDA	Q9GZW0	100%	226	405
  				SELENOPROTEIN).
  HE2CC17	566806	1140	WUblastx.64	(Q9HBW6) NAG13.	Q9HBW6	69%	996	694
  						59%	731	516
  						75%	347	180
  						42%	1037	933
  						70%	540	352
  HE2CJ53	637592	1141	WUblastx.64	(Q9NUM3) CDNA FLJ11274 FIS,	Q9NUM3	100%	123	266
  				CLONE PLACE1009368, WEAKLY		100%	266	412
  				SIMILAR TO MET
  HE2DI16	604029	1145	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	66%	1350	1201
  				CLONE KAIA0536.		76%	1483	1331
  HE2DJ84	746643	1146	WUblastx.64	(P82673) MITOCHONDRIAL 28S	P82673	100%	2	313
  				RIBOSOMAL PROTEIN S28 (MRP-
  				S28).
  HE2DY23	877491	1147	WUblastx.64	(Q9VW97) CG17149 PROTEIN.	Q9VW97	48%	1018	1284
  HE2EH45	581520	1150	WUblastx.64	(Q9H3F4) MSTP030.	Q9H3F4	73%	690	815
  						63%	8	250
  						75%	97	735
  HE2FE89	780314	1151	WUblastx.64	(Q9QYN1) ENH3.	Q9QYN1	95%	254	541
  HE2FR49	714456	1152	WUblastx.64	(Q9NUP7) CDNA FLJ11219 FIS,	Q9NUP7	64%	63	104
  				CLONE PLACE1008122.		100%	92	400
  HE2GB19	566808	1153	WUblastx.64	(O95245) ENVELOPE PROTEIN	O95245	41%	397	519
  				(FRAGMENT).		39%	246	362
  						63%	140	247
  HE2HB61	798113	1155	WUblastx.64	(Q9BS71) UNKNOWN (PROTEIN	Q9BS71	100%	261	365
  				FOR MGC: 12301).
  HE2HF76	637594	1157	WUblastx.64	(Q9HAZ1) PROTEIN SERINE	Q9HAZ1	100%	11	859
  				THREONINE KINASE CLK4.
  HE21E66	830436	1159	WUblastx.64	(AAH22050) Hypothetical 61.5 kDa	AAH22050	98%	98	523
  				protein (Fragment)
  HE2NW57	638615	1160	WUblastx.64	(Q9UI58) PRO0483 PROTEIN.	Q9UI58	57%	191	15
  HE2OA95	637595	1161	WUblastx.64	(Q9UIK5) TMEFF2 PROTEIN	Q9UIK5	99%	1647	1231
  				PRECURSOR (TRANSMEMBRANE
  				PROTEIN TENB2) (TPEF
  HE2PB61	847379	1163	WUblastx.64	(Q9BWY7) DJ639P13.1 (NOVEL	Q9BWY7	92%	3	719
  				PROTEIN SIMILAR TO RAT
  				TRANSPORTER-LIKE PR
  HE2PI43	847380	1164	WUblastx.64	(Q9H8E8) CDNA FLJ13697 FIS,	Q9H8E8	100%	234	407
  				CLONE PLACE2000172.
  HE6CJ41	604908	1166	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	54%	898	1050
  				PROTEIN.
  HE6EI30	847381	1169	WUblastx.64	(Q9BRG1) SIMILAR TO RIKEN	Q9BRG1	100%	603	523
  				CDNA 1110020N13 GENE.
  HE8BP64	847059	1176	WUblastx.64	(Q9DBS0) HIPPOCAMPUS	Q9DBS0	71%	10	1299
  				ABUNDANT GENE TRANSCRIPT
  				1.
  HE8BQ49	589443	1177	WUblastx.64	hypothetical protein - human	pir|S72482|S72482	75%	343	474
  				transposon MER37		64%	105	248
  HE8BR18	731933	1178	WUblastx.64	(Q9NW38) CDNA FLJ10335 FIS,	Q9NW38	100%	654	1091
  				CLONE NT2RM2000669.
  HE8BT58	806144	1180	WUblastx.64	(Q9BX67) JUNCTIONAL	Q9BX67	91%	80	937
  				ADHESION MOLECULE 3
  				PRECURSOR.
  HE8CC34	753266	1183	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	72%	2116	1829
  				CLONE COL04765.
  HE8DK52	658676	1186	WUblastx.64	(Q9D1U5) D730039F16RIK	Q9D1U5	66%	778	813
  				PROTEIN.		64%	843	935
  HE8DZ94	877630	1187	WUblastx.64	(Q9Y3C2) CGI-119 PROTEIN.	Q9Y3C2	91%	1295	1657
  HE8EX86	823176	1189	WUblastx.64	(AAK53401) Elongation factor G2.	AAK53401	100%	1410	457
  						96%	2790	2593
  						88%	2579	1362
  HE8FC10	693908	1190	HMMER	PFAM: Zinc finger, C2H2 type	PF00096	72.8	606	674
  			2.1.1
  			WUblastx.64	(Q9H8L4) CDNA FLJ13479 FIS,	Q9H8L4	84%	300	731
  				CLONE PLACE1003738, WEAKLY		98%	6	173
  				SIMILAR TO ZIN		45%	438	731
  						45%	438	674
  						44%	438	674
  						39%	438	674
  						36%	438	674
  						40%	444	674
  						37%	438	674
  						37%	444	674
  						51%	6	134
  						48%	6	161
  						46%	6	134
  						46%	6	134
  						47%	9	134
  						48%	6	134
  						47%	9	134
  						40%	9	134
  						44%	48	134
  HE8FG15	799520	1191	WUblastx.64	(O43491) PROTEIN 4.1-G.	O43491	84%	226	1197
  HE8FG24	733560	1192	WUblastx.64	(Q9HAQ1) GLYCEROL 3-	Q9HAQ1	90%	11	76
  				PHOSPHATE PERMEASE.		86%	94	522
  HE8FK78	852180	1193	WUblastx.64	(Q9H8A3) CDNA FLJ13837 FIS,	Q9H8A3	100%	1	75
  				CLONE THYRO1000748,		80%	72	266
  				MODERATELY SIMILAR TO		88%	269	1372
  HE8FR53	843807	1196	WUblastx.64	(Q9H373) PRO1107.	Q9H373	98%	3411	3181
  HE8MQ01	886800	1199	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	71%	2692	2483
  HE8MS43	799521	1200	WUblastx.64	(Q9D7J4) 2310005N03RIK	Q9D7J4	88%	1023	946
  				PROTEIN.		83%	1247	1155
  						77%	175	44
  HE8NC81	862015	1202	WUblastx.64	(Q9H6B2) CDNA: FLJ22418 FIS,	Q9H6B2	100%	74	106
  				CLONE HRC08590.		88%	168	923
  HE8NC81	1096692	3106	HMMER	PFAM: Immunoglobulin domain	PF00047	28.3	563	814
  			2.1.1
  			WUblastx.64	(Q9H6B2) CDNA: FLJ22418 FIS,	Q9H6B2	99%	419	1264
  				CLONE HRC08590.
  HE8QU21	838391	1204	WUblastx.64	(Q9BGZ4) HYPOTHETICAL 11.6 KDA	Q9BGZ4	44%	1391	1125
  				PROTEIN.
  HE8SH74	833053	1205	WUblastx.64	(Q9H387) PRO2550.	Q9H387	69%	2476	2441
  						50%	3129	3034
  						76%	3017	2868
  HE8UX34	838170	1206	WUblastx.64	(Q9H8Q9) CDNA FLJ13310 FIS,	Q9H8Q9	67%	7	162
  				CLONE OVARC1001453.
  HE9CI81	734918	1209	WUblastx.64	(Q9DB52) 2900009I07RIK	Q9DB52	82%	2273	2016
  				PROTEIN.
  HE9CJ38	565082	1210	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	64%	1180	1079
  				PROTEIN.		54%	1380	1243
  HE9CN58	562768	1212	WUblastx.64	(Q99710) YY1-ASSOCIATED	Q99710	44%	6	353
  				FACTOR 2.
  HE9CV59	847061	1213	WUblastx.64	(Q9CYF7) 5730494N06RIK	Q9CYF7	76%	73	408
  				PROTEIN.
  HE9DG54	821326	1214	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	53%	1508	1197
  				PRODUCT.
  HE9DH59	886059	1215	WUblastx.64	(AAK49519) Putative mitochondrial	AAK49519	100%	54	419
  				solute carrier sp		94%	1534	2085
  HE9EM54	560627	1218	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	57%	611	688
  				CLONE COL03536.		67%	756	902
  HE9FH28	633737	1219	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	58%	965	591
  				CLONE COL04765.
  HE9HE13	868829	1220	WUblastx.64	(Q9P183) PRO2160.	Q9P183	100%	434	183
  HE9HE13	824328	1221	WUblastx.64	(Q9UI56) PROTEIN PRO0518.	P518_HUMAN	100%	665	462
  HE9HF59	664485	1222	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	45%	1593	1276
  				PROTEIN.
  HE9TA42	834972	1227	WUblastx.64	(P53992) PROTEIN TRANSPORT	S24C_HUMAN	100%	1423	1674
  				PROTEIN SEC24C (SEC24-		98%	2099	2359
  				RELATED PR		85%	1674	2105
  						29%	1833	1943
  						38%	58	135
  						98%	8	1435
  HEAAC21	581048	1228	WUblastx.64	(Q9NV24) CDNA FLJ10980 FIS,	Q9NV24	100%	100	246
  				CLONE PLACE1001570.
  HEAAD63	603321	1231	WUblastx.64	(Q9H965) CDNA FLJ12983 FIS,	Q9H965	84%	613	575
  				CLONE NT2RP3000002.		68%	789	613
  HEAAM96	562769	1234	WUblastx.64	(Q9H4S5) DJ336K20B.1 (NOVEL	Q9H4S5	83%	541	1044
  				PROTEIN BASED ON FGENESH)
  				(FRAGMENT).
  HEAAN52	862020	1235	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	54%	796	1104
  				PROTEIN.
  HEAAW94	847340	1238	WUblastx.64	(Q9UEV9) ACTIN-BINDING	Q9UEV9	94%	285	890
  				PROTEIN HOMOLOG ABP-278.		41%	285	884
  						38%	285	884
  						36%	285	872
  						35%	285	884
  						34%	285	884
  						34%	285	878
  						32%	303	887
  						33%	285	881
  						33%	285	878
  						31%	285	869
  						29%	288	887
  						30%	279	884
  						31%	285	863
  						36%	414	848
  						30%	288	884
  						32%	429	857
  						31%	309	824
  						26%	321	824
  						38%	285	539
  						37%	552	824
  HEBAT05	637602	1240	WUblastx.64	(Q9NWW0) CDNA FLJ20568 FIS,	Q9NWW0	96%	111	380
  				CLONE REC00775.		87%	84	131
  HEBCN80	596808	1243	WUblastx.64	(Q9BGX7) HYPOTHETICAL 13.0 KDA	Q9BGX7	57%	782	865
  				PROTEIN.		53%	666	788
  HEEAF49	880521	1254	WUblastx.64	(Q9P147) PRO2822.	Q9P147	79%	2117	1896
  HEEAJ46	637623	1255	WUblastx.64	(AAK55521) PRO0764.	AAK55521	86%	2358	2314
  						66%	1626	1582
  						55%	2336	2163
  						73%	1601	1368
  HEGAI20	825818	1256	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	63%	893	759
  				PRODUCT.
  HEIAC52	826611	1257	WUblastx.64	(Q9H6A7) CDNA: FLJ22429 FIS,	Q9H6A7	75%	40	915
  				CLONE HRC09084.
  HELAT58	732199	1259	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	56%	1807	1881
  				CLONE COL03536.		60%	1731	1805
  						59%	1880	2035
  HELAW94	834796	1260	WUblastx.64	(AAH02585) RAB7, member RAS	AAH02585	100%	159	551
  				oncogene family-like 1.
  HELDF80	799522	1261	WUblastx.64	N-ras upstream protein NRU - human	pir|S29815|S29815	99%	3	1289
  						28%	6	1061
  						28%	435	1118
  HELDK79	532597	1263	WUblastx.64	L6 surface protein - human	pir|A42926|A42926	78%	71	676
  HELDQ42	695716	1264	WUblastx.64	(O60629) BLADDER CANCER-	BC10_HUMAN	100%	289	549
  				ASSOCIATED PROTEIN
  				(BLADDER CANCER
  HELEL76	862490	1266	WUblastx.64	(Q9BRX8) SIMILAR TO RIKEN	Q9BRX8	93%	171	818
  				CDNA 5730469M10 GENE.
  HELEL76	839470	1267	WUblastx.64	(Q9BRX8) SIMILAR TO RIKEN	Q9BRX8	98%	545	694
  				CDNA 5730469M10 GENE.		90%	48	506
  HELEO45	847067	1268	WUblastx.64	(Q9H6Q6) CDNA: FLJ21982 FIS,	Q9H6Q6	92%	1385	1423
  				CLONE HEP06188.		88%	7	1392
  HELFA57	845981	1269	WUblastx.64	(O95761) WUGSC: H_DJ0771P04.2	O95761	95%	340	1362
  				PROTEIN (FRAGMENT).		95%	9	137
  						78%	62	409
  HELHN47	872795	1273	WUblastx.64	(Q9NWC8) HYPOTHETICAL 17.6 KDA	Q9NWC8	91%	1233	772
  				PROTEIN.
  HELHP11	638064	1274	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	76%	1429	1313
  				CLONE COL03536.
  HELHP11	851116	1275	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	79%	1441	1313
  				CLONE COL03536.
  HEMBV40	847069	1277	WUblastx.64	NADH dehydrogenase (ubiquinone)	pir|JC5822|JC5822	77%	798	1055
  				(EC 1.6.5.3) CI-B12 chain - human
  HEMCJ80	765041	1278	WUblastx.64	(Q9NXF8) CDNA FLJ20279 FIS,	Q9NXF8	98%	489	710
  				CLONE HEP03229.		73%	3	206
  						79%	155	487
  HEMDB07	617137	1280	WUblastx.64	(Q9H387) PRO2550.	Q9H387	82%	400	660
  HEMGK71	847374	1282	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	80%	1383	1258
  				CLONE KAIA0536.		64%	1576	1352
  HEOMU44	778085	1287	WUblastx.64	(Q9N307) HYPOTHETICAL	Q9N307	52%	823	1392
  				PROTEIN Y61A9LA.B.
  HEONI85	566833	1288	WUblastx.64	hypothetical 43.5K protein - mouse	pir|JU0319|JU0319	77%	236	421
  						90%	382	672
  HEONK04	658682	1289	WUblastx.64	(Q9UKN4) BRIDGING	Q9UKN4	72%	267	1070
  				INTEGRATOR-2.
  HEONP08	601691	1290	WUblastx.64	(Q9Y349) HYPOTHETICAL 15.7 KDA	Q9Y349	100%	924	523
  				PROTEIN.
  HEPBC23	799985	1292	WUblastx.64	(Q9H1V4) DJ1182A14.3 (SIMILAR	Q9H1V4	92%	4	303
  				TO MST1 (MACROPHAGE
  				STIMULATING 1 (HEPA
  HEPCU48	805585	1295	WUblastx.64	(Q9UNT2) PROTEIN O-	Q9UNT2	79%	315	1490
  				MANNOSYL-TRANSFERASE 1.		95%	79	324
  						100%	15	32
  HEQAH47	877634	1296	WUblastx.64	hypothetical protein	pir|T08772|T08772	100%	630	1085
  				DKFZp586M121.1 - human		87%	12	476
  				(fragment)		30%	90	308
  						30%	189	305
  						29%	1104	1355
  						28%	30	245
  						63%	257	289
  HEQBJ01	861786	1300	WUblastx.64	(Q9LVQ7) ZINC FINGER PROTEIN.	Q9LVQ7	34%	424	849
  HEQBJ01	876546	1301	WUblastx.64	(Q9LVQ7) ZINC FINGER PROTEIN.	Q9LVQ7	34%	424	849
  HEQBM94	580823	1302	WUblastx.64	(Q9BT25) UNKNOWN (PROTEIN	Q9BT25	100%	470	559
  				FOR IMAGE: 3636299)		72%	583	843
  				(FRAGMENT).		77%	7	471
  HESAG57	855936	1307	WUblastx.64	(O43432) EIF4GII.	O43432	82%	936	7
  						100%	1221	1030
  						100%	1365	1336
  						27%	1382	1254
  						28%	1545	1327
  HETBJ88	855937	1310	WUblastx.64	(Q9H1Y3) DJ317G22.2	Q9H1Y3	83%	10	864
  				(ENCEPHALOPSIN) (PANOPSIN).
  HETCM67	861909	1311	HMMER	PFAM: Disintegrin	PF00200	112.2	239	466
  			2.1.1
  			WUblastx.64	(Q9UKQ2) ADAM 28 PRECURSOR	AD28_HUMAN	96%	11	1318
  				(EC 3.4.24.—) (A DISINTEGRIN AND
  HETDD61	800668	1312	WUblastx.64	(Q9Y6B7) ADAPTER-RELATED	A4B1_HUMAN	96%	789	1085
  				PROTEIN COMPLEX 4 BETA 1		85%	363	569
  				SUBUNIT (		96%	1315	1761
  HETDD61	852381	1313	WUblastx.64	(Q9Y6B7) ADAPTER-RELATED	A4B1_HUMAN	96%	1314	1760
  				PROTEIN COMPLEX 4 BETA 1		96%	789	1085
  				SUBUNIT (		85%	363	569
  HETDP76	782836	1316	WUblastx.64	(Q9BUK6) HYPOTHETICAL 61.8 KDA	Q9BUK6	100%	29	394
  				PROTEIN.		92%	700	1620
  						80%	1895	2116
  						53%	1428	1466
  						100%	465	653
  HETFO57	844190	1317	WUblastx.64	(Q9H229) RAD50-INTERACTING	Q9H229	96%	2	1363
  				PROTEIN 1.
  HFAAI17	607563	1322	WUblastx.64	(Q9H387) PRO2550.	Q9H387	61%	426	518
  						73%	281	403
  HFADF41	607557	1324	WUblastx.64	B-cell growth factor precursor - human	pir|A47582|A47582	56%	600	737
  HFADM09	899435	1325	WUblastx.64	ISOFORM 8 OF Q9Y4J8	sp_vs|Q9Y4J8-	100%	382	480
  					02|Q9Y4J8
  HFAUA23	636071	1326	WUblastx.64	(Q9HAL3) CDNA FLJ11393 FIS,	Q9HAL3	25%	243	91
  				CLONE HEMBA1000591, WEAKLY		45%	466	263
  				SIMILAR TO PTB
  HFCAG75	834834	1327	WUblastx.64	(AAK55521) PRO0764.	AAK55521	80%	745	701
  						78%	720	484
  HFCAI40	866441	1328	HMMER	PFAM: TPR Domain	PF00515	52.6	167	253
  			2.1.1
  			WUblastx.64	(Q9V532) CG8777 PROTEIN.	Q9V532	36%	20	1021
  HFCAQ17	695720	1329	WUblastx.64	NADH dehydrogenase (ubiquinone)	pir|JE0379|JE0379	78%	20	238
  				(EC 1.6.5.3) chain NDUFA3 - human
  HFCBC16	559312	1330	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	55%	998	792
  				CLONE COL03536.
  HFCBT29	654842	1334	WUblastx.64	(Q9H9H1) CDNA FLJ12756 FIS,	Q9H9H1	73%	80	280
  				CLONE NT2RP2001295, WEAKLY
  				SIMILAR TO ZIN
  HFCDN13	722221	1336	WUblastx.64	(Q9P0T7) HSPC186	Q9P0T7	90%	2	187
  				(HYPOTHETICAL 20.6 KDA
  				PROTEIN).
  HFCDT67	598900	1337	WUblastx.64	(Q9H0N3) HYPOTHETICAL 107.1 KDA	Q9H0N3	82%	9	455
  				PROTEIN.		77%	845	910
  						45%	373	438
  						67%	498	902
  HFCDY36	834512	1338	WUblastx.64	catalase (EC 1.11.1.6) -	pir|I40767|I40767	93%	279	190
  				Campylobacter jejuni
  HFCEC45	847384	1339	WUblastx.64	(Q9D3Q4) 5033428A16RIK	Q9D3Q4	98%	68	349
  				PROTEIN.
  HFCET43	596813	1340	WUblastx.64	(Q9Z1M7)	Q9Z1M7	100%	7	144
  				ACETYLGLUCOSAMINYLTRANSFERASE-
  				LIKE PROTEIN.
  HFEAG55	838142	1341	WUblastx.64	(Q9EP97) SENTRIN/SUMO-	Q9EP97	100%	12	200
  				SPECIFIC PROTEASE (SMT3
  				ISOPEPTIDASE 1).
  HFEAU63	790190	1342	WUblastx.64	ISOFORM EYA1B OF Q99502	sp_vs|Q99502-	90%	2180	2028
  					01|Q99502
  HFEBA88	684297	1343	WUblastx.64	(BAB55210) CDNA FLJ14675 fis,	BAB55210	100%	205	297
  				clone NT2RP2003952, w		44%	70	327
  HFEBO15	831772	1345	WUblastx.64	(Q9HC46) HYPOTHETICAL 42.8 KDA	Q9HC46	85%	226	477
  				PROTEIN.		53%	150	332
  						51%	2	298
  HFEBO17	852218	1346	WUblastx.64	(BAB55130) CDNA FLJ14559 fis,	BAB55130	100%	523	624
  				clone NT2RM2001998.		91%	606	809
  HFFAE46	654843	1347	WUblastx.64	(O14597) NON-FUNCTIONAL	O14597	100%	608	658
  				FOLATE BINDING PROTEIN.		96%	703	801
  						78%	386	442
  						58%	602	649
  						57%	402	632
  HFFAH01	843438	1348	WUblastx.64	hypothetical protein	pir|T43460|T43460	91%	510	241
  				DKFZp434P1721.1 - human		98%	259	11
  				(fragment)		95%	818	513
  HFFAL70	827570	1349	WUblastx.64	(AAH07384) Unknown (protein for	AAH07384	84%	921	466
  				IMAGE: 3677194) (Fra
  HFFAV61	664486	1350	WUblastx.64	(BAA92711) Adaptor protein	BAA92711	100%	1555	1184
  				p130Cas.		50%	577	536
  HFGAN63	708053	1353	WUblastx.64	(Q9D4E9) 4932701A20RIK	Q9D4E9	34%	159	542
  				PROTEIN.		52%	561	617
  HFIAB78	581054	1355	WUblastx.64	(Q9UKA1) F-BOX PROTEIN FBL5	Q9UKA1	100%	672	872
  				(FRAGMENT).		100%	14	667
  						58%	608	658
  						32%	461	661
  						36%	690	836
  HFIIK29	843587	1360	WUblastx.64	(Q9H705) CDNA: FLJ21611 FIS,	Q9H705	94%	48	218
  				CLONE COL07344.
  HFIIK29	852308	1361	WUblastx.64	(Q9H705) CDNA: FLJ21611 FIS,	Q9H705	94%	48	218
  				CLONE COL07344.
  HFIIK29	873681	1362	WUblastx.64	(Q9H705) CDNA: FLJ21611 FIS,	Q9H705	94%	48	218
  				CLONE COL07344.
  HFIIK29	885007	1363	WUblastx.64	(Q9H705) CDNA: FLJ21611 FIS,	Q9H705	94%	48	218
  				CLONE COL07344.
  HFIIZ61	855939	1366	WUblastx.64	(Q62786) PROSTAGLANDIN F2-	FPRP_RAT	92%	35	229
  				ALPHA RECEPTOR REGULATORY
  				PROTEIN
  HFIUT21	794323	1372	WUblastx.64	(AAH07934) DKFZP434A043	AAH07934	100%	6	320
  				protein.
  HFIXC39	683053	1374	WUblastx.64	hypothetical protein 1 - rat (fragment)	pir|S33477|S33477	36%	1816	1541
  HFIXC69	704080	1375	WUblastx.64	(Q9NRX3) NADH: UBIQUINONE	Q9NRX3	87%	32	292
  				OXIDOREDUCTASE MLRQ
  				SUBUNIT HOMOLOG.
  HFIXE39	844408	1376	WUblastx.64	(CAC18649) Lipoyl-containing	CAC18649	100%	1394	1651
  				component X precursor.
  HFIYP15	688044	1377	WUblastx.64	(Q9H324) ZINC	Q9H324	49%	430	1074
  				METALLOENDOPEPTIDASE		35%	406	915
  				(FRAGMENT).		31%	553	1110
  						36%	712	966
  						33%	577	738
  						34%	481	558
  HFKBC47	847076	1383	WUblastx.64	(O95302) FK506-BINDING	O95302	99%	1177	323
  				PROTEIN (FRAGMENT).		54%	1174	404
  						47%	997	323
  HFKDX53	731867	1384	WUblastx.64	(Q9BTV6) UNKNOWN (PROTEIN	Q9BTV6	100%	407	763
  				FOR IMAGE: 3502107)		65%	204	371
  				(FRAGMENT).		100%	766	1296
  HFKEB14	709663	1385	WUblastx.64	(Q9NVY4) CDNA FLJ10436 FIS,	Q9NVY4	97%	8	127
  				CLONE NT2RP1000574, HIGHLY
  				SIMILAR TO HOM
  HFKEU17	815554	1389	WUblastx.64	(AAH00465) Growth arrest and DNA-	AAH00465	97%	226	330
  				damage-inducible,		86%	473	784
  HFKEV77	850678	1390	WUblastx.64	hypothetical protein	pir|T14797|T14797	89%	238	567
  				DKFZp564B167.1 - human
  HFKFI15	775846	1391	WUblastx.64	(Q12948) FORKHEAD BOX	FXC1_HUMAN	100%	2	166
  				PROTEIN C1 (FORKHEAD-
  				RELATED PROTEIN
  HFKFK49	847077	1393	WUblastx.64	(Q9UHR0) MAP KINASE-	Q9UHR0	100%	277	366
  				INTERACTING KINASE.
  HFOXD49	844524	1398	WUblastx.64	(O15194) YA22 PROTEIN (HYA22).	O15194	99%	7	435
  HFOXE28	847385	1399	WUblastx.64	(Q9H0E0) HYPOTHETICAL 13.4 KDA	Q9H0E0	47%	1011	1286
  				PROTEIN.		39%	135	476
  						34%	360	671
  						60%	837	881
  HFOYR54	743196	1403	WUblastx.64	(O00566) U3 SMALL NUCLEOLAR	MP10_HUMAN	78%	118	1365
  				RIBONUCLEOPROTEIN PROTEIN		63%	136	168
  				MPP10
  HFPBQ55	608171	1409	WUblastx.64	(Q24333) ELASTIN LIKE PROTEIN	Q24333	100%	45	116
  				(FRAGMENT).
  HFPCM32	608215	1411	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	48%	719	441
  				CLONE COL03536.
  HFPCU47	741049	1414	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	59%	1645	1580
  				CLONE COL04765.		58%	1598	1356
  HFPCY66	825718	1415	WUblastx.64	(Q9H387) PRO2550.	Q9H387	72%	1371	1252
  						72%	1555	1424
  HFPDC65	839208	1416	WUblastx.64	(Q9H5G8) CDNA: FLJ23451 FIS,	Q9H5G8	100%	72	605
  				CLONE HSI06456.
  HFPDE42	844402	1417	WUblastx.64	(Q99PQ3) TRIPARTITE MOTIF	Q99PQ3	97%	3	218
  				PROTEIN TRIM9 (FRAGMENT).
  HFPDP70	745433	1420	WUblastx.64	(Q9H919) CDNA FLJ13078 FIS,	Q9H919	83%	937	990
  				CLONE NT2RP3002002.		88%	879	905
  						58%	980	1096
  HFPDX08	746379	1422	WUblastx.64	(Q9H9Y3) CDNA FLJ12474 FIS,	Q9H9Y3	100%	3	311
  				CLONE NT2RM1000927.
  HFPEP69	835459	1423	WUblastx.64	(Q9P0X1) LIPOPOLYSACCHARIDE	Q9P0X1	96%	654	749
  				SPECIFIC RESPONSE-5 PROTEIN
  				(FRAGMENT).
  HFRAU40	701986	1424	WUblastx.64	hypothetical protein 3 - human	pir|E41925|E41925	34%	228	109
  						57%	511	428
  						45%	428	252
  HFSAY91	828067	1426	WUblastx.64	hypothetical protein	pir|T46933|T46933	100%	1280	1197
  				DKFZp434M035.1 - human		92%	1096	437
  HFSBC10	638315	1427	WUblastx.64	(O95025) SEMAPHORIN 3D	SM3D_HUMAN	82%	1437	532
  				PRECURSOR.		82%	626	231
  						54%	742	443
  						100%	234	67
  HFSBE94	600398	1428	WUblastx.64	(Q9P195) PRO1722.	Q9P195	75%	705	610
  						73%	853	695
  HFTAN11	638316	1429	WUblastx.64	(Q9H387) PRO2550.	Q9H387	54%	676	614
  						82%	771	667
  						68%	537	490
  						83%	617	489
  HFTBL17	844488	1434	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	95%	967	710
  				PROTEIN (FRAGMENT).
  HFTBL17	863152	1435	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	95%	969	712
  				PROTEIN (FRAGMENT).
  HFTCI85	664487	1437	WUblastx.64	(O60448) NEURONAL THREAD	O60448	63%	1140	1075
  				PROTEIN AD7C-NTP.		92%	1070	1032
  						38%	1139	1032
  						67%	1285	1130
  						59%	1067	1002
  						33%	877	707
  						65%	1275	1129
  						77%	1283	1257
  						72%	1276	1019
  HFTCJ32	604911	1438	WUblastx.64	(Q9H387) PRO2550.	Q9H387	59%	669	586
  						71%	889	689
  HFTCO17	844354	1439	WUblastx.64	(Q9QY01) SERINE/THREONINE	Q9QY01	83%	1	231
  				KINASE UNC51.2.		95%	216	611
  HFTCW07	604987	1440	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	75%	798	589
  				CLONE LNG09295.
  HFTDF32	600360	1441	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	73%	1017	1310
  				CLONE COL04765.
  HFTDF79	827459	1442	WUblastx.64	hypothetical protein XF0051	pir|G82853|G82853	40%	873	691
  				[imported]- Xylella fastidiosa (strain		32%	872	627
  				9a5c)		51%	1202	1113
  						42%	813	589
  						32%	961	716
  						28%	792	424
  						62%	1184	1113
  						62%	1184	1113
  						42%	873	619
  HFTDK11	837382	1443	WUblastx.64	(Q9Y6D7) ES18 (FRAGMENT).	Q9Y6D7	100%	2	67
  						100%	558	650
  						63%	97	618
  HFTDU08	825715	1444	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	70%	885	775
  				CLONE KAIA0536.
  HFVGK67	569778	1445	WUblastx.64	(Q9H387) PRO2550.	Q9H387	83%	1269	985
  HFVHD38	826709	1446	WUblastx.64	SHORT ISOFORM OF O00339	sp_vs|O00339-	98%	2163	550
  					01|O00339	56%	2163	1387
  						50%	2163	1387
  						49%	2163	1387
  						48%	2142	1387
  						40%	1386	820
  HFVIC33	799524	1448	HMMER	PFAM: Enoyl-CoA	PF00378	34.7	462	680
  			2.1.1	hydratase/isomerase family
  			WUblastx.64	(Q9NTX5) DJ351K20.2.1 (NOVEL	Q9NTX5	96%	694	975
  				ENOYL COA/ACYL COA		99%	72	716
  				HYDRATASE/DEHYDROGENA
  HFXAK32	638319	1449	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	40%	1403	1104
  				PROTEIN.
  HFXAK59	831141	1450	WUblastx.64	(Q9BTC8) SIMILAR TO	Q9BTC8	95%	1537	431
  				METASTASIS ASSOCIATED 1.
  HFXBM52	737672	1453	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	80%	708	631
  				CLONE MAMMA1000472.		50%	123	64
  						68%	569	465
  HFXBV67	748230	1455	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	52%	1618	1319
  				human
  HFXCI42	606806	1458	WUblastx.64	(Q9GMI7) HYPOTHETICAL 9.0 KDA	Q9GMI7	43%	542	297
  				PROTEIN.		72%	534	406
  HFXCL59	825713	1459	WUblastx.64	(Q9NSI6) WD-REPEAT PROTEIN 9	WDR9_HUMAN	90%	1008	397
  				(FRAGMENT).		76%	53	3
  						35%	1002	385
  						28%	993	604
  						24%	960	556
  						29%	349	182
  						100%	367	182
  						88%	180	154
  HFXCS53	828903	1462	WUblastx.64	(Q9H8H0) CDNA FLJ13640 FIS,	Q9H8H0	99%	1107	442
  				CLONE PLACE1011221.
  HFXDB37	569805	1463	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	58%	1116	1208
  				PROTEIN.		57%	991	1131
  HFXDL76	821571	1466	WUblastx.64	probable pol polyprotein-related	pir|S21348|S21348	63%	1811	1746
  				protein 4 - rat		48%	1520	1446
  						64%	1740	1519
  HFXDP44	590269	1469	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	64%	531	761
  				CLONE KAT08285.		50%	918	1115
  						88%	745	825
  						36%	1005	1103
  						30%	660	779
  HFXDR08	621342	1470	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	64%	506	700
  				PROTEIN.
  HFXDR28	621300	1471	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	68%	652	717
  				CLONE LNG09295.		70%	501	653
  HFXDR28	873859	1472	WUblastx.64	(Q9H5X2) CDNA: FLJ22865 FIS,	Q9H5X2	98%	853	314
  				CLONE KAT02171.
  HFXDR47	846477	1473	WUblastx.64	(Q9H387) PRO2550.	Q9H387	100%	977	957
  						92%	1051	1010
  						65%	957	733
  HFXDZ03	846323	1474	WUblastx.64	(Q9UN78) HYPOTHETICAL 40.1 KDA	Q9UN78	32%	1065	739
  				PROTEIN.		52%	528	385
  						24%	1161	877
  						39%	757	521
  HFXEE88	838822	1476	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	56%	1310	1116
  				CLONE KAT08285.
  HFXGR32	534607	1477	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	57%	24	272
  				CLONE MAMMA1000472.
  HFXGT51	638174	1478	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	74%	1126	989
  				CLONE KAIA0536.		78%	1232	1107
  HFXHI33	874255	1481	WUblastx.64	(Q9HBS7) HYPOTHETICAL 14.2 KDA	Q9HBS7	66%	1535	1278
  				PROTEIN.
  HFXHL21	581363	1482	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	56%	1441	1373
  				PROTEIN.		64%	1601	1443
  HFXHM93	858720	1485	WUblastx.64	(AAK55521) PRO0764.	AAK55521	66%	803	1027
  HFXHN89	786196	1486	WUblastx.64	(O60448) NEURONAL THREAD	O60448	61%	1063	1218
  				PROTEIN AD7C-NTP.		59%	1208	1273
  						65%	1072	1332
  HFXJB21	743165	1487	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	73%	1269	1003
  				CLONE COL04765.
  HFXJN93	596815	1488	WUblastx.64	(Q9D4B1) 4933405A16RIK	Q9D4B1	97%	501	298
  				PROTEIN.
  HFXJS15	800866	1489	WUblastx.64	(Q9ESN6) NEURAL ACTIVITY-	Q9ESN6	97%	1	447
  				RELATED RING FINGER PROTEIN		32%	37	438
  				(TRIPARTITE MOTI		33%	10	435
  						31%	10	516
  HFXJT53	823591	1490	WUblastx.64	(AAL50053) Viperin.	AAL50053	94%	111	1193
  HFXKL60	895307	1492	WUblastx.64	(Q9H3C0) PRO0898.	Q9H3C0	37%	1749	1889
  						67%	1865	2029
  HFXLG08	885514	1493	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	60%	1303	1067
  				PROTEIN.
  HFXLK91	827288	1494	WUblastx.64	(Q9NWT2) CDNA FLJ20623 FIS,	Q9NWT2	87%	3	827
  				CLONE KAT04793.
  HGBBR29	823106	1497	WUblastx.64	(O43592) EXPORTIN T.	O43592	100%	183	1118
  HGBDV35	581058	1499	WUblastx.64	(AAK53702) ADMP.	AAK53702	42%	91	252
  HGBHE23	832303	1502	WUblastx.64	hypothetical protein	pir|T12479|T12479	86%	24	455
  				DKFZp564N1362.1 - human
  				(fragment)
  HGBHI15	608170	1503	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	63%	663	565
  				PROTEIN.		65%	559	380
  HGLAG32	668271	1505	WUblastx.64	(Q9BRZ3) UNKNOWN (PROTEIN	Q9BRZ3	66%	2	154
  				FOR MGC: 2724).
  HGLAH86	603525	1507	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	55%	824	618
  				CLONE KAT08285.
  HHEBP28	775744	1517	WUblastx.64	(Q9BRL7) SIMILAR TO VESICLE	Q9BRL7	100%	3	233
  				TRAFFICKING PROTEIN.
  HHECR10	621371	1519	WUblastx.64	(Q9H7S6) CDNA FLJ14310 FIS,	Q9H7S6	64%	972	766
  				CLONE PLACE3000271.
  HHEMC55	566796	1520	WUblastx.64	(O60448) NEURONAL THREAD	O60448	64%	605	417
  				PROTEIN AD7C-NTP.		42%	659	417
  						63%	1063	896
  						48%	912	808
  						41%	575	405
  						47%	1063	965
  						40%	1017	781
  						42%	976	878
  						61%	1060	827
  						32%	1063	812
  						50%	896	771
  						80%	349	320
  						65%	656	597
  						40%	606	325
  						43%	516	343
  						45%	941	885
  						65%	606	445
  HHEMP35	821328	1523	WUblastx.64	(Q9Y6X2) PROTEIN INHIBITOR OF	Q9Y6X2	93%	302	931
  				ACTIVATIED STAT3 (PROTEIN		60%	75	308
  				INHIBITOR OF
  HHEMZ08	840026	1524	WUblastx.64	(Q9NZ80) UNCHARACTERIZED	Q9NZ80	77%	845	1351
  				BONE MARROW PROTEIN BM042.
  HHENR74	825671	1527	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	70%	1005	1085
  				CLONE COL04765.		77%	811	876
  						76%	878	1003
  HHENY07	638321	1529	WUblastx.64	(O00311) CELL DIVISION CYCLE	CDC7_HUMAN	88%	8	136
  				7-RELATED PROTEIN KINASE (EC 2		98%	121	744
  HHEOK77	841099	1530	WUblastx.64	(Q9Z1W6) P80 (FRAGMENT).	Q9Z1W6	76%	238	276
  						87%	942	1127
  						23%	510	764
  						80%	315	977
  HHEPE72	621254	1531	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	53%	1015	749
  				CLONE COL04765.
  HHEPE81	840614	1532	WUblastx.64	(O95373) RANBP7/IMPORTIN 7.	O95373	86%	3	389
  HHEQY60	799535	1535	WUblastx.64	(O60448) NEURONAL THREAD	O60448	68%	703	566
  				PROTEIN AD7C-NTP.		62%	731	555
  						68%	568	503
  						47%	570	502
  						58%	489	439
  						65%	731	429
  HHFEB79	1300768	1541	WUblastx.64	(Q92545) RW1 PROTEIN	RW1_HUMAN	74%	708	2387
  				(FRAGMENT).		80%	6	590
  						32%	1984	2148
  HHFEB79	863749	3107	WUblastx.64	Similar to a C. elegans protein encoded	dbj|BAA13387.1|	69%	1303	2400
  				in cosmid C27F2 (U40419) [Homo		80%	601	1185
  				sapiens]
  HHFEN34	701992	1543	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	63%	709	644
  				CLONE HEMBA1006036.		57%	942	691
  HHFFZ01	847388	1544	WUblastx.64	(Q9H6Y7) CDNA: FLJ21676 FIS,	Q9H6Y7	89%	835	1080
  				CLONE COL09164.		91%	1077	1763
  HHFGI71	663531	1545	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	75%	1174	1272
  				PROTEIN.		67%	1424	1269
  HHFGJ54	731870	1546	WUblastx.64	(Q9H906) CDNA FLJ13099 FIS,	Q9H906	99%	1232	1645
  				CLONE NT2RP3002248.		96%	510	1259
  HHFGL38	833063	1547	WUblastx.64	(Q9H387) PRO2550.	Q9H387	53%	2764	2465
  HHFGZ23	721653	1549	WUblastx.64	hypothetical protein 3 - human	pir|E41925|E41925	64%	978	1136
  HHFHM47	638181	1551	WUblastx.64	(O60448) NEURONAL THREAD	O60448	76%	680	618
  				PROTEIN AD7C-NTP.		40%	804	670
  						65%	802	671
  						84%	611	573
  						72%	681	616
  						60%	77	3
  						62%	838	557
  HHGAA76	767745	1552	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	49%	1301	870
  				PROTEIN (FRAGMENT).		69%	499	431
  						68%	59	3
  						74%	1455	1294
  						66%	888	505
  HHGAD46	812646	1553	WUblastx.64	(Q9UGT4) BK65A6.2 (NOVEL	Q9UGT4	85%	148	783
  				SUSHI DOMAIN (SCR REPEAT)		67%	3	203
  				CONTAINING PROTEIN
  HHGBC21	840455	1555	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	54%	1118	858
  				CLONE COL03536.
  HHGBW55	638183	1559	WUblastx.64	hypothetical protein [imported] -	pir|T50835|T50835	97%	3	143
  				human
  HHGDR05	610266	1564	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	63%	821	690
  				CLONE HEMBA1006036.
  HHGDR92	840454	1565	WUblastx.64	(Q9Y512) PROTEIN CGI-51.	CG51_HUMAN	99%	10	1104
  HHGDS56	663379	1566	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	57%	1318	1187
  				CLONE KAIA0536.		62%	1476	1309
  HHGDW65	825652	1567	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	56%	463	726
  				PROTEIN.
  HHPDE28	877640	1571	WUblastx.64	(Q9H620) CDNA: FLJ22673 FIS,	Q9H620	98%	393	596
  				CLONE HSI10503.
  HHPEB61	658693	1575	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	50%	777	935
  				CLONE COL03536.		54%	622	804
  HHPFP26	753269	1576	WUblastx.64	(Q9BQG8) HYPOTHETICAL 32.5 KDA	Q9BQG8	100%	906	1742
  				PROTEIN.
  HHPFS11	570807	1577	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	50%	402	587
  				CLONE COL04765.
  HHPFS18	829347	1579	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	75%	2558	2427
  				CLONE COL04765.
  HHPGH34	535780	1580	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	68%	1019	972
  				FOR MGC: 5149).		64%	835	794
  						68%	963	850
  HHPGU87	658694	1582	WUblastx.64	SHORT ISOFORM OF O60658	sp_vs|O60658-	100%	1050	1274
  					01|O60658
  HHPSE55	735562	1585	WUblastx.64	(Q9CQ15) 0610011N22RIK	Q9CQ15	79%	67	846
  				PROTEIN (RIKEN CDNA
  				0610011N22 GENE).
  HHPSF70	709665	1586	WUblastx.64	(AAH18471) Similar to nitrogen	AAH18471	81%	1	96
  				fixation gene 1 (S.
  HHPSH74	638185	1587	WUblastx.64	hypothetical protein DKFZp566E044.1 -	pir|T46284|T46284	72%	323	1339
  				human		99%	10	321
  						54%	10	321
  						53%	323	571
  						20%	752	1030
  						87%	1272	1730
  HHPTF26	857654	1590	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	66%	1006	752
  				PROTEIN.
  HHSAE74	839473	1592	WUblastx.64	(Q9P1J1) PRO1546.	Q9P1J1	89%	451	365
  						87%	588	442
  HHSAG62	847392	1593	WUblastx.64	(Q9H876) CDNA FLJ13898 FIS,	Q9H876	99%	2	460
  				CLONE THYRO1001738, WEAKLY		99%	600	1052
  				SIMILAR TO TUB		100%	533	604
  HHSBJ92	838604	1595	WUblastx.64	(Q9BTM2) SIMILAR TO RIKEN	Q9BTM2	100%	509	718
  				CDNA 1300006O23 GENE
  				(FRAGMENT).
  HHSDB43	812510	1600	HMMER	PFAM: Zinc finger, C2H2 type	PF00096	117.4	877	945
  			2.1.1
  			WUblastx.64	hypothetical protein	pir|T42688|T42688	92%	25	1344
  				DKFZp434H0127.1 - human
  				(fragment)
  HHSDL07	899430	1601	WUblastx.64	(Q9UJH9) C380A1.2.1 (NOVEL	Q9UJH9	85%	9	317
  				PROTEIN (ISOFORM 1))
  				(UNKNOWN) (PROTEIN FO
  HHSDX07	847393	1602	WUblastx.64	(Q9H9W2) CDNA FLJ12510 FIS,	Q9H9W2	86%	3	512
  				CLONE NT2RM2001723.		79%	434	1066
  HHSFF54	836149	1603	WUblastx.64	(Q9BS18) UNKNOWN (PROTEIN	Q9BS18	100%	129	350
  				FOR MGC: 12537).
  HIABC70	838605	1607	WUblastx.64	(Q9NPJ9) APOLIPOPROTEIN B48	Q9NPJ9	77%	15	851
  				RECEPTOR.		100%	1047	1160
  						34%	522	821
  						25%	303	842
  						24%	306	818
  						24%	312	839
  HIBCO70	853360	1609	HMMER	PFAM: Myelin proteolipid protein	PF01275	32.3	127	192
  			2.1.1	(PLP or lipophilin)
  			WUblastx.64	(Q99NH0) GENE TRAP ANKYRIN	Q99NH0	95%	176	1564
  				REPEAT CONTAINING PROTEIN.		38%	410	1414
  						39%	413	1408
  						37%	419	1411
  						39%	413	1408
  						38%	410	1360
  						37%	443	1441
  						38%	425	1420
  						37%	608	1411
  						38%	407	1141
  						33%	887	1411
  HIBDA41	603909	1611	WUblastx.64	(Q9H387) PRO2550.	Q9H387	80%	404	697
  HIBEC45	822854	1612	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	63%	1023	1283
  				PROTEIN.
  HILBW03	841377	1613	WUblastx.64	(Q9NVS4) CDNA FLJ10546 FIS,	Q9NVS4	97%	705	1163
  				CLONE NT2RP2001721.
  HISAE16	845233	1614	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	85%	7	90
  				CLONE COL04765.
  HISAG53	843902	1615	WUblastx.64	(BAB55426) CDNA FLJ14972 fis,	BAB55426	100%	84	122
  				clone THYRO1000715.		83%	952	1614
  						78%	127	993
  HISAN63	797658	1616	WUblastx.64	(O00363) PUTATIVE P150.	O00363	33%	1609	1427
  						29%	1299	1045
  						59%	1454	1392
  						35%	716	384
  HISAT78	840961	1617	WUblastx.64	(Q9NUD5) DJ1103G7.7 (PUTATIVE	Q9NUD5	66%	155	280
  				NOVEL PROTEIN).		96%	13	108
  						29%	594	767
  						100%	114	194
  						32%	591	785
  						50%	538	567
  						100%	576	743
  						98%	280	567
  HISBA38	561711	1618	WUblastx.64	(Q9H387) PRO2550.	Q9H387	53%	919	836
  						53%	996	907
  						51%	842	687
  HISBB66	839808	1619	WUblastx.64	(O95627) POTENTIAL	O95627	88%	2	451
  				TRANSCRIPTIONAL REPRESSOR
  				NOT4HP.
  HISEJ52	886777	1623	WUblastx.64	(Q9Y6J0) CALCINEURIN-BINDING	CABI_HUMAN	94%	480	1697
  				PROTEIN CABIN 1 (CALCINEURIN I
  HJABC58	753272	1624	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	68%	993	928
  				CLONE COL03536.		73%	1186	998
  HJABS31	825607	1627	WUblastx.64	(AAG49400) Ring-H2 protein.	AAG49400	90%	1	1221
  HJACE25	835517	1629	WUblastx.64	(O75921) RNA POLYMERASE II	O75921	95%	381	662
  				TERMINATION FACTOR.		91%	64	390
  HJACK21	664506	1630	WUblastx.64	(Q9CYB5) 5730552M22RIK	Q9CYB5	98%	530	1123
  				PROTEIN.
  HJBCG74	839361	1631	WUblastx.64	(Q9UNW1) MULTIPLE INOSITOL	Q9UNW1	88%	33	560
  				POLYPHOSPHATE		99%	560	1492
  				PHOSPHATASE.
  HJBCO21	695727	1632	WUblastx.64	(Q9Y6E5) HSPC024-ISO.	Q9Y6E5	84%	9	482
  HJBCQ40	831119	1633	WUblastx.64	(BAB55245) CDNA FLJ14722 fis,	BAB55245	84%	58	1068
  				clone NT2RP3001621.
  HJBDM36	773168	1634	WUblastx.64	(O15498) SNARE PROTEIN YKT6.	O15498	92%	127	720
  HJMAZ60	844796	1637	WUblastx.64	(Q9D9R1) ADULT MALE TESTIS	Q9D9R1	87%	482	1054
  				CDNA, RIKEN FULL-LENGTH		88%	14	484
  				ENRICHED LIBRARY,
  HJMBB20	824264	1638	WUblastx.64	ISOFORM GAC OF O94925	sp_vs|O94925-	99%	3	527
  					02|O94925
  HJMBB20	842673	1639	WUblastx.64	ISOFORM GAC OF O94925	sp_vs|O94925-	100%	4	1461
  					02|O94925
  HJMBB20	844890	1640	WUblastx.64	ISOFORM GAC OF O94925	sp_vs|O94925-	100%	4	1461
  					02|O94925
  HJMBK59	664490	1641	WUblastx.64	(O75935) DYNACTIN SUBUNIT.	O75935	100%	2	286
  HJMBP01	638190	1642	WUblastx.64	(AAH00573) HSPC163 protein.	AAH00573	100%	13	174
  HJPAF69	589794	1646	WUblastx.64	(Q9H387) PRO2550.	Q9H387	77%	439	531
  						73%	278	424
  HJPAQ19	566832	1647	WUblastx.64	hypothetical protein	pir|T08694|T08694	78%	9	869
  				DKFZp564O092.1 - human (fragment)
  HJPAZ35	845379	1648	WUblastx.64	(Q9Y262) HSPC025.	Q9Y262	95%	126	1745
  HJPBI77	602875	1649	WUblastx.64	(Q9V756) CG10155 PROTEIN.	Q9V756	42%	16	249
  HJPBN96	610087	1650	WUblastx.64	(Q9NX09) CDNA FLJ20500 FIS,	Q9NX09	100%	106	288
  				CLONE KAT09159.		100%	70	108
  HJPBU47	824063	1651	WUblastx.64	(Q9NPM0) 8D6 ANTIGEN	Q9NPM0	100%	240	377
  				(FRAGMENT).		77%	315	380
  						73%	261	329
  						80%	329	844
  HJPDK61	844034	1654	WUblastx.64	(Q9V7R7) CG8311 PROTEIN.	Q9V7R7	41%	466	1194
  HKABI53	805966	1655	WUblastx.64	(Q9BUP3) TAT-INTERACTING	Q9BUP3	93%	126	212
  				PROTEIN (30 KD).		99%	212	778
  HKABN63	566840	1656	WUblastx.64	(Q9BRZ8) SIMILAR TO RIKEN	Q9BRZ8	85%	40	306
  				CDNA 1810009K13 GENE.		98%	263	700
  HKACA25	824087	1657	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	64%	1493	1543
  				PROTEIN.		66%	1276	1488
  HKACO64	897591	1658	WUblastx.64	(Q9P195) PRO1722.	Q9P195	61%	694	593
  						58%	930	748
  HKACX90	604042	1660	WUblastx.64	(Q9CZH5) 2700094L05RIK	Q9CZH5	69%	13	510
  				PROTEIN.
  HKADI27	1028177	1661	WUblastx.64	(O35390) ENDO-ALPHA-D-	O35390	65%	302	562
  				MANNOSIDASE.		51%	531	602
  						87%	574	1512
  HKADN26	802131	1662	WUblastx.64	(Q24333) ELASTIN LIKE PROTEIN	Q24333	48%	57	263
  				(FRAGMENT).
  HKADT55	668243	1664	WUblastx.64	(Q9CS80) 5730420G12RIK PROTEIN	Q9CS80	97%	454	708
  				(FRAGMENT).		93%	13	57
  HKAEK58	695729	1665	HMMER	PFAM: Acyl-CoA dehydrogenase	PF00441	449.8	160	1269
  			2.1.1
  			WUblastx.64	(Q9UKU7) ACYL-COENZYME A	Q9UKU7	100%	39	113
  				DEHYDROGENASE-8.		99%	112	1284
  HKAEK72	840457	1666	HMMER	PFAM: Sugar (and other) transporter	PF00083	70.8	270	575
  			2.1.1
  			WUblastx.64	(Q9UGQ3) SUGAR TRANSPORTER	Q9UGQ3	99%	9	602
  				(GLUCOSE TRANSPORTER 9).
  HKAFQ41	847281	1668	WUblastx.64	(O94771) TRANSCRIPTION	O94771	84%	36	896
  				FACTOR-LIKE 5.
  HKAHH71	846826	1669	WUblastx.64	(Q9VAA6) CG7950 PROTEIN.	Q9VAA6	57%	31	348
  HKAKU90	838608	1671	WUblastx.64	(Q9H2T6) ANTIGEN ART1/P17.	Q9H2T6	85%	19	1230
  HKFAA15	753275	1673	WUblastx.64	(Q9P195) PRO1722.	Q9P195	67%	1748	1593
  						76%	1605	1465
  HKGAJ81	581063	1676	WUblastx.64	(Q9H387) PRO2550.	Q9H387	81%	445	335
  						48%	1240	1166
  						70%	620	429
  HKGAK45	809093	1677	WUblastx.64	(Q9H387) PRO2550.	Q9H387	63%	1946	1815
  						68%	2088	1945
  HKGAP57	841627	1679	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	54%	770	639
  				CLONE KAIA0536.		72%	907	767
  HKGAW41	858745	1680	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	68%	1458	1327
  				CLONE COL04765.		71%	1622	1431
  HKGBA21	822870	1681	WUblastx.64	(O00367) L1 ELEMENT L1.19 P40	O00367	55%	1697	1783
  				PROTEIN.		30%	810	1064
  						50%	1279	1731
  HKGBC33	823368	1682	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	76%	783	1001
  HKGBC73	608169	1683	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	54%	74	9
  				PROTEIN.		78%	133	77
  HKGBF61	658697	1684	WUblastx.64	(AAK55521) PRO0764.	AAK55521	70%	978	919
  						39%	1700	1587
  						59%	138	73
  						70%	1171	980
  HKGBH54	588482	1685	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	61%	2206	2114
  						43%	1994	1884
  						54%	2135	1959
  HKGBP52	899282	1686	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	68%	1562	1440
  				CLONE KAIA0536.		76%	1726	1556
  HKGCE23	564825	1687	WUblastx.64	(Q9D445) 4933414E04RIK	Q9D445	60%	1456	1749
  				PROTEIN.
  HKGCE62	638200	1688	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	63%	995	897
  						61%	1131	985
  HKGCK41	559327	1689	WUblastx.64	(Q9NQ89) MIB001 PROTEIN.	Q9NQ89	93%	20	583
  HKGCK41	795256	1690	WUblastx.64	(Q9NQ89) MIB001 PROTEIN.	Q9NQ89	93%	31	591
  HKGDA95	581064	1693	WUblastx.64	(Q9BYC7) MITOCHONDRIAL	Q9BYC7	98%	77	313
  				RIBOSOMAL PROTEIN BMRP36A.		98%	618	782
  						82%	428	625
  HKGDO12	857225	1694	WUblastx.64	(O60448) NEURONAL THREAD	O60448	55%	1223	1170
  				PROTEIN AD7C-NTP.		72%	1429	1277
  						57%	1437	1222
  						48%	1218	1129
  						56%	1289	1146
  						79%	1441	1223
  HKIME53	636982	1695	WUblastx.64	(Q9H3S2) HCCA2 PROTEIN.	Q9H3S2	100%	4	30
  						100%	29	70
  						89%	63	446
  HKIMG23	738671	1696	WUblastx.64	(O75896) FUS1 PROTEIN.	FUS1_HUMAN	76%	217	255
  						100%	131	172
  						100%	246	458
  HKMLF77	604743	1704	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	91%	673	638
  				CLONE KAIA0536.		88%	737	684
  						52%	627	457
  HKMLM32	826523	1705	WUblastx.64	(Q9P0E3) HSPC093 (FRAGMENT).	Q9P0E3	57%	861	1001
  HKMLR17	847432	1706	HMMER	PFAM: CUB domain	PF00431	145	425	760
  			2.1.1
  			WUblastx.64	(Q9UKZ9) PROCOLLAGEN C-	Q9UKZ9	94%	8	1210
  				TERMINAL PROTEINASE
  				ENHANCER PROTEIN 2.
  HKMLT89	826167	1707	WUblastx.64	(Q9HD86) NAG18.	Q9HD86	87%	1141	1233
  HKMLV05	663924	1708	WUblastx.64	(Q9H387) PRO2550.	Q9H387	81%	1559	1290
  HKMLV25	695731	1709	WUblastx.64	(Q9BT42) SIMILAR TO LR8	Q9BT42	97%	328	474
  				PROTEIN.		40%	266	340
  						99%	2	334
  HKMMD91	847394	1712	WUblastx.64	(AAH07729) Similar to RIKEN cDNA	AAH07729	100%	1150	752
  				2810021O14 gene.
  HKMMP90	746876	1713	WUblastx.64	(O40947) ORF 73.	O40947	30%	237	398
  						28%	240	443
  						30%	210	317
  						24%	1054	1569
  						25%	1054	1659
  						24%	1054	1659
  						25%	1054	1659
  						25%	1060	1662
  						23%	1054	1677
  HKPAC10	527238	1715	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	47%	970	677
  				CLONE HEMBA1006036.
  HKPAC50	830367	1716	WUblastx.64	(O95139) NADH-UBIQUINONE	NB7M_HUMAN	61%	106	324
  				OXIDOREDUCTASE B17 SUBUNIT
  				(EC 1.6
  HKTAC18	831292	1718	WUblastx.64	(Q9Y5W8) SORTING NEXIN 13	SNXD_HUMAN	88%	3	53
  				(FRAGMENT).		96%	38	400
  HL1SA89	839594	1719	WUblastx.64	(Q9Y399) MITOCHONDRIAL 28S	Q9Y399	99%	63	641
  				RIBOSOMAL PROTEIN S2 (MRP-
  				S2).
  HL2AB60	722226	1720	WUblastx.64	SHORT ISOFORM OF Q9Y679	sp_vs|Q9Y679-	91%	64	1293
  					01|Q9Y679
  HL3AF32	753468	1722	WUblastx.64	(Q9CZ73) 2810049G06RIK	Q9CZ73	76%	6	527
  				PROTEIN.
  HLDAV70	623603	1723	WUblastx.64	(Q9UJW0) DYNACTIN P62	Q9UJW0	100%	175	285
  				SUBUNIT (CDNA FLJ20292 FIS,
  				CLONE HEP05374).
  HLDBL62	899498	1724	WUblastx.64	(Q9NYC7) HEPATOCELLULAR	Q9NYC7	78%	36	740
  				CARCINOMA-ASSOCIATED
  				ANTIGEN 112.
  HLDBV18	805684	1725	WUblastx.64	(Q9H7D5) CDNA: FLJ21022 FIS,	Q9H7D5	94%	284	1198
  				CLONE CAE06383.
  HLDBV54	570813	1726	HMMER	PFAM: Sec1 family	PF00995	18.7	180	332
  			2.1.1
  HLDNF18	821599	1730	WUblastx.64	(Q9NWI8) CDNA FLJ20823 FIS,	Q9NWI8	78%	17	583
  				CLONE ADSE00282 (SIMILAR TO
  				TRANSLOCASE O
  HLDNN84	612801	1731	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	51%	751	849
  						59%	600	755
  HLDOD77	610208	1732	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	71%	513	617
  				PROTEIN.		62%	395	523
  HLDOL74	812765	1733	WUblastx.64	(Q9P0A2) HSPC279 (FRAGMENT).	Q9P0A2	94%	57	671
  HLDRU08	701995	1735	WUblastx.64	(Q9NVD7) CDNA FLJ10793 FIS,	Q9NVD7	99%	13	729
  				CLONE NT2RP4000588 (ALPHA-
  				PARVIN).
  HLDXF43	514022	1736	WUblastx.64	cytochrome-c oxidase (EC 1.9.3.1)	pir|A00472|OBHU2	52%	164	559
  				chain II - human mitochondrion
  HLEAA10	824265	1737	WUblastx.64	(Q13579) MARINER	Q13579	85%	842	741
  				TRANSPOSASE.		77%	738	13
  HLHAE14	865625	1739	WUblastx.64	(Q9H7S6) CDNA FLJ14310 FIS,	Q9H7S6	66%	1412	1528
  				CLONE PLACE3000271.		44%	935	1060
  HLHAE14	886763	1740	WUblastx.64	(Q9H7S6) CDNA FLJ14310 FIS,	Q9H7S6	66%	1412	1528
  				CLONE PLACE3000271.		44%	935	1060
  HLHBS54	837503	1741	WUblastx.64	(Q9NZV5) SELENOPROTEIN N	SELN_HUMAN	92%	1	1455
  				PRECURSOR.
  HLHCG24	847398	1744	WUblastx.64	(Q9BZE5) PGC-1 RELATED CO-	Q9BZE5	82%	10	615
  				ACTIVATOR.
  HLHCN51	836150	1746	WUblastx.64	(Q9D1H8) 1110007K17RIK	Q9D1H8	72%	1588	1514
  				PROTEIN.		48%	1514	1275
  HLHCT96	886178	1747	WUblastx.64	(Q9H6G8) CDNA: FLJ22294 FIS,	Q9H6G8	90%	1976	1917
  				CLONE HRC04426.		68%	2152	1955
  HLHDJ05	841732	1750	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	68%	812	708
  				PROTEIN.		66%	708	547
  HLHDL37	847399	1751	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	78%	690	571
  				CLONE KAT08285.
  HLHDL69	883335	1752	WUblastx.64	(O09014) PEPTIDE/HISTIDINE	O09014	88%	305	592
  				TRANSPORTER.		72%	598	1314
  HLHDL69	850691	1753	WUblastx.64	(O09014) PEPTIDE/HISTIDINE	O09014	75%	305	340
  				TRANSPORTER.		78%	330	1484
  HLHDL69	843836	1754	WUblastx.64	(O09014) PEPTIDE/HISTIDINE	O09014	75%	305	340
  				TRANSPORTER.		78%	330	1484
  HLHDL69	855906	1755	WUblastx.64	(O09014) PEPTIDE/HISTIDINE	O09014	75%	305	340
  				TRANSPORTER.		78%	330	1484
  HLHDM38	566843	1756	WUblastx.64	hypothetical protein	pir|T46471|T46471	45%	6	326
  				DKFZp434L0130.1 - human		39%	181	339
  						61%	476	886
  HLHDR92	692150	1757	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	77%	1252	1172
  				CLONE KAIA0536.		58%	1181	1095
  HLHDY94	847121	1758	WUblastx.64	(O00572) HYPOTHETICAL 43.0 KDA	O00572	56%	637	876
  				PROTEIN.		61%	18	752
  HLHEI72	874180	1761	HMMER	PFAM: Eukaryotic protein kinase	PF00069	106.7	101	403
  			2.1.1	domain
  			WUblastx.64	(Q9Y6S4) SIMILARITY IS TO	Q9Y6S4	81%	589	621
  				SERINE/THREONINE-PROTEIN		97%	89	526
  				KINASE (FRAGMENT).
  HLHEX62	608285	1762	WUblastx.64	(Q9H387) PRO2550.	Q9H387	70%	1102	833
  HLHFP09	805967	1764	WUblastx.64	ISOFORM 2 OF O95460	sp_vs|O95460-	98%	3	881
  					01|O95460	38%	102	740
  HLHGG78	638210	1765	WUblastx.64	(O95989) DIPHOSPHOINOSITOL	O95989	100%	10	291
  				POLYPHOSPHATE
  				PHOSPHOHYDROLASE.
  HLHSQ35	570814	1767	WUblastx.64	(Q9JIX6) RNA BINDING PROTEIN	Q9JIX6	100%	671	712
  				NAPOR-3 (FRAGMENT).		87%	711	827
  HLHTP55	847322	1769	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	50%	320	433
  				CLONE COL04765.		56%	462	635
  HLIBD74	604914	1770	WUblastx.64	(Q9H797) CDNA: FLJ21128 FIS,	Q9H797	98%	12	290
  				CLONE CAS06258.
  HLIBE41	772762	1771	WUblastx.64	(Q9D0Q3) 1300007B24RIK	Q9D0Q3	58%	71	409
  				PROTEIN.
  HLLAX64	827292	1775	WUblastx.64	(BAB55004) CDNA FLJ14357 fis,	BAB55004	96%	1020	1502
  				clone HEMBA1000005, h		91%	27	416
  						77%	364	417
  						25%	996	1196
  						97%	395	1057
  						32%	761	862
  						28%	752	973
  						28%	1257	1478
  						45%	611	676
  						45%	1428	1499
  						17%	1002	1229
  HLLAX95	588405	1776	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	52%	541	603
  				PROTEIN.		65%	381	521
  HLMCT51	772579	1780	WUblastx.64	(Q9DAD1) 1700013E09RIK	Q9DAD1	55%	443	883
  				PROTEIN.
  HLMCT95	570815	1781	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	53%	1414	1121
  				PRODUCT.
  HLMDD65	566891	1782	WUblastx.64	(P20591) INTERFERON-	MX1_HUMAN	100%	489	575
  				REGULATED RESISTANCE GTP-		70%	580	720
  				BINDING PROTEI
  HLMFB62	604915	1785	WUblastx.64	(Q9H700) CDNA: FLJ21617 FIS,	Q9H700	68%	154	2
  				CLONE COL07481.
  HLMFG52	602166	1786	WUblastx.64	(Q9UHT5) PRO1848.	Q9UHT5	71%	837	941
  HLMFU53	801936	1787	WUblastx.64	(Q9ESD6) LNV.	Q9ESD6	67%	1141	620
  HLMHG68	580850	1788	WUblastx.64	(Q9HAI5) CDNA FLJ11583 FIS,	Q9HAI5	92%	305	775
  				CLONE HEMBA1003680, WEAKLY
  				SIMILAR TO PUT
  HLMHN06	853606	1789	WUblastx.64	probable transposase - human	pir|S72481|S72481	59%	456	286
  				transposon MER37		80%	262	107
  HLMIM84	588487	1791	WUblastx.64	(Q9Y3B0) CGI-105 PROTEIN.	Q9Y3B0	84%	296	334
  						98%	645	806
  HLMIW76	570816	1794	WUblastx.64	(O60448) NEURONAL THREAD	O60448	85%	531	490
  				PROTEIN AD7C-NTP.		57%	528	472
  						59%	600	535
  						57%	528	472
  						77%	744	718
  						63%	601	536
  						66%	752	591
  						69%	736	590
  						70%	737	474
  HLQAD72	853613	1798	HMMER	PFAM: Pancreatic ribonucleases	PF00074	213.4	208	564
  			2.1.1
  			WUblastx.64	ribonuclease 4 (EC 3.1.—.—) precursor -	pir|I52489|I52489	90%	124	564
  				human
  HLQAM30	609880	1799	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	76%	648	610
  						57%	490	398
  						73%	617	480
  HLQAM59	560662	1800	WUblastx.64	(AAH08373) Similar to hypothetical	AAH08373	82%	879	829
  				protein PRO1722.		54%	754	722
  						63%	1018	881
  HLQBB23	745364	1801	WUblastx.64	(Q9CYS6) 2810459M11RIK	Q9CYS6	44%	38	286
  				PROTEIN.
  HLQCY09	745384	1804	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	57%	884	843
  				CLONE KAT08285.		58%	759	505
  HLQDK45	621414	1807	WUblastx.64	(Q9CPU5) 2700019M19RIK	Q9CPU5	100%	615	737
  				PROTEIN.
  HLQDM47	743237	1808	WUblastx.64	giant protein p619 - human	pir|S71752|S71752	92%	377	616
  HLTDI20	610269	1817	WUblastx.64	(Q9H6C9) CDNA: FLJ22385 FIS,	Q9H6C9	70%	5	76
  				CLONE HRC07610.		95%	58	186
  HLTDI65	740756	1818	WUblastx.64	(O60448) NEURONAL THREAD	O60448	41%	1290	1382
  				PROTEIN AD7C-NTP.		50%	1456	1623
  						46%	1881	2027
  						47%	1881	2018
  						54%	1958	2029
  						54%	1878	1949
  						41%	1513	1605
  						57%	2095	2157
  						55%	1955	2038
  						60%	1275	1484
  						54%	1372	1464
  						47%	2095	2163
  						42%	2008	2157
  						64%	1472	1513
  						61%	1263	1463
  						36%	1939	2079
  						32%	1296	1460
  						50%	1363	1470
  						61%	2015	2149
  						46%	1315	1608
  HLTDK30	834810	1819	WUblastx.64	(Q9HA72) CDNA FLJ12133 FIS,	Q9HA72	100%	511	747
  				CLONE MAMMA1000278		97%	231	506
  				(SIMILAR TO HYPOTHETIC		97%	740	1180
  HLTDL37	638213	1820	WUblastx.64	(Q9Y383) CGI-74 PROTEIN.	Q9Y383	82%	2	754
  						66%	1084	1155
  						37%	974	1078
  HLTDU35	827293	1821	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	44%	1118	954
  				PRODUCT.		62%	954	820
  HLTEH84	782094	1823	WUblastx.64	(Q9H5P9) CDNA: FLJ23188 FIS,	Q9H5P9	97%	15	491
  				CLONE LNG12038.
  HLTEL39	853615	1824	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	39%	1391	1218
  				CLONE COL03536.		70%	1536	1387
  HLTEW52	663148	1826	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	70%	1498	1244
  				CLONE KAIA0536.
  HLTEZ36	821676	1827	WUblastx.64	(Q9H387) PRO2550.	Q9H387	76%	7	171
  HLTGG14	824068	1828	WUblastx.64	(Q9N032) UNNAMED PROTEIN	Q9N032	70%	1142	1020
  				PRODUCT.
  HLUAF94	794116	1829	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	75%	1438	1473
  						72%	1256	1444
  HLWAH33	855954	1830	WUblastx.64	(Q9NSI3) PRED43 PROTEIN	Q9NSI3	100%	1660	1761
  				(FRAGMENT).		96%	25	102
  HLWAO11	885339	1831	WUblastx.64	(Q9ES77) POLYDOM PROTEIN	Q9ES77	76%	14	2656
  				PRECURSOR.		32%	14	2371
  						32%	2	2383
  						29%	152	2635
  						28%	14	2371
  						27%	14	1699
  						28%	506	1408
  						30%	2	628
  						29%	38	640
  						27%	161	787
  						28%	1469	2020
  						31%	2084	2617
  						34%	2267	2659
  						33%	14	373
  						32%	80	448
  						32%	2021	2635
  						35%	1019	1297
  						29%	1472	1969
  						22%	494	1990
  						33%	1745	2074
  						25%	14	478
  						27%	1067	1597
  						28%	1886	2179
  						26%	170	895
  						24%	1310	1771
  						26%	2249	2638
  						30%	1430	1654
  						22%	1142	1609
  						35%	1145	1288
  						36%	2003	2110
  						18%	1382	2194
  						35%	95	187
  						24%	455	709
  						27%	2354	2560
  						25%	440	673
  						33%	173	271
  HLWAX50	809094	1833	WUblastx.64	(AAC08702) Meltrin-L precursor.	AAC08702	92%	628	1629
  						93%	1587	2273
  						64%	178	255
  						39%	1553	1669
  						97%	257	661
  HLWBK16	638217	1835	WUblastx.64	(Q9UEV9) ACTIN-BINDING	Q9UEV9	94%	679	1191
  				PROTEIN HOMOLOG ABP-278.		28%	572	700
  						52%	590	658
  						57%	605	658
  						58%	593	658
  						34%	673	750
  						29%	682	1185
  						50%	593	652
  						37%	593	721
  						33%	727	1164
  						37%	853	1125
  						30%	664	1179
  						53%	590	775
  						31%	593	775
  						34%	730	1188
  						34%	691	1185
  						32%	745	1125
  						32%	590	775
  						37%	706	1185
  						32%	691	1170
  						32%	688	1179
  						37%	688	1173
  						37%	703	1185
  						31%	703	1182
  						28%	703	1176
  						36%	715	1149
  						30%	703	1188
  						32%	949	1173
  						32%	691	1158
  						28%	691	1185
  						41%	706	1185
  						36%	593	775
  						32%	593	775
  						26%	551	775
  						31%	691	1179
  						34%	593	775
  						30%	748	1125
  HLYAJ79	608289	1839	WUblastx.64	(Q9B2U5) ATP SYNTHASE 6.	Q9B2U5	63%	301	807
  HLYAT54	581067	1842	WUblastx.64	(Q9H9F0) CDNA FLJ12802 FIS,	Q9H9F0	93%	35	220
  				CLONE NT2RP2002124, WEAKLY
  				SIMILAR TO UBI
  HLYBC81	588490	1843	WUblastx.64	(Q9HBW6) NAG13.	Q9HBW6	43%	1373	1711
  HLYBD09	610019	1844	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	69%	1546	1644
  				CLONE KAIA0536.		65%	1391	1513
  HLYBL67	834538	1845	WUblastx.64	(Q9NRE8) PADI-H PROTEIN.	Q9NRE8	64%	335	294
  						61%	304	158
  HLYBN23	667995	1847	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	83%	1397	1450
  						80%	1333	1395
  HLYBT28	608152	1850	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	54%	1082	774
  				PRODUCT.
  HLYBY04	596819	1852	WUblastx.64	(Q9H387) PRO2550.	Q9H387	83%	1565	1530
  						71%	1533	1270
  HLYCE15	621407	1853	WUblastx.64	(Q9P147) PRO2822.	Q9P147	75%	518	420
  HLYCH04	608295	1854	WUblastx.64	(O62658) LINE-1 ELEMENT ORF2.	O62658	47%	106	273
  						45%	7	39
  HLYDE38	735987	1856	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	47%	987	835
  				PRODUCT.		68%	1091	1005
  HLYDG55	835625	1857	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	73%	638	423
  				CLONE LNG09295.
  HLYEA60	685469	1859	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	50%	104	181
  						70%	27	116
  HLYEJ14	853362	1860	WUblastx.64	(O95498) VASCULAR NON-	VNN2_HUMAN	97%	94	1653
  				INFLAMMATORY MOLECULE 2
  				PRECURSOR (VA
  HLYEJ44	838149	1861	WUblastx.64	(Q9H400) DJ583P15.4.1 (NOVEL	Q9H400	100%	124	153
  				PROTEIN (TRANSLATION OF		80%	273	1007
  				CDNA FLJ20406 (E
  HLYEU51	607815	1862	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	67%	691	563
  				CLONE KAIA0536.		78%	857	708
  HLYGV19	826334	1863	WUblastx.64	(Q9H0W4) HYPOTHETICAL 19.2 KDA	Q9H0W4	72%	195	644
  				PROTEIN.
  HMABK52	853363	1864	WUblastx.64	(Q9N032) UNNAMED PROTEIN	Q9N032	62%	1136	1002
  				PRODUCT.
  HMACL77	853365	1866	WUblastx.64	(O95564) HYPOTHETICAL 30.9 KDA	O95564	100%	8	256
  				PROTEIN.
  HMACT74	843744	1867	WUblastx.64	gamma-interferon-inducible protein	pir|A43708|A43708	94%	403	639
  				IP-30 precursor - human
  HMADJ14	843725	1868	WUblastx.64	(Q9H295) DC-SPECIFIC	Q9H295	96%	871	945
  				TRANSMEMBRANE PROTEIN.		90%	47	880
  HMADJ74	795479	1869	WUblastx.64	(Q9H295) DC-SPECIFIC	Q9H295	100%	1010	1393
  				TRANSMEMBRANE PROTEIN.		93%	186	1067
  HMAEA58	872563	1870	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	36%	584	519
  				CLONE KAIA0536.		59%	1339	1172
  HMAGF01	837235	1871	WUblastx.64	(Q9UJ41) RAB5 GDP/GTP	Q9UJ41	89%	71	1543
  				EXCHANGE FACTOR
  				HOMOLOGUE.
  HMCED78	829071	1873	WUblastx.64	(Q9NWY5) CDNA FLJ20533 FIS,	Q9NWY5	98%	380	862
  				CLONE KAT10931.
  HMCFN86	886182	1874	WUblastx.64	(Q9BVB2) UNKNOWN (PROTEIN	Q9BVB2	100%	22	294
  				FOR IMAGE: 3459631)
  				(FRAGMENT).
  HMCGJ47	699850	1875	WUblastx.64	(AAK29328) Serine	AAK29328	91%	11	511
  				palmitoyltransferase.		91%	495	1430
  HMCGK88	654854	1876	WUblastx.64	(Q9BT48) SIMILAR TO	Q9BT48	100%	506	330
  				HYPOTHETICAL PROTEIN		92%	982	506
  				FLJ20116.
  HMCIH27	862038	1877	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	70%	1293	1063
  				CLONE COL04765.
  HMDAE88	638222	1881	WUblastx.64	(Q9H3C0) PRO0898.	Q9H3C0	57%	1001	843
  						81%	844	734
  HMDAM39	602907	1885	WUblastx.64	(Q9P195) PRO1722.	Q9P195	66%	648	388
  HMEAA41	587304	1886	WUblastx.64	(Q9H6H3) CDNA: FLJ22282 FIS,	Q9H6H3	100%	9	419
  				CLONE HRC03861.
  HMEEH21	603187	1888	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	65%	280	504
  				PRODUCT.
  HMEEZ07	638225	1890	WUblastx.64	(Q9UK97) F-BOX ONLY PROTEIN	FBX9_HUMAN	100%	795	917
  				9.
  HMEIH57	840370	1892	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	74%	238	11
  				PROTEIN (FRAGMENT).		58%	1348	1190
  						50%	1236	256
  HMEIJ21	767276	1893	WUblastx.64	(Q9H067) HYPOTHETICAL 66.5 KDA	Q9H067	61%	2388	1777
  				PROTEIN.		50%	828	709
  						56%	1676	840
  HMEJC96	761218	1895	WUblastx.64	(O60836) MEMBRANE	O60836	86%	193	669
  				GLYCOPROTEIN GP36
  				PRECURSOR.
  HMEJK28	825591	1897	WUblastx.64	(O00479) NON-HISTONE	O00479	54%	273	485
  				CHROMOSOMAL PROTEIN (HIGH-
  				MOBILITY GROUP (NONHIS
  HMEKW44	825499	1899	WUblastx.64	(O18975) HYPOTHETICAL 16.6 KDA	O18975	71%	7	198
  				PROTEIN (FRAGMENT).
  HMEKW71	855955	1900	WUblastx.64	(Q9UHF1) NOTCH4-LIKE PROTEIN	Q9UHF1	96%	579	758
  				(HYPOTHETICAL 29.6 KDA
  				PROTEIN).
  HMHBI09	847402	1903	WUblastx.64	(AAH07644) Similar to RIKEN cDNA	AAH07644	93%	39	683
  				9430029K10 gene (F
  HMHBI93	844729	1904	WUblastx.64	(Q9Z1S4) TGFB1-INDUCED ANTI-	TIAF_MOUSE	90%	1391	1735
  				APOPTOTIC FACTOR 1 (12 KDA
  				TGF-
  HMIAC52	825484	1906	WUblastx.64	(AAH00713) Neighbor of A-kinase	AAH00713	30%	941	1201
  				anchoring protein 9		52%	988	1056
  						75%	112	927
  HMIAG42	846339	1908	WUblastx.64	(AAK54122) RGS20 ret splice variant	AAK54122	78%	1071	1730
  				1.
  HMIAG55	736020	1909	WUblastx.64	(AAD20460) Ribosomal protein L11.	AAD20460	67%	424	609
  HMIAG72	824091	1910	WUblastx.64	(O60519) CRE BINDING PROTEIN-	O60519	85%	318	677
  				LIKE 2.
  HMIAO82	833070	1912	WUblastx.64	hypothetical protein	pir|T46340|T46340	58%	32	358
  				DKFZp434B0814.1 - human		50%	443	742
  				(fragment)
  HMIAR42	809096	1913	WUblastx.64	(Q9BVE4) SIMILAR TO	Q9BVE4	87%	1046	1165
  				INTERFERON-RELATED		91%	831	1052
  				DEVELOPMENTAL REGULATOR		99%	7	849
  				1.
  HMIAV33	668252	1914	WUblastx.64	(Q9UN80) HYPOTHETICAL 149.0 KDA	Q9UN80	34%	1051	1182
  				PROTEIN.		48%	1167	1337
  						40%	603	430
  						41%	138	10
  						28%	1015	611
  						38%	431	162
  HMIBE95	804597	1917	WUblastx.64	probable transposase - human	pir|S72481|S72481	60%	1932	2075
  				transposon MER37		64%	1221	1355
  						42%	1355	1948
  HMKAN71	884161	1920	WUblastx.64	(Q96G03) Unknown (protein for	Q96G03	100%	12	1385
  				MGC: 19508).
  HMKBA33	596949	1921	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	47%	1147	1010
  				PRODUCT.		44%	1248	1162
  						75%	1441	1382
  						57%	1314	1147
  						33%	383	261
  						65%	1691	1482
  HMKCK32	561576	1923	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	53%	619	335
  				PRODUCT.
  HMKDG69	588407	1927	WUblastx.64	pro-pol-dUTPase polyprotein - murine	pir|T29097|T29097	59%	104	24
  				endogenous retrovirus ERV-L		34%	700	116
  				(fragment)
  HMKDM80	608168	1928	WUblastx.64	(Q9H6G8) CDNA: FLJ22294 FIS,	Q9H6G8	74%	178	11
  				CLONE HRC04426.
  HMKEG88	634245	1929	WUblastx.64	(O00378) PUTATIVE P150.	O00378	47%	294	4
  HMMAA09	822859	1930	WUblastx.64	(AAK55521) PRO0764.	AAK55521	68%	1305	1249
  						67%	1271	1014
  HMMAK92	610100	1931	WUblastx.64	(Q9CZ33) 2810408E11RIK	Q9CZ33	86%	15	152
  				PROTEIN.
  HMMBF22	780406	1934	WUblastx.64	(Q9H6V7) CDNA: FLJ21825 FIS,	Q9H6V7	56%	1228	1341
  				CLONE HEP01348.		93%	407	499
  						58%	824	1159
  HMMBH94	607463	1936	WUblastx.64	(AAK55521) PRO0764.	AAK55521	80%	857	813
  						71%	835	593
  HMMBK55	608179	1937	WUblastx.64	(Q9H397) PRO2852.	Q9H397	72%	521	468
  						36%	480	361
  						70%	691	527
  HMMBT47	804594	1941	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	66%	1238	1203
  				CLONE KAIA0536.		54%	1206	874
  HMMCD35	607840	1942	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	59%	813	676
  				CLONE COL04765.		58%	661	512
  HMMCD95	603202	1943	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	58%	1498	1397
  				PRODUCT.		63%	1643	1494
  HMPAB26	580854	1944	WUblastx.64	(P78560) DEATH DOMAIN	CRAD_HUMAN	100%	66	365
  				CONTAINING PROTEIN CRADD
  				(CASPASE AND
  HMQAI38	589964	1946	WUblastx.64	immune-responsive gene 1 - mouse	pir|I54546|I54546	71%	11	1363
  				(fragment)
  HMQAT69	799551	1947	WUblastx.64	(Q9Y5X1) SORTING NEXIN 9 (SH3	SNX9_HUMAN	94%	739	1842
  				AND PX DOMAIN-CONTAINING		82%	660	809
  				PROT		93%	56	748
  HMQBL90	600363	1948	WUblastx.64	pre-B cell Ig lambda-like omega light	pir|A33911|A33911	52%	879	1091
  				chain (non-rearranging) 14.1 - human		76%	974	1309
  HMQBV82	826522	1949	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	61%	739	876
  				PROTEIN.		72%	969	868
  						40%	651	592
  HMQCA75	767086	1950	WUblastx.64	(AAK55521) PRO0764.	AAK55521	62%	1235	1071
  						72%	1072	944
  HMQCB37	732168	1951	WUblastx.64	(AAK55521) PRO0764.	AAK55521	85%	426	446
  						71%	1087	1149
  						50%	872	1042
  						79%	503	904
  HMQCX41	603388	1953	WUblastx.64	(O60448) NEURONAL THREAD	O60448	43%	192	260
  				PROTEIN AD7C-NTP.		55%	16	204
  HMQDM09	664494	1954	WUblastx.64	(Q9NZV6) SELENOPROTEIN X 1	SELX_HUMAN	98%	38	385
  				(PROTEIN HSPC270).
  HMSAP33	731969	1956	WUblastx.64	(Q9P147) PRO2822.	Q9P147	83%	1526	1398
  HMSAZ48	877650	1957	WUblastx.64	probable pol polyprotein-related	pir|S21348|S21348	46%	793	1218
  				protein 4 - rat		66%	722	784
  HMSBN18	847405	1958	WUblastx.64	(O95789) ZNF258.	O95789	65%	165	329
  HMSBS25	826487	1959	WUblastx.64	(Q9Y642) K: CL COTRANSPORTER	Q9Y642	77%	1019	1189
  				3.		81%	337	384
  						100%	257	346
  HMSBU14	844443	1960	WUblastx.64	hypothetical protein	pir|T47135|T47135	41%	954	1184
  				DKFZp761L0812.1 - human		63%	1192	1488
  				(fragment)
  HMSCB94	847406	1962	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	45%	1690	1589
  				CLONE KAT08285.		80%	1931	1683
  HMSCK12	825475	1963	WUblastx.64	(Q9P195) PRO1722.	Q9P195	62%	771	613
  						81%	622	491
  HMSCV75	838530	1965	WUblastx.64	(Q9NRE8) PADI-H PROTEIN.	Q9NRE8	40%	1565	1461
  						67%	1438	1301
  HMSCW44	716249	1967	WUblastx.64	(Q9P195) PRO1722.	Q9P195	47%	1251	1102
  						66%	1112	942
  HMSCZ19	847408	1968	WUblastx.64	(O60427) BC269730_2	O60427	84%	621	1124
  				(HYPOTHETICAL 52.0 KDA		94%	1	675
  				PROTEIN) (FATTY ACID DESAT
  HMSDI67	827298	1969	WUblastx.64	probable pol polyprotein-related	pir|S21348|S21348	55%	1255	1121
  				protein 4-rat		38%	1748	1284
  HMSDR28	801948	1971	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	76%	1097	1035
  				CLONE COL04765.		72%	1317	1102
  HMSFT25	581069	1972	WUblastx.64	(AAK55521) PRO0764.	AAK55521	43%	1088	1198
  						61%	918	1025
  HMSFW52	603177	1973	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	67%	1380	1631
  				CLONE KAIA0536.
  HMSGU30	847409	1975	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	55%	1718	1494
  				PROTEIN.
  HMSHB42	792385	1976	WUblastx.64	(Q9NR81) RHOGEF	Q9NR81	97%	4	867
  				(HYPOTHETICAL 59.8 KDA
  				PROTEIN).
  HMSHB42	600401	1977	WUblastx.64	(Q9NR81) RHOGEF	Q9NR81	99%	4	1203
  				(HYPOTHETICAL 59.8 KDA
  				PROTEIN).
  HMSHN72	855917	1978	WUblastx.64	(Q98SS5) DOUBLECORTIN.	Q98SS5	91%	650	754
  HMSHW73	704099	1980	WUblastx.64	(Q9P195) PRO1722.	Q9P195	62%	2214	2134
  						65%	2131	2063
  						71%	2072	1935
  HMSII36	825453	1982	WUblastx.64	(AAK55521) PRO0764.	AAK55521	61%	23	61
  						72%	51	161
  HMSIT42	581070	1983	WUblastx.64	(Q9P147) PRO2822.	Q9P147	80%	1160	936
  HMSJB08	877651	1984	WUblastx.64	(Q9D2Y4) 9130019I15RIK	Q9D2Y4	56%	1037	612
  				PROTEIN.		58%	1987	1028
  HMSJR44	638098	1987	WUblastx.64	(Q9P195) PRO1722.	Q9P195	73%	798	721
  						43%	1168	1037
  						82%	853	803
  						36%	1633	1511
  						64%	995	843
  HMSKQ91	825450	1988	WUblastx.64	(Q9HBN2) HYPOTHETICAL 15.8 KDA	Q9HBN2	68%	864	742
  				PROTEIN.
  HMTAF92	740784	1990	WUblastx.64	(Q9Y6I4) UBIQUITIN CARBOXYL-	UBP3_HUMAN	93%	2211	2167
  				TERMINAL HYDROLASE 3 (EC		84%	2053	644
  				3.1.2.
  HMTAT36	811885	1991	WUblastx.64	(Q9H6Z9) CDNA: FLJ21620 FIS,	Q9H6Z9	100%	1	117
  				CLONE COL07838.
  HMUAD65	809097	1993	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	67%	1524	1213
  				CLONE COL03536.
  HMUBK53	553624	1997	WUblastx.64	(Q9H8X7) CDNA FLJ13156 FIS,	Q9H8X7	86%	18	398
  				CLONE NT2RP3003490, WEAKLY
  				SIMILAR TO HOM
  HMUBY57	844800	2001	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	78%	1827	1609
  				CLONE LNG09295.
  HMUBZ15	609837	2002	WUblastx.64	(Q9BU61) SIMILAR TO NUCLEAR	Q9BU61	60%	968	1012
  				PROTEIN E3-3 ORF1.		100%	412	963
  HMVAL15	822860	2003	WUblastx.64	A-kinase anchor protein 95 - human	pir|T13161|T13161	83%	1	348
  HMVBC84	743191	2004	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	47%	1019	957
  				CLONE COL04765.		76%	1135	1019
  HMVBD68	831130	2005	WUblastx.64	catalase (EC 1.11.1.6) -	pir|I40767|I40767	85%	207	103
  				Campylobacter jejuni
  HMVCG17	846161	2006	WUblastx.64	(Q9NZS9) APOPTOSIS	Q9NZS9	96%	282	458
  				REGULATOR.		33%	89	151
  						100%	204	290
  HMVDT89	831127	2011	WUblastx.64	(Q9HAT2) SIALIC ACID-SPECIFIC	Q9HAT2	90%	2	385
  				ACETYLESTERASE II.		98%	384	809
  HMVDT89	875313	2012	WUblastx.64	(Q9HAT2) SIALIC ACID-SPECIFIC	Q9HAT2	90%	2	385
  				ACETYLESTERASE II.		98%	384	809
  HMWAO65	839268	2013	WUblastx.64	(Q9P0A1) HSPC280 (FRAGMENT).	Q9P0A1	100%	30	371
  HMWAO82	825427	2014	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	84%	461	366
  				CLONE KAT08285.
  HMWBK35	566888	2016	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	76%	956	918
  				CLONE KAT08285.		64%	919	650
  HMWBL38	602940	2018	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	76%	743	615
  				CLONE KAT08285.		65%	916	719
  HMWBM48	566828	2019	WUblastx.64	ribosomal protein L3 precursor,	pir|A27294|R5HUL3	80%	609	379
  				mitochondrial - human		100%	794	609
  HMWCG28	847413	2020	WUblastx.64	(Q9P1S9) KINASE DEFICIENT	Q9P1S9	84%	35	892
  				PROTEIN KDP (FRAGMENT).
  HMWCP85	757974	2021	WUblastx.64	Na+-dependent vitamin C (L-ascorbic	pir|JC7182|JC7182	64%	37	297
  				acid) transporter SVCT1 - human		79%	1181	1507
  						61%	698	1264
  						86%	369	737
  HMWDG30	623758	2022	WUblastx.64	(Q9VQ76) CG4263 PROTEIN.	Q9VQ76	43%	1364	1411
  						37%	30	668
  HMWDU20	695743	2023	WUblastx.64	(Q9NXB8) CDNA FLJ20335 FIS,	Q9NXB8	89%	918	1034
  				CLONE HEP11429 (FRAGMENT).		75%	347	454
  						93%	1004	1819
  HMWDZ63	608146	2025	WUblastx.64	(P41565) ISOCITRATE	IDHG_RAT	33%	399	301
  				DEHYDROGENASE [NAD]		100%	169	74
  				SUBUNIT GAMMA, MITO
  HMWEA77	839221	2026	WUblastx.64	(Q9BGX7) HYPOTHETICAL 13.0 KDA	Q9BGX7	60%	1066	1110
  				PROTEIN.		70%	1100	1282
  HMWEC03	598902	2027	WUblastx.64	(Q9P0X2) HEAT SHOCK PROTEIN	Q9P0X2	100%	1066	1365
  				HSP60.		91%	573	1064
  						99%	105	572
  HMWEF46	825433	2028	WUblastx.64	(Q9H0G8) HYPOTHETICAL 14.0 KDA	Q9H0G8	100%	1113	1226
  				PROTEIN.
  HMWEK43	847414	2029	WUblastx.64	(Q9H8K0) CDNA FLJ13520 FIS,	Q9H8K0	46%	940	1167
  				CLONE PLACE1005828.
  HMWEM23	863105	2030	WUblastx.64	(Q96QF0) SSX2 interacting protein	Q96QF0	96%	1086	1532
  				hRabin3A, isoform alpha2.		96%	128	1114
  HMWEM23	859387	2031	WUblastx.64	(Q9H673) CDNA: FLJ22548 FIS,	Q9H673	95%	1086	1532
  				CLONE HSI00519.		96%	152	1114
  HMWEU96	1310888	2033	WUblastx.64	(Q9UH62) HYPOTHETICAL 42.5 KDA	Q9UH62	88%	91	1227
  				PROTEIN (ALEX3) (ALEX3
  				PROTEIN).
  HMWEU96	601698	3110	WUblastx.64	(AB039669) ALEX3 [Homo sapiens]	dbj|BAA94602.1|	88%	86	1222
  HMWEX02	596821	2034	WUblastx.64	(Q9H7H4) FLJ00116 PROTEIN	Q9H7H4	67%	509	1000
  				(FRAGMENT).
  HMWFB65	638102	2035	WUblastx.64	(O95427) MCD4P HOMOLOG.	O95427	74%	37	591
  						95%	894	959
  						100%	611	898
  						68%	9	59
  HMWFD77	610105	2036	WUblastx.64	(Q9P1N7) PRO0974.	Q9P1N7	83%	459	494
  						55%	293	433
  HMWFO25	566855	2037	WUblastx.64	(Q9HA89) CDNA FLJ12060 FIS,	Q9HA89	100%	742	816
  				CLONE HEMBB1002142.
  HMWGM41	847415	2039	WUblastx.64	(Q9H8P0) CDNA FLJ13352 FIS,	Q9H8P0	84%	33	986
  				CLONE OVARC1002165, WEAKLY
  				SIMILAR TO 3-O
  HMWGO95	582078	2040	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	82%	1446	1396
  				CLONE COL04765.		68%	1390	1145
  HMWGV85	847416	2041	WUblastx.64	(Q9H8H3) CDNA FLJ13631 FIS,	Q9H8H3	100%	41	298
  				CLONE PLACE1011090, HIGHLY
  				SIMILAR TO HOM
  HMWGZ42	824259	2042	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	50%	262	309
  				FOR MGC: 5149).		42%	1316	1534
  HMWIQ26	722234	2045	WUblastx.64	(Q9NGC3) CENTAURIN GAMMA	Q9NGC3	48%	844	395
  				1A PROTEIN.		47%	298	5
  						43%	221	162
  						21%	388	218
  HMWIU49	800894	2046	WUblastx.64	(O95831) PROGRAMED CELL	PCD8_HUMAN	98%	3	407
  				DEATH PROTEIN 8,		38%	1384	1500
  				MITOCHONDRIAL PREC		94%	379	1362
  HMWJJ62	838612	2047	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	71%	657	875
  				PROTEIN.
  HMWJJ64	581071	2048	WUblastx.64	(Q9D0Q7) 2600005P05RIK	Q9D0Q7	78%	12	878
  				PROTEIN.
  HNAAD76	596801	2049	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	65%	1650	1528
  				PRODUCT.		59%	1807	1646
  HNALD94	844722	2051	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	70%	764	817
  				CLONE KAIA0536.		72%	816	926
  HNALE44	785045	2052	WUblastx.64	(Q9Y503) FILAMIN, MUSCLE	Q9Y503	100%	12	86
  				ISOFORM.		30%	916	1170
  						72%	12	86
  						62%	12	83
  						52%	12	62
  						31%	783	896
  						60%	18	86
  						51%	12	95
  						45%	12	77
  						36%	18	92
  						36%	9	95
  						95%	79	1170
  						31%	916	1170
  						28%	49	795
  						36%	1096	1170
  						33%	1127	1207
  						35%	901	1170
  						31%	112	801
  						30%	1823	1927
  						29%	925	1170
  						39%	916	1170
  						30%	916	1170
  						27%	217	504
  						29%	18	98
  						46%	15	86
  						29%	910	1170
  						30%	901	1170
  						34%	79	792
  						42%	79	798
  						25%	940	1170
  						30%	79	792
  						32%	916	1170
  						38%	340	864
  						29%	88	855
  						30%	88	792
  						32%	109	795
  						33%	16	798
  						26%	103	855
  						29%	916	1170
  						25%	925	1170
  						33%	85	792
  						30%	262	795
  						29%	109	816
  						34%	916	1164
  						32%	520	792
  						26%	751	1170
  						29%	430	792
  						33%	940	1170
  						36%	12	77
  						31%	82	792
  						34%	79	783
  HNEAK38	631279	2056	WUblastx.64	(Q26195) PVA1 GENE.	Q26195	71%	1204	1100
  						47%	1383	1333
  						59%	1358	1197
  HNEAK65	604991	2057	WUblastx.64	(Q9P195) PRO1722.	Q9P195	72%	570	427
  						82%	715	560
  HNEBY79	841769	2059	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	58%	944	1153
  						72%	1270	1488
  HNECL75	782493	2061	WUblastx.64	(Q9HCX0) PELLINO.	Q9HCX0	96%	215	649
  						100%	627	1268
  HNECX90	834776	2062	WUblastx.64	(Q9UJL8) HYPOTHETICAL 43.5 KDA	Q9UJL8	84%	81	1265
  				PROTEIN.
  HNECX90	881133	2063	WUblastx.64	(Q9UJL8) HYPOTHETICAL 43.5 KDA	Q9UJL8	84%	81	1265
  				PROTEIN.
  HNEDP75	581072	2065	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	63%	910	722
  				CLONE KAIA0536.
  HNEDQ02	846061	2066	WUblastx.64	(Q9HA25) CDNA FLJ12374 FIS,	Q9HA25	76%	941	1063
  				CLONE MAMMA1002470,		93%	1018	2013
  				WEAKLY SIMILAR TO PRO
  HNEDU46	695745	2067	WUblastx.64	(Q9H3P1) 6-PHOSPHOFRUCTO-2-	Q9H3P1	100%	343	420
  				KINASE HEART ISOFORM.		95%	137	208
  						95%	766	825
  HNFAD50	839369	2068	WUblastx.64	(Q9H175) HYPOTHETICAL 59.6 KDA	Q9H175	47%	128	859
  				PROTEIN.
  HNFAD50	843242	2069	WUblastx.64	(Q9H175) HYPOTHETICAL 59.6 KDA	Q9H175	54%	50	379
  				PROTEIN.
  HNFAG67	804546	2070	WUblastx.64	(O60759) CYTOHESIN BINDING	O60759	73%	327	857
  				PROTEIN HE.
  HNFCJ77	886185	2071	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	74%	2342	2250
  				FOR MGC: 5149).		63%	1819	1763
  						73%	1763	1719
  						61%	2034	1792
  HNFCY57	877653	2073	WUblastx.64	(AAL12497) Cryopyrin.	AAL12497	91%	8	2203
  HNFDL89	823362	2074	WUblastx.64	(Q9BT07) UNKNOWN (PROTEIN	Q9BT07	46%	2098	2193
  				FOR MGC: 4033).		100%	1964	2101
  HNFDU92	602933	2076	WUblastx.64	(O60448) NEURONAL THREAD	O60448	60%	703	407
  				PROTEIN AD7C-NTP.		48%	721	425
  						88%	435	409
  						74%	570	388
  						56%	764	627
  						56%	522	388
  						43%	571	503
  						43%	495	388
  						57%	745	683
  						48%	757	677
  HNFDY09	843300	2077	WUblastx.64	(Q9BZ41) BA476I15.3 (NOVEL	Q9BZ41	92%	1243	1281
  				PROTEIN SIMILAR TO SEPTIN)		75%	1280	1363
  				(FRAGMENT).		53%	1104	1238
  HNFDY31	724955	2078	WUblastx.64	(O00571) DEAD-BOX PROTEIN 3	DDX3_HUMAN	64%	169	621
  				(HELICASE-LIKE PROTEIN 2)		98%	693	1028
  				(HLP2
  HNFEA17	753242	2079	WUblastx.64	(Q9BRG9) UNKNOWN (PROTEIN	Q9BRG9	94%	965	1318
  				FOR MGC: 11314).
  HNFEP55	722236	2080	WUblastx.64	(Q9NW80) HYPOTHETICAL 73.6 KDA	Q9NW80	41%	877	966
  				PROTEIN.		65%	158	862
  HNFET12	824170	2081	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	75%	1217	1182
  				CLONE COL03536.		75%	1182	952
  HNFFR59	634649	2082	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	65%	1613	1485
  				CLONE MAMMA1000472.
  HNFGR15	844044	2084	WUblastx.64	(Q99770) HYPOTHETICAL 15.4 KDA	Q99770	53%	1128	1172
  				PROTEIN.		90%	824	886
  HNFGW53	589525	2086	WUblastx.64	(Q9NUM6) CDNA FLJ11267 FIS,	Q9NUM6	53%	777	938
  				CLONE PLACE1009174.
  HNFHV68	567445	2089	WUblastx.64	pro-pol-dUTPase polyprotein - murine	pir|T29097|T29097	44%	380	24
  				endogenous retrovirus ERV-L
  				(fragment)
  HNFIE15	898153	2090	WUblastx.64	(Q9H387) PRO2550.	Q9H387	72%	2374	2300
  						62%	2576	2370
  HNFIE29	852233	2091	WUblastx.64	(Q9HBS7) HYPOTHETICAL 14.2 KDA	Q9HBS7	42%	1484	1407
  				PROTEIN.		54%	1419	1168
  HNFJE27	825400	2093	WUblastx.64	(Q9NUQ9) CDNA FLJ11197 FIS,	Q9NUQ9	93%	986	1123
  				CLONE PLACE1007690
  				(HYPOTHETICAL 36.7 KDA
  HNGAC71	695746	2095	WUblastx.64	(Q9GKW4) HYPOTHETICAL 12.6 KDA	Q9GKW4	77%	64	11
  				PROTEIN.		85%	138	79
  HNGAX06	841890	2099	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	100%	22	108
  				PROTEIN (FRAGMENT).
  HNGBE44	825391	2103	WUblastx.64	(O00437) AXONEMAL DYNEIN	O00437	71%	323	502
  				HEAVY CHAIN (FRAGMENT).
  HNGBI83	566815	2105	WUblastx.64	(O60448) NEURONAL THREAD	O60448	69%	322	420
  				PROTEIN AD7C-NTP.		53%	122	160
  						73%	331	420
  						74%	13	87
  						59%	10	87
  						51%	12	104
  						55%	22	102
  						61%	42	80
  						55%	28	81
  HNGCF29	580201	2111	WUblastx.64	(Q9H387) PRO2550.	Q9H387	71%	1112	1074
  						60%	1069	842
  HNGCF64	823147	2112	WUblastx.64	(AAH07558) Unknown (protein for	AAH07558	48%	1893	1765
  				MGC: 15483).		55%	1763	1629
  HNGDF54	702002	2113	WUblastx.64	(Q9N8T2) POSSIBLE SRPA.	Q9N8T2	29%	13	654
  						27%	14	679
  						27%	14	679
  						29%	13	654
  HNGDH27	581074	2115	WUblastx.64	(Q92722) RETINOBLASTOMA 1.	Q92722	45%	290	99
  HNGDN07	847081	2116	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	80%	1091	966
  HNGEE06	689506	2122	WUblastx.64	(Q9H960) CDNA FLJ12988 FIS,	Q9H960	43%	66	1
  				CLONE NT2RP3000080.		60%	1040	909
  HNGEN32	621308	2128	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	76%	361	399
  				CLONE LNG09295.		80%	408	557
  HNGET33	825378	2131	WUblastx.64	(Q9H387) PRO2550.	Q9H387	78%	459	755
  HNGEZ02	825376	2134	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	81%	840	938
  				CLONE KAIA0536.
  HNGEZ90	634858	2135	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	72%	1119	1045
  				CLONE COL04765.		71%	956	810
  HNGFD30	612782	2138	WUblastx.64	(O75423)	O75423	72%	264	100
  				ORF3, SPLICEVARIANT_A.
  HNGFD31	663532	2139	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	76%	574	624
  				CLONE MAMMA1000472.		78%	491	574
  HNGFG04	772548	2141	WUblastx.64	(Q9Y485) X-LIKE 1 PROTEIN.	Q9Y485	100%	423	533
  HNGFI21	581075	2145	WUblastx.64	(Q9EQC8) PAPILLARY RENAL	Q9EQC8	58%	117	296
  				CELL CARCINOMA-ASSOCIATED
  				PROTEIN.
  HNGFT70	581079	2150	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	76%	1073	855
  				CLONE LNG09295.
  HNGGF13	638117	2153	WUblastx.64	hypothetical protein hc1 - mouse	pir|S26689|S26689	38%	13	183
  				(fragment)		37%	13	186
  HNGGK63	822862	2154	WUblastx.64	(Q9QZ22) OLFACTORY	Q9QZ22	59%	1224	1003
  				RECEPTOR.		88%	985	278
  HNGGT10	866179	2159	HMMER	PFAM: 3-beta hydroxysteroid	PF01073	263.9	19	468
  			2.1.1	dehydrogenase/isomerase family
  			WUblastx.64	(Q9BSN9) 3 BETA-HYDROXY-	Q9BSN9	75%	10	480
  				DELTA 5-C27-STEROID
  				OXIDOREDUCTASE.
  HNGHB89	722238	2161	WUblastx.64	(O88748) E-STOP PROTEIN.	O88748	100%	153	37
  HNGHD07	608149	2162	WUblastx.64	(Q9BRA2) UNKNOWN (PROTEIN	Q9BRA2	51%	588	755
  				FOR MGC: 14353).		70%	746	943
  HNGIK07	825360	2168	WUblastx.64	(Q9H3I6) BRAIN MY040 PROTEIN.	Q9H3I6	76%	1139	912
  HNGIM40	747699	2169	WUblastx.64	(Q9H5W7) CDNA: FLJ22921 FIS,	Q9H5W7	87%	2354	2208
  				CLONE KAT06711.
  HNGIM83	751714	2170	HMMER	PFAM: HCO3-transporter family	PF00955	117.9	72	290
  			2.1.1
  			WUblastx.64	(Q9UIB9) BICARBONATE	Q9UIB9	74%	9	290
  				TRANSPORTER.
  HNGIO93	897527	2171	HMMER	PFAM: Ribosomal L27 protein	PF01016	71.7	−21	−233
  			2.1.1
  			WUblastx.64	(Q9P0M9) HSPC250	Q9P0M9	88%	151	594
  				(MITOCHONDRIAL RIBOSOMAL
  				PROTEIN L27) (L27MT) (HYPOT
  HNGIU16	826717	2173	WUblastx.64	(Q9H387) PRO2550.	Q9H387	78%	2322	2215
  						76%	2505	2467
  						78%	2467	2315
  HNGIX91	564574	2174	WUblastx.64	(AAH07609) Similar to hypothetical	AAH07609	100%	956	982
  				protein PRO1722.		77%	885	950
  						76%	739	891
  HNGJU60	825336	2182	WUblastx.64	(Q9GW02) EXTREMELY	Q9GW02	51%	166	258
  				CYSTEINE/VALINE RICH		50%	166	261
  				PROTEIN (FRAGMENT).		46%	166	261
  						56%	169	258
  						48%	169	261
  						45%	169	261
  						48%	169	261
  						40%	290	487
  						44%	287	487
  						41%	290	487
  						41%	169	261
  						44%	287	490
  						46%	169	258
  						55%	231	257
  						44%	290	487
  						45%	287	487
  						42%	287	487
  						44%	290	487
  						44%	290	490
  						48%	163	261
  						46%	166	261
  						48%	169	261
  						48%	163	261
  						44%	287	487
  						40%	172	261
  						45%	169	261
  						43%	287	487
  						48%	169	261
  HNGKW35	899408	2184	WUblastx.64	(O00627) HYPOTHETICAL 11.0 KDA	O00627	53%	292	477
  				PROTEIN (ORF2).
  HNGKY94	835021	2185	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	66%	581	501
  				CLONE COL04765.		69%	777	583
  HNHAB38	609892	2188	WUblastx.64	(Q9H387) PRO2550.	Q9H387	68%	635	339
  HNHAD34	612845	2190	WUblastx.64	(Q9N032) UNNAMED PROTEIN	Q9N032	64%	1056	922
  				PRODUCT.
  HNHAY26	704789	2198	WUblastx.64	(Q9H397) PRO2852.	Q9H397	85%	2098	1919
  HNHAZ20	839696	2200	WUblastx.64	(Q9C074) HYPOTHETICAL 73.9 KDA	Q9C074	81%	1213	11
  				PROTEIN (FRAGMENT).
  HNHBE38	562730	2202	WUblastx.64	(AAK55521) PRO0764.	AAK55521	65%	1131	1072
  						72%	1097	855
  HNHBG18	604924	2203	WUblastx.64	(O00365) L1 ELEMENT L1.15 P40	O00365	29%	717	271
  				PROTEIN.
  HNHBM16	566866	2205	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	87%	80	33
  				CLONE COL03536.		71%	310	80
  HNHCH78	694657	2206	WUblastx.64	(AAH07609) Similar to hypothetical	AAH07609	100%	270	290
  				protein PRO1722.		88%	194	271
  						75%	49	195
  HNHCT47	634691	2210	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	46%	434	396
  				CLONE COL04765.		56%	621	448
  HNHDC52	812424	2213	WUblastx.64	(O00365) L1 ELEMENT L1.15 P40	O00365	32%	1038	910
  				PROTEIN.		68%	904	269
  HNHDI17	738043	2216	WUblastx.64	(O95662) POT. ORF VI	O95662	80%	265	327
  				(FRAGMENT).		56%	5	295
  HNHDR57	638121	2219	WUblastx.64	(O60448) NEURONAL THREAD	O60448	58%	1193	1095
  				PROTEIN AD7C-NTP.		63%	496	440
  						56%	485	393
  						50%	503	426
  						64%	1195	1124
  						33%	658	440
  						41%	1186	1043
  						40%	402	322
  						61%	1065	874
  						51%	1177	923
  						36%	486	331
  						31%	1090	455
  						53%	437	393
  						60%	1168	863
  HNHDW34	607867	2222	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	62%	1175	1017
  				CLONE COL04765.		56%	961	671
  HNHEF70	610115	2228	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	80%	481	437
  				CLONE COL04765.		77%	417	352
  						50%	650	462
  HNHEL22	607421	2231	WUblastx.64	(Q9NUL2) CDNA FLJ11292 FIS,	Q9NUL2	71%	280	342
  				CLONE PLACE1009665.		72%	363	656
  HNHEN70	854712	2232	WUblastx.64	olfactory receptor OR18 - rat	pir|S29710|S29710	67%	34	567
  HNHEP41	638124	2234	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	41%	915	730
  				CLONE KAIA0536.		73%	736	635
  HNHEZ76	618544	2238	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	72%	359	466
  				CLONE MAMMA1000472.
  HNHFF81	732075	2240	WUblastx.64	(Q9GMP5) HYPOTHETICAL 6.6 KDA	Q9GMP5	71%	1208	1333
  				PROTEIN.
  HNHFR42	638127	2242	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	75%	522	427
  HNHGD95	609905	2244	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	46%	430	392
  				CLONE COL04765.		56%	608	444
  HNHGR82	617117	2245	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	48%	125	39
  				PROTEIN.		56%	262	110
  HNHGY77	638128	2247	WUblastx.64	(Q9BZV1) UBX DOMAIN-	Q9BZV1	53%	292	248
  				CONTAINING PROTEIN 1.		100%	212	90
  HNHHA47	658743	2248	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	65%	983	864
  				CLONE KAIA0536.		66%	1138	1001
  HNHKI74	777856	2252	WUblastx.64	(Q9BGX7) HYPOTHETICAL 13.0 KDA	Q9BGX7	64%	350	541
  				PROTEIN.
  HNHLD80	839255	2254	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	44%	563	414
  				CLONE MAMMA1000472.		72%	700	572
  HNHLS76	853372	2255	WUblastx.64	(O15410) CAGH45.	O15410	74%	146	316
  HNHMY76	838256	2259	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	75%	576	647
  				CLONE KAIA0536.		83%	520	555
  						75%	659	778
  HNKAA76	802009	2264	WUblastx.64	(Q9BTI4) SIMILAR TO RIKEN	Q9BTI4	84%	323	1492
  				CDNA 8430408O15 GENE.		60%	353	421
  HNTAF42	824082	2265	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	63%	380	673
  				CLONE KAT08285.
  HNTCG32	897776	2266	HMMER	PFAM: Sodium/hydrogen exchanger	PF00999	244.7	178	789
  			2.1.1	family
  			WUblastx.64	(AAK54508) Nonselective sodium	AAK54508	60%	13	837
  				potassium/proton exc		52%	772	1089
  HNTNY89	886188	2267	WUblastx.64	(Q9H400) DJ583P15.4.1 (NOVEL	Q9H400	90%	338	1006
  				PROTEIN (TRANSLATION OF		100%	158	187
  				CDNA FLJ20406 (E		68%	307	354
  HNTRQ40	809100	2269	WUblastx.64	(Q9UBC2) EPIDERMAL GROWTH	Q9UBC2	64%	1498	1857
  				FACTOR RECEPTOR SUBSTRATE		76%	11	451
  				EPS15R.		92%	492	1604
  						66%	1810	1845
  						57%	1567	1608
  						36%	32	178
  						40%	202	486
  						40%	1540	1623
  						48%	1567	1686
  						41%	1398	1544
  						33%	1395	1589
  						50%	1452	1544
  						34%	1377	1547
  						36%	495	695
  						35%	20	196
  						40%	519	623
  						23%	750	1610
  HOAAJ09	654862	2273	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	82%	560	510
  				FOR MGC: 5149).		77%	769	572
  HOAAL10	566867	2274	WUblastx.64	(Q9P1N7) PRO0974.	Q9P1N7	42%	1113	1217
  						58%	929	1129
  HOABC12	801922	2276	WUblastx.64	(Q13391) HYPOTHETICAL	Q13391	100%	208	405
  				PROTEIN 384D8_6.		97%	5	208
  HOABH36	740758	2277	WUblastx.64	(Q9BSY5) UNKNOWN (PROTEIN	Q9BSY5	94%	147	1073
  				FOR IMAGE: 3831362)		86%	1078	1353
  				(FRAGMENT).		83%	10	177
  HOBNA89	604999	2278	WUblastx.64	microtubule-associated	pir|A54602|A54602	51%	369	7
  				serine/threonine protein kinase
  				MAST205 - mouse
  HOBNF51	580861	2279	WUblastx.64	(O00410) IMPORTIN BETA-3	IMB3_HUMAN	95%	10	414
  				SUBUNIT (KARYOPHERIN BETA-3		20%	271	375
  				SUBUNI		99%	395	1051
  HODAH24	656887	2280	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	68%	1326	1189
  				FOR MGC: 5149).
  HODAH46	862551	2281	WUblastx.64	(Q9H6Y7) CDNA: FLJ21676 FIS,	Q9H6Y7	82%	264	608
  				CLONE COL09164.
  HODAV25	787471	2282	WUblastx.64	(Q9UHS9) PRO1914 PROTEIN.	Q9UHS9	100%	72	1
  HODAW64	840069	2283	WUblastx.64	(Q9H7T2) CDNA FLJ14295 FIS,	Q9H7T2	93%	634	1263
  				CLONE PLACE1008426, WEAKLY
  				SIMILAR TO RES
  HODAY17	806341	2284	WUblastx.64	(Q9H7L0) FLJ00062 PROTEIN	Q9H7L0	90%	492	1478
  				(FRAGMENT).		100%	77	487
  HODBF86	605001	2289	WUblastx.64	(Q9BRM8) UNKNOWN (PROTEIN	Q9BRM8	43%	189	410
  				FOR MGC: 13219).
  HODDJ25	824592	2299	WUblastx.64	(Q9C0K7) AMYOTROPHIC	Q9C0K7	100%	266	1519
  				LATERAL SCLEROSIS 2.
  HODDQ06	834824	2302	WUblastx.64	(Q9NZK6) PDZ-BINDING KINASE.	Q9NZK6	94%	200	1165
  HODEA20	840376	2303	WUblastx.64	(Q9BXY9) RALBP1.	Q9BXY9	84%	141	1229
  						68%	6	179
  HOEBI94	795312	2306	WUblastx.64	(Q9Y4J5) RIBONUCLEOPROTEIN	Q9Y4J5	74%	823	999
  				(HNRNP 2H9).		100%	695	820
  						33%	710	820
  						100%	167	469
  HOEBJ70	836143	2307	WUblastx.64	(AAH09229) Unknown (protein for	AAH09229	100%	560	646
  				MGC: 16480).
  HOECB33	840378	2308	WUblastx.64	(Q9H6D8) CDNA: FLJ22362 FIS,	Q9H6D8	100%	10	525
  				CLONE HRC06544.
  HOECX21	842866	2309	WUblastx.64	(Q9HC05) CD003 PROTEIN.	Q9HC05	100%	368	225
  HOEDE27	825262	2310	WUblastx.64	(Q9P195) PRO1722.	Q9P195	55%	914	759
  						67%	780	649
  HOEEK81	844390	2311	WUblastx.64	(BAB22091) Adult male kidney	BAB22091	60%	51	596
  				cDNA, RIKEN full-lengt
  HOEEZ62	638833	2312	WUblastx.64	(Q9T9V8) NADH	Q9T9V8	88%	66	173
  				DEHYDROGENASE SUBUNIT 3.		83%	303	338
  HOEFJ26	847422	2313	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	77%	1198	995
  				CLONE LNG09295.
  HOFMF63	847423	2315	WUblastx.64	L6 surface protein - human	pir|A42926|A42926	82%	409	690
  						77%	86	409
  HOFMJ65	664498	2316	WUblastx.64	(P70560) COLLAGEN ALPHA 1(XII)	CA1C_RAT	74%	987	1160
  				CHAIN (FRAGMENT).
  HOFMO16	596835	2318	HMMER	PFAM: 3-beta hydroxysteroid	PF01073	68.3	149	346
  			2.1.1	dehydrogenase/isomerase family
  			WUblastx.64	(Q9BSN9) 3 BETA-HYDROXY-	Q9BSN9	75%	11	184
  				DELTA 5-C27-STEROID		83%	349	438
  				OXIDOREDUCTASE.		94%	290	346
  HOFMV22	812864	2321	WUblastx.64	(Q9P039) HSPC113.	Q9P039	60%	180	293
  						50%	4	198
  HOFNY15	668259	2323	WUblastx.64	(Q9H9J2) CDNA FLJ12701 FIS,	Q9H9J2	73%	69	326
  				CLONE NT2RP1000730.		83%	331	999
  HOFNY28	603911	2324	WUblastx.64	interferon gamma receptor accessory	pir|I38500|I38500	59%	13	654
  				factor-1 precursor - human
  HOGAA41	843499	2326	WUblastx.64	(Q9D3B1) 6330408J20RIK	Q9D3B1	96%	580	1257
  				PROTEIN.
  HOGAB51	825278	2327	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	74%	919	1119
  				CLONE LNG09295.
  HOGAH40	790978	2328	WUblastx.64	(Q9NX63) CDNA FLJ20420 FIS,	Q9NX63	93%	185	844
  				CLONE KAT02462.
  HOGAP06	823364	2329	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	70%	1445	1669
  				PROTEIN.
  HOGAR71	722242	2331	HMMER	PFAM: PMP-22/EMP/MP20/Claudin	PF00822	336	96	632
  			2.1.1	family
  			WUblastx.64	(O95471) CLAUDIN-7.	CLD7_HUMAN	100%	87	719
  HOGCC26	834792	2332	WUblastx.64	(Q9HCN4) XPA BINDING PROTEIN	Q9HCN4	87%	19	1140
  				1.
  HOGCD78	610217	2333	WUblastx.64	(Q9H6Q7) CDNA: FLJ21979 FIS,	Q9H6Q7	95%	428	487
  				CLONE HEP06065 (FRAGMENT).		100%	217	321
  HOGCK03	847427	2334	WUblastx.64	(Q99LE1) UNKNOWN (PROTEIN	Q99LE1	53%	310	405
  				FOR MGC: 7036).		78%	369	860
  HOGCL01	846353	2335	HMMER	PFAM: KOW motif	PF00467	40.2	332	442
  			2.1.1
  			WUblastx.64	(Q96A35) Similar to mitochondrial	Q96A35	100%	167	814
  				ribosomal protein L24.
  HOHBB36	847428	2336	WUblastx.64	myosin I alpha chain - mouse	pir|A45438|A45438	90%	2093	3
  						93%	2237	2103
  HOHBC57	813392	2337	WUblastx.64	(Q9VEL9) CG4090 PROTEIN.	Q9VEL9	21%	2162	1767
  						22%	4081	3632
  						31%	1961	1836
  						18%	2582	1737
  						17%	4772	4509
  						20%	1793	1374
  						20%	2582	2376
  						26%	1897	1652
  						21%	3583	2564
  						22%	3550	2534
  						21%	2087	1809
  						24%	1970	1704
  						22%	4084	2534
  						38%	575	498
  						20%	2021	1773
  						21%	3556	2534
  HOHBO66	853375	2338	WUblastx.64	(Q9NY61) DED PROTEIN	Q9NY61	94%	1322	828
  				(APOPTOSIS ANTAGONIZING		88%	844	212
  				TRANSCRIPTION FACTOR).
  HONAH67	821419	2342	WUblastx.64	(Q9H7T6) CDNA FLJ14272 FIS,	Q9H7T6	97%	1192	1338
  				CLONE PLACE1004793, WEAKLY		96%	513	1172
  				SIMILAR TO RET
  HOOAC84	604043	2343	WUblastx.64	(Q9UPN3) ACTIN CROSS-LINKING	ACF7_HUMAN	84%	1324	1482
  				FAMILY PROTEIN 7		100%	325	444
  				(MACROPHIN) (		100%	592	690
  						29%	1381	1461
  						36%	1387	1461
  HOPBP13	825243	2344	WUblastx.64	(Q9P195) PRO1722.	Q9P195	63%	2105	1851
  HORBI80	877660	2346	WUblastx.64	(Q9NY33) DIPEPTIDYL-	DPP3_HUMAN	100%	30	92
  				PEPTIDASE III (EC 3.4.14.4) (DPP		99%	92	2239
  				III) (
  HORBL77	852099	2347	WUblastx.64	(Q9CWX4) 2410001E19RIK	Q9CWX4	27%	852	1034
  				PROTEIN.		31%	212	784
  HOSEM81	846354	2350	WUblastx.64	(O75872) RAB3-GAP	O75872	100%	1012	803
  				REGULATORY DOMAIN.
  HOSEO83	847083	2351	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	68%	356	412
  				CLONE KAIA0536.		73%	310	354
  						53%	551	769
  HOSFR35	845994	2352	WUblastx.64	(Q9NXV7) CDNA FLJ20035 FIS,	Q9NXV7	63%	9	320
  				CLONE COL00213.		67%	316	780
  HOUBC29	872565	2354	WUblastx.64	GTP-binding protein rab2 - rat	pir|B39963|B39963	94%	763	876
  HOUBG39	601696	2355	WUblastx.64	(Q9H6G8) CDNA: FLJ22294 FIS,	Q9H6G8	79%	1603	1403
  				CLONE HRC04426.
  HOUCD12	605006	2356	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	69%	250	555
  				CLONE COL04765.
  HOUDB17	603417	2357	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	66%	672	367
  				PRODUCT.
  HOUIG68	793749	2363	WUblastx.64	(Q9NVC9) CDNA FLJ10808 FIS,	Q9NVC9	100%	2	355
  				CLONE NT2RP4000879, WEAKLY
  				SIMILAR TO UBI
  HOVAJ68	741098	2369	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	55%	1588	1421
  				CLONE MAMMA1000472.
  HOVAW46	892162	2370	WUblastx.64	(O00363) PUTATIVE P150.	O00363	41%	395	345
  						39%	1030	773
  						37%	1359	1009
  						71%	759	697
  						45%	697	359
  HOVBB19	843768	2371	WUblastx.64	catalase (EC 1.11.1.6) -	pir|I40767|I40767	97%	307	197
  				Campylobacter jejuni
  HOVBI16	581097	2374	WUblastx.64	(AAG50170) Tripartite motif protein	AAG50170	63%	83	115
  				TRIM28 alpha.		83%	202	702
  HOVCO53	564632	2380	WUblastx.64	(Q9H8N2) CDNA FLJ13381 FIS,	Q9H8N2	52%	1321	1127
  				CLONE PLACE1001010.
  HPBDE33	853660	2384	WUblastx.64	hypothetical protein	pir|T12458|T12458	64%	257	985
  				DKFZp564O0823.1 - human
  HPBDE33	897372	2385	WUblastx.64	hypothetical protein	pir|T12458|T12458	65%	257	985
  				DKFZp564O0823.1 - human
  HPCAG17	757836	2387	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	64%	1306	1013
  				CLONE COL03536.
  HPCAG17	863943	2388	WUblastx.64	(Q9H387) PRO2550.	Q9H387	71%	1141	1007
  						71%	1008	850
  HPDDQ28	566793	2391	WUblastx.64	(O95357) PUTATIVE G PROTEIN-	O95357	98%	231	440
  				COUPLED RECEPTOR (CDNA		100%	10	231
  				FLJ10899 FIS, CLON
  HPDDT14	580867	2392	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	71%	193	5
  				PROTEIN.
  HPFCP75	581083	2399	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	53%	656	949
  				CLONE KAT08285.
  HPFDB66	829318	2400	WUblastx.64	(Q9NTM9) BA483F11.3 (CGI-32	Q9NTM9	100%	102	920
  				PROTEIN).
  HPFDD28	824856	2401	WUblastx.64	(Q9P195) PRO1722.	Q9P195	66%	672	628
  						70%	622	551
  						69%	815	660
  HPIAK27	852658	2404	WUblastx.64	(AAH06621) RIKEN cDNA	AAH06621	83%	16	1050
  				2810403A07 gene.		70%	1286	1864
  						95%	1162	1293
  						40%	1162	1290
  						34%	1201	1269
  						44%	1174	1245
  HPIAL55	847429	2405	WUblastx.64	(Q9D0W4) 1110060O18RIK	Q9D0W4	96%	506	592
  				PROTEIN.		90%	1088	1150
  						72%	694	1002
  						100%	82	231
  						90%	1778	1807
  HPIAT18	877663	2406	WUblastx.64	(Q9H397) PRO2852.	Q9H397	84%	3823	3728
  						74%	3735	3571
  HPIAZ52	801924	2407	WUblastx.64	(Q9H387) PRO2550.	Q9H387	68%	2082	1987
  						67%	1988	1833
  HPIBA07	886191	2408	WUblastx.64	(Q99ML9) ARKADIA.	Q99ML9	73%	61	1482
  HPIBA24	840379	2409	WUblastx.64	(Q9ULZ5) GONADOTROPIN	Q9ULZ5	50%	27	1865
  				INDUCIBLE TRANSCRIPTION		56%	378	1793
  				REPRESSOR-4.		57%	420	1805
  						53%	549	1868
  						53%	330	1544
  HPIBI40	588944	2410	WUblastx.64	(O43264)	ZW10_HUMAN	99%	1	339
  				CENTROMERE/KINETOCHORE		98%	336	860
  				PROTEIN ZW10 HOMOLOG.
  HPJAB75	841653	2413	WUblastx.64	(Q9BZ63) FKSG60.	Q9BZ63	80%	1581	1273
  HPJAN76	826185	2414	WUblastx.64	(O43194) PUTATIVE G PROTEIN-	GP39_HUMAN	89%	328	828
  				COUPLED RECEPTOR GPR39.
  HPJAN76	854893	2415	WUblastx.64	(O43194) PUTATIVE G PROTEIN-	GP39_HUMAN	89%	328	828
  				COUPLED RECEPTOR GPR39.
  HPJAU94	826719	2416	WUblastx.64	(O00371) L1 ELEMENT L1.21 P40	O00371	83%	3944	4159
  				PROTEIN.		56%	3529	3921
  HPJAW78	812766	2417	WUblastx.64	(Q9D1W2) C030013D06RIK	Q9D1W2	44%	87	254
  				PROTEIN.		38%	96	266
  						40%	77	262
  						45%	87	260
  						47%	86	256
  						43%	243	85
  						40%	259	86
  						39%	249	67
  						41%	261	88
  HPJBS16	608307	2418	WUblastx.64	(CAC39435) Epigen protein precursor.	CAC39435	64%	31	357
  HPJBU04	798101	2419	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	72%	830	567
  				CLONE COL03536.		47%	2315	2265
  HPJCP75	886192	2421	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	61%	1459	1704
  				PROTEIN.
  HPJCV35	827317	2422	WUblastx.64	(Q9Y5S2) CDC42-BINDING	Q9Y5S2	92%	2685	2951
  				PROTEIN KINASE BETA.		100%	533	610
  HPJCX13	852869	2423	HMMER	PFAM: Reverse transcriptase (RNA-	PF00078	307.8	3321	4148
  			2.1.1	dependent DNA polymerase)
  			WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	96%	4105	266
  				human
  HPMBT05	658715	2427	WUblastx.64	(Q14287) HYPOTHETICAL	Q14287	73%	491	592
  				PROTEIN (FRAGMENT).		85%	403	483
  HPMDD27	830748	2432	WUblastx.64	(Q9NZX0) HSPC068.	Q9NZX0	96%	2	1417
  HPMDF45	638148	2433	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	67%	1678	1857
  				CLONE KAIA0536.		72%	1856	1987
  HPMDP57	567303	2434	WUblastx.64	(Q9H387) PRO2550.	Q9H387	64%	1286	1074
  						77%	1447	1211
  HPMEG72	795709	2435	WUblastx.64	(O00549) ORF2-LIKE PROTEIN	O00549	55%	1844	2056
  				(FRAGMENT).
  HPMFM70	756931	2436	WUblastx.64	(Q9H387) PRO2550.	Q9H387	81%	2108	1998
  						61%	2286	2248
  						68%	2248	2105
  HPMFP48	597457	2437	WUblastx.64	(Q9P195) PRO1722.	Q9P195	66%	987	853
  						69%	1130	975
  HPMFW01	844865	2438	WUblastx.64	(Q9NP48) PUTATIVE LIPID	Q9NP48	97%	973	1113
  				KINASE (CDNA FLJ10842 FIS,
  				CLONE NT2RP4001343
  HPMGW43	830452	2440	WUblastx.64	vesicle-associated membrane protein-	pir|JG0186|JG0186	100%	31	72
  				associated protein B - human		95%	59	427
  HPMKB09	900362	2442	WUblastx.64	(Q9UJU6) SRC HOMOLOGY 3	Q9UJU6	92%	798	1406
  				DOMAIN-CONTAINING PROTEIN		92%	117	656
  				HIP-55 (DREBRIN F).
  HPMSH26	780109	2443	WUblastx.64	(Q9NQC3) NOGO-A PROTEIN.	Q9NQC3	69%	1592	1014
  						70%	1748	1578
  HPMSH96	610023	2444	WUblastx.64	(AAH00127) Glutathione-S-	AAH00127	88%	1203	1505
  				transferase like, glutathi
  HPQAJ27	782957	2446	WUblastx.64	peptidyl-prolyl cis-trans isomerase	pir|B81216|B81216	54%	1370	987
  				NMB0281 [imported] - Neisseria		77%	973	707
  				meningitidis (strain MC58 serogroup
  				B)
  HPQAN50	788813	2447	WUblastx.64	(O00549) ORF2-LIKE PROTEIN	O00549	35%	381	10
  				(FRAGMENT).		32%	1151	222
  						38%	1285	590
  HPRAD30	805969	2458	WUblastx.64	(Q9P1C6) PRO2738.	Q9P1C6	52%	2465	2265
  HPRCC91	638151	2459	WUblastx.64	(Q9H1R7) BA534G20.4	Q9H1R7	100%	2	97
  				(SUPERVILLIN) (FRAGMENT).
  HPRCF40	834808	2460	WUblastx.64	(AAH07482) Similar to conserved	AAH07482	97%	3	221
  				membrane protein at
  HPRCF50	638153	2461	WUblastx.64	(Q9H387) PRO2550.	Q9H387	66%	1787	1912
  						58%	1614	1790
  HPRCM72	813512	2463	WUblastx.64	(Q9D3K9) 2810468K05RIK	Q9D3K9	45%	296	526
  				PROTEIN.
  HPRCS59	601523	2464	WUblastx.64	(Q61787) ORF 2.	Q61787	30%	746	189
  HPRCT73	610024	2465	WUblastx.64	(Q9NPZ5)	B3G2_HUMAN	96%	1231	1145
  				GALACTOSYLGALACTOSYLXYL		89%	1476	1300
  				OSYLPROTEIN 3-BETA-
  				GLUCURON
  HPTRE80	884167	2467	WUblastx.64	(O43819) SCO2 PROTEIN	SCO2_HUMAN	85%	779	39
  				HOMOLOG PRECURSOR.
  HPTRI42	655362	2468	WUblastx.64	(Q9BVA7) UNKNOWN (PROTEIN	Q9BVA7	85%	3	611
  				FOR MGC: 5621).
  HPTTT62	561954	2473	WUblastx.64	(Q9H3X5) HYPOTHETICAL 85.5 KDA	Q9H3X5	100%	8	169
  				PROTEIN (FRAGMENT).
  HPTVH24	831983	2474	WUblastx.64	(Q9H7Z7) CDNA FLJ14038 FIS,	Q9H7Z7	90%	3	974
  				CLONE HEMBA1005206.
  HPTVI96	636064	2477	WUblastx.64	(Q9H5X3) HYPOTHETICAL 13.8 KDA	Q9H5X3	77%	42	416
  				PROTEIN.
  HPVAA15	783074	2478	WUblastx.64	(AAK54355) ATP-binding cassette	AAK54355	40%	4	483
  				transporter family		33%	7	477
  						31%	487	870
  						30%	487	828
  HPWAS27	536008	2484	WUblastx.64	(Q9P0E3) HSPC093 (FRAGMENT).	Q9P0E3	40%	1042	1146
  						65%	512	625
  HPWAV82	830084	2486	WUblastx.64	(Q9H387) PRO2550.	Q9H387	79%	3	89
  						69%	104	229
  HPWBA36	840380	2487	WUblastx.64	(Q9H387) PRO2550.	Q9H387	54%	1183	1073
  						51%	1238	1152
  						62%	1380	1219
  HPWTF23	843700	2488	HMMER	PFAM: TSC-22/dip/bun family	PF01166	146.4	442	621
  			2.1.1
  			WUblastx.64	(Q99576) GLUCOCORTICOID-	GILZ_HUMAN	94%	271	672
  				INDUCED LEUCINE ZIPPER
  				PROTEIN (DEL
  HPWTF23	844775	2489	HMMER	PFAM: TSC-22/dip/bun family	PF01166	146.4	442	621
  			2.1.1
  			WUblastx.64	(Q99576) GLUCOCORTICOID-	GILZ_HUMAN	94%	271	672
  				INDUCED LEUCINE ZIPPER
  				PROTEIN (DEL
  HPWTF53	844737	2490	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	48%	1855	2082
  				PROTEIN.
  HRAAC36	798104	2494	WUblastx.64	(Q26195) PVA1 GENE.	Q26195	65%	835	758
  						62%	750	565
  						41%	2229	2119
  HRAAF59	847086	2495	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	56%	1519	1247
  				CLONE COL04765.
  HRAAG89	621271	2496	WUblastx.64	(Q9GMW5) HYPOTHETICAL 45.1 KDA	Q9GMW5	93%	2	187
  				PROTEIN.
  HRAAZ12	834637	2498	WUblastx.64	(AAG41897) Neuropilin-2a(17).	AAG41897	100%	1361	1435
  						92%	863	901
  						56%	385	579
  						96%	579	878
  						79%	901	1356
  HRABP28	823344	2500	WUblastx.64	(AAK55521) PRO0764.	AAK55521	44%	1136	975
  						44%	1245	1111
  HRABU56	621381	2501	WUblastx.64	(O75876) RNA-BINDING PROTEIN.	O75876	90%	105	320
  HRABZ80	562230	2502	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	64%	1166	1116
  				CLONE COL03536.		54%	1104	1000
  HRACB01	637647	2503	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	52%	1423	1349
  				PROTEIN.		52%	1344	1135
  HRACI39	840461	2504	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	59%	1346	1146
  				PRODUCT.
  HRADU15	801926	2505	WUblastx.64	(Q9H7S6) CDNA FLJ14310 FIS,	Q9H7S6	75%	645	586
  				CLONE PLACE3000271.		74%	787	638
  HRDAH04	651356	2506	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	38%	1336	1229
  				CLONE COL04765.		60%	1478	1329
  HRDBA20	637709	2507	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	80%	1113	1069
  				CLONE MAMMA1000472.		71%	1049	1008
  						79%	1267	1106
  HRDBD32	637710	2508	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	80%	1113	1069
  				CLONE MAMMA1000472.		71%	1049	1008
  						79%	1267	1106
  HRDBL01	631169	2509	WUblastx.64	(Q9P195) PRO1722.	Q9P195	65%	934	755
  HRDDM85	799542	2510	WUblastx.64	(O95662) POT. ORF VI	O95662	60%	1034	1096
  				(FRAGMENT).		56%	538	1077
  HRDEJ86	695755	2512	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	74%	827	627
  				CLONE KAIA0536.
  HRDFE30	750872	2514	WUblastx.64	(Q9NWU2) BA305P22.1.	Q9NWU2	89%	533	823
  						95%	141	548
  HRDFT83	606799	2515	WUblastx.64	(O60448) NEURONAL THREAD	O60448	60%	442	377
  				PROTEIN AD7C-NTP.		84%	584	528
  						60%	439	380
  						53%	578	459
  						61%	932	768
  						30%	778	641
  						68%	779	714
  						70%	942	769
  						58%	387	337
  						47%	948	892
  						42%	584	294
  						100%	691	668
  						62%	948	664
  HRGCA01	589970	2516	WUblastx.64	(Q9H387) PRO2550.	Q9H387	57%	1318	1016
  HRGCA06	866189	2517	HMMER	PFAM: Ribosomal protein S16	PF00886	69.1	143	325
  			2.1.1
  			WUblastx.64	(Q9Y3D3) 28S RIBOSOMAL	RT16_HUMAN	99%	74	445
  				PROTEIN S16, MITOCHONDRIAL
  				PRECURSOR
  HRGSE38	898233	2518	WUblastx.64	ATPase inhibitor precursor,	pir|JC7175|JC7175	83%	77	367
  				mitochondrial - human
  HRLME03	610614	2520	WUblastx.64	(Q9H3B9) PRO0956.	Q9H3B9	39%	220	122
  						68%	111	46
  HROAP64	835467	2522	WUblastx.64	(Q24333) ELASTIN LIKE PROTEIN	Q24333	100%	46	117
  				(FRAGMENT).
  HROAS35	827304	2523	WUblastx.64	cytochrome-c oxidase (EC 1.9.3.1)	pir|A00482|OTHU3	77%	483	881
  				chain III - human 1
  HROBJ10	836368	2525	WUblastx.64	(Q9Y5P3) RETINOIC ACID-	RAI2_HUMAN	36%	1719	1441
  				INDUCED PROTEIN 2.		96%	1817	1719
  						96%	1054	671
  						100%	1445	1044
  HRTAE88	822964	2529	WUblastx.64	(Q13579) MARINER	Q13579	76%	2579	2845
  				TRANSPOSASE.
  HRTAP63	780698	2530	WUblastx.64	(Q9Y3C9) CGI-127 PROTEIN.	Q9Y3C9	100%	498	860
  HSAAN03	599334	2532	WUblastx.64	(Q9H387) PRO2550.	Q9H387	70%	924	832
  						72%	676	602
  						77%	1094	921
  HSAAS05	703244	2533	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	55%	1475	1416
  				PRODUCT.		55%	1419	1216
  HSAAW13	821334	2534	WUblastx.64	(AAK57641) CAMP-specific cyclic	AAK57641	99%	1	1101
  				nucleotide phosphod
  HSATA61	801927	2538	WUblastx.64	(Q9CR30) 1110007C05RIK	Q9CR30	73%	6	224
  				PROTEIN.
  HSATG66	824903	2539	WUblastx.64	(Q9JKP5) MUSCLEBLIND.	Q9JKP5	100%	561	590
  						52%	13	213
  						83%	1	465
  HSATI91	838829	2540	WUblastx.64	hypothetical protein (L1H 3′ region) -	pir|B34087|B34087	43%	345	127
  				human		35%	808	515
  						30%	828	580
  						50%	527	375
  						32%	128	45
  HSATR50	826398	2541	WUblastx.64	(AAH02742) U6 snRNA-associated	AAH02742	100%	397	483
  				Sm-like protein LSm8
  HSATT82	566784	2542	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	57%	1004	891
  				CLONE HEMBA1006036.		68%	915	766
  HSATW19	637658	2543	WUblastx.64	(Q9UI50) PRO0657 (FRAGMENT).	Q9UI50	75%	448	669
  HSATW67	604938	2544	WUblastx.64	pro-pol-dUTPase polyprotein - murine	pir|T29097|T29097	63%	653	441
  				endogenous retrovirus ERV-L		82%	436	119
  				(fragment)
  HSATZ02	490895	2545	WUblastx.64	(Q9H387) PRO2550.	Q9H387	75%	964	857
  						76%	858	742
  HSAUB89	600369	2547	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	62%	359	631
  				PROTEIN.
  HSAUI53	866191	2548	WUblastx.64	retrovirus-related reverse transcriptase	pir|B25313|GNLRL1	42%	362	9
  				pseudogene - slow loris		32%	562	365
  						44%	942	868
  						44%	1037	957
  						38%	570	436
  						37%	827	723
  HSAUV74	866193	2549	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	62%	827	693
  HSAUX39	823161	2550	WUblastx.64	(Q62510) ZINC FINGER PROTEIN	Q62510	33%	960	1040
  				62 (FRAGMENT).		41%	1319	1645
  						43%	1244	1975
  						50%	1343	1975
  						52%	1349	1894
  						59%	1412	1975
  						45%	1235	1975
  						63%	1385	1972
  						52%	1349	1966
  						63%	1388	1966
  						48%	1130	1810
  						49%	1244	1975
  						64%	1367	1972
  						56%	1223	1975
  HSAVE52	600370	2552	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	55%	1425	1366
  				CLONE COL04765.		56%	1384	1127
  HSAVH32	603367	2553	WUblastx.64	(AAK55521) PRO0764.	AAK55521	73%	811	767
  						56%	789	547
  HSAVO11	566467	2555	WUblastx.64	(O60448) NEURONAL THREAD	O60448	41%	1599	1207
  				PROTEIN AD7C-NTP.		66%	1597	1439
  						54%	1257	1192
  						64%	1582	1301
  HSAVO17	738007	2556	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	72%	1407	1129
  				CLONE COL03536.
  HSAVQ13	606818	2557	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	66%	494	538
  				PROTEIN.		72%	406	471
  HSAVR85	786185	2558	WUblastx.64	(Q9H396) PRO2870.	Q9H396	100%	3791	3546
  HSAVY92	606808	2559	WUblastx.64	(Q9H5T7) CDNA: FLJ23054 FIS,	Q9H5T7	100%	19	123
  				CLONE LNG03193.
  HSAVZ05	690151	2560	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	65%	57	257
  				CLONE COL03536.		56%	256	303
  						67%	1529	1212
  HSAWB58	738018	2561	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	75%	1386	1607
  				PROTEIN.
  HSAWH36	581087	2562	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	71%	1109	867
  				CLONE KAT08285.
  HSAWM20	606840	2563	WUblastx.64	(Q9P147) PRO2822.	Q9P147	67%	488	387
  						57%	363	322
  						73%	591	490
  HSAWM74	866195	2564	WUblastx.64	(O95166) MM46 (HT004 PROTEIN)	O95166	100%	8	85
  				(MAP1 LIGHT CHAIN 3 RELATED
  				PROTEIN).
  HSAWX70	872498	2565	WUblastx.64	(O00369) L1 ELEMENT L1.20 P40	O00369	43%	1309	1088
  				PROTEIN.		73%	1028	300
  HSAXI10	598726	2567	WUblastx.64	(Q9H387) PRO2550.	Q9H387	79%	643	512
  						75%	811	776
  						71%	776	621
  HSAXL49	606826	2568	WUblastx.64	(Q9H387) PRO2550.	Q9H387	63%	742	572
  						83%	863	717
  HSAXL82	566801	2569	WUblastx.64	(Q9CXK1) 5730406I15RIK	Q9CXK1	96%	533	613
  				PROTEIN.
  HSAXS06	668260	2572	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	50%	955	824
  				CLONE COL03536.		62%	1121	954
  HSAYL24	608642	2574	WUblastx.64	(Q9UI59) PRO0478 PROTEIN.	Q9UI59	73%	132	7
  HSBAJ47	842694	2578	WUblastx.64	(Q9H189) SPHINGOSINE-1-	Q9H189	100%	1	237
  				PHOSPHATASE.
  HSDDC55	663277	2580	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	82%	743	793
  				CLONE KAIA0536.		57%	490	741
  HSDEA26	822812	2581	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	76%	1325	1387
  						75%	1140	1322
  HSDGH56	853379	2587	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	94%	865	815
  				CLONE COL04765.		61%	1096	1058
  						70%	1058	879
  HSDGM01	608310	2588	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	85%	722	781
  				PROTEIN.		53%	529	726
  HSDGM42	870143	2589	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	92%	360	2639
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HSDGM42	824916	2590	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	91%	359	2638
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HSDGM42	852473	2591	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	95%	1544	606
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HSDGM42	861925	2592	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	92%	420	2699
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HSDGM42	865828	2593	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	92%	420	2699
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HSDGM42	886748	2594	WUblastx.64	(Q9BT21) SIMILAR TO RIKEN	Q9BT21	92%	420	2699
  				CDNA 2610005L19 GENE
  				(FRAGMENT).
  HSDIK31	847087	2597	WUblastx.64	(O15121) PUTATIVE FATTY ACID	O15121	100%	19	369
  				DESATURASE MLD		97%	354	881
  				(DEGENERATIVE SPERMATOCYT
  HSDJC96	753284	2599	WUblastx.64	(Q9Y6A3) HYPOTHETICAL 5.6 KDA	Q9Y6A3	100%	1946	1800
  				PROTEIN (FRAGMENT).
  HSDJF04	695756	2601	WUblastx.64	(O95890) UNKNOWN.	O95890	94%	2	382
  HSDJG47	847325	2602	WUblastx.64	(Q9H6U6) CDNA: FLJ21857 FIS,	Q9H6U6	84%	59	1180
  				CLONE HEP02294.
  HSDJH72	805971	2603	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	66%	1500	1447
  				FOR MGC: 5149).		71%	1709	1485
  HSDJL07	895387	2604	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	64%	764	898
  				CLONE LNG09295.		82%	1481	1290
  HSDJR49	741079	2605	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	63%	1000	1122
  				CLONE COL04765.		70%	893	1015
  						68%	786	872
  						62%	1124	1210
  						73%	1000	1044
  						67%	569	733
  HSDJV24	806246	2606	WUblastx.64	(Q9CWT1) 2410004N11RIK	Q9CWT1	72%	539	1429
  				PROTEIN.
  HSDJV40	623716	2607	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	69%	1105	977
  				CLONE KAIA0536.		59%	1360	1295
  						38%	1583	1401
  						60%	1277	1104
  HSDKA64	600372	2608	WUblastx.64	(Q9H387) PRO2550.	Q9H387	80%	1340	1278
  						72%	1548	1342
  HSDKF96	839730	2610	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	63%	1672	1412
  				PRODUCT.
  HSDZO08	827460	2611	WUblastx.64	(Q9UES6) I-1 RECEPTOR	Q9UES6	74%	1	393
  				CANDIDATE PROTEIN.		30%	1	156
  						92%	449	1648
  HSEBB18	877813	2613	WUblastx.64	(Q9NXX4) CDNA FLJ20005 FIS,	Q9NXX4	89%	476	898
  				CLONE ADKA02526.
  HSFAM19	691371	2614	WUblastx.64	(O18966) EAG CHANNEL.	O18966	83%	76	510
  HSHAG54	834781	2615	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	51%	1028	738
  				CLONE LNG09295.
  HSHAS72	835991	2616	HMMER	PFAM: G-protein gamma subunit.	PF00631	139.1	225	422
  			2.1.1
  			WUblastx.64	(AAK53385) G protein gamma 12	AAK53385	100%	207	422
  				subunit.
  HSHAX04	812178	2617	WUblastx.64	peptidylprolyl isomerase (EC 5.2.1.8)	pir|S66681|S66681	96%	14	916
  				A - human
  HSHBT15	581088	2618	WUblastx.64	(O95863) ZINC FINGER PROTEIN	SNAI_HUMAN	100%	570	680
  				SNAI1 (SNAIL PROTEIN		79%	10	570
  				HOMOLOG)
  HSHCE85	855969	2619	WUblastx.64	(Q9Y546) DJ167A19.4 (NOVEL	Q9Y546	100%	202	1224
  				PROTEIN).
  HSIAC81	783076	2620	WUblastx.64	(Q9H3T4) KLOTHO-RELATED	Q9H3T4	99%	8	1261
  				PROTEIN 1.
  HSIAP01	897538	2622	WUblastx.64	(Q9NXP8) CDNA FLJ20124 FIS,	Q9NXP8	92%	590	673
  				CLONE COL06056.		28%	218	535
  						100%	685	732
  						89%	71	613
  HSIDZ25	658721	2625	WUblastx.64	(Q9HBN2) HYPOTHETICAL 15.8 KDA	Q9HBN2	37%	1366	1280
  				PROTEIN.		80%	1689	1597
  HSIEB64	651359	2626	WUblastx.64	(Q9P1J1) PRO1546.	Q9P1J1	70%	1718	1747
  						60%	1860	1949
  						53%	1741	1875
  HSIFO61	845026	2628	WUblastx.64	(O95831) PROGRAMED CELL	PCD8_HUMAN	96%	95	1933
  				DEATH PROTEIN 8,
  				MITOCHONDRIAL PREC
  HSIFO61	852715	2629	WUblastx.64	(O95831) PROGRAMED CELL	PCD8_HUMAN	96%	183	2021
  				DEATH PROTEIN 8,
  				MITOCHONDRIAL PREC
  HSIGC63	877486	2630	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	58%	1734	1354
  				CLONE KAIA0536.
  HSIGM95	840382	2631	WUblastx.64	(Q9H397) PRO2852.	Q9H397	80%	818	759
  						75%	754	707
  						77%	942	823
  HSJAN83	825132	2633	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	86%	1063	977
  				CLONE HEMBA1006036.		63%	963	898
  						57%	827	771
  						87%	839	768
  						77%	960	841
  HSJAQ10	853383	2634	WUblastx.64	(AAH05957) Solute carrier family 25	AAH05957	100%	11	187
  				(mitochondrial
  HSJAR59	846364	2635	WUblastx.64	(Q9NZS6) GLUCOCORTICOID	Q9NZS6	100%	16	138
  				RECEPTOR AF-1 SPECIFIC
  				ELONGATION FACTOR (FRA
  HSJAU93	702019	2636	WUblastx.64	(Q9BUK4) SIMILAR TO	Q9BUK4	74%	123	728
  				HYPOTHETICAL PROTEIN
  				FLJ10709.
  HSKHV81	841590	2646	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	60%	935	801
  				CLONE MAMMA1000472.		26%	308	219
  HSKYR49	806548	2648	WUblastx.64	(Q9BTV7) UNKNOWN (PROTEIN	Q9BTV7	100%	10	456
  				FOR IMAGE: 3357127)
  				(FRAGMENT).
  HSKYU81	899335	2649	WUblastx.64	(Q9Y2W2) SH3 DOMAIN-BINDING	Q9Y2W2	84%	950	1276
  				PROTEIN SNP70 (NPW38-BINDING		100%	536	565
  				PROTEIN NPWB		31%	240	365
  						72%	6	533
  HSKYY92	853384	2650	WUblastx.64	(Q9P0E3) HSPC093 (FRAGMENT).	Q9P0E3	57%	1455	1351
  						50%	1348	1193
  HSLAB11	823825	2651	WUblastx.64	(Q9CVT0) 1700040C17RIK	Q9CVT0	99%	915	1427
  				PROTEIN (FRAGMENT).		34%	313	453
  						89%	187	915
  HSLAS96	740764	2652	WUblastx.64	(Q9H387) PRO2550.	Q9H387	62%	1173	1075
  						73%	1378	1175
  HSLAW59	637669	2653	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	52%	578	423
  				CLONE HEMBA1006036.		70%	427	287
  HSLCH54	562018	2654	WUblastx.64	(P51805) PLEXIN A3 PRECURSOR	PLX4_HUMAN	97%	445	570
  				(PLEXIN 4) (TRANSMEMBRANE		81%	9	446
  				PROT
  HSLCH57	899415	2655	WUblastx.64	SREBP cleavage activating protein -	pir|T18526|T18526	92%	1408	2205
  				Chinese hamster		39%	658	774
  						47%	352	459
  HSLCI86	737626	2656	WUblastx.64	estrogen sulfotransferase (EC 2.8.2.—) -	pir|JC2229|JC2229	33%	493	555
  				human		100%	704	1090
  HSLCS31	604046	2657	HMMER	PFAM: PPR repeat	PF01535	22.6	493	597
  			2.1.1
  			WUblastx.64	(Q9H9R0) CDNA FLJ12598 FIS,	Q9H9R0	98%	454	828
  				CLONE NT2RM4001384.		100%	826	1047
  HSLCS34	751324	2658	WUblastx.64	(Q9NVE5) CDNA FLJ10785 FIS,	Q9NVE5	70%	40	576
  				CLONE NT2RP4000457, WEAKLY		86%	375	1007
  				SIMILAR TO UBI
  HSLCV16	772948	2659	WUblastx.64	(Q9H0J7) HYPOTHETICAL 53.4 KDA	Q9H0J7	99%	1	552
  				PROTEIN.		87%	1537	1698
  HSLDW54	853386	2660	WUblastx.64	probable pol polyprotein-related	pir|S21348|S21348	52%	732	631
  				protein 4 - rat		44%	625	452
  						34%	1115	738
  HSLEC18	722249	2661	WUblastx.64	(Q96RP7) Galbetal-3GalNAc 3′-	Q96RP7	97%	60	1319
  				sulfotransferase.
  HSLEG59	637671	2662	WUblastx.64	(Q14287) HYPOTHETICAL	Q14287	44%	968	807
  				PROTEIN (FRAGMENT).		20%	745	614
  						50%	1150	941
  HSLFR59	853388	2663	WUblastx.64	hypothetical protein	pir|T46471|T46471	86%	877	987
  				DKFZp434L0130.1 - human		93%	987	1481
  HSLGD91	883491	2664	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	70%	764	817
  				CLONE KAIA0536.		72%	816	926
  HSNAQ52	600402	2672	WUblastx.64	(Q9BGZ4) HYPOTHETICAL 11.6 KDA	Q9BGZ4	85%	197	156
  				PROTEIN.		81%	142	95
  HSNAW06	580248	2674	WUblastx.64	(Q9GMI2) HYPOTHETICAL 9.4 KDA	Q9GMI2	61%	219	413
  				PROTEIN.
  HSNBQ36	784994	2678	WUblastx.64	(Q9NUU5) CDNA FLJ11128 FIS,	Q9NUU5	98%	358	732
  				CLONE PLACE1006236.
  HSNBS39	617125	2679	WUblastx.64	retrovirus-related hypothetical protein	pir|S23650|S23650	76%	313	263
  				II - human 1		47%	82	26
  						38%	275	93
  HSOAT44	847357	2681	WUblastx.64	(Q9GZY9) CDNA: FLJ20877 FIS,	Q9GZY9	100%	10	195
  				CLONE ADKA02965 (CDNA:
  				FLJ20871 FIS, CLO
  HSOBH11	794000	2683	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	58%	657	743
  				PROTEIN.		59%	508	603
  HSOBW65	778388	2685	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	64%	160	110
  				PROTEIN (FRAGMENT).		67%	636	142
  HSPAA89	825099	2686	WUblastx.64	(P05142) PROLINE-RICH PROTEIN	PRP2_MOUSE	42%	210	148
  				MP-2 PRECURSOR.		37%	1144	641
  HSQBL20	780518	2696	WUblastx.64	(Q9UBQ5) MRNA OF MUSCLE	Q9UBQ5	100%	183	836
  				SPECIFIC GENE M9, COMPLETE
  				CDS (ARG134 PROTEI
  HSQCY74	886110	2698	WUblastx.64	(Q9H455) DJ383J4.4 (A NOVEL	Q9H455	99%	10	1455
  				PROTEIN SIMILAR TO
  				ASPARTYL-TRNA SYNTHETA
  HSRAA81	695759	2705	WUblastx.64	(CAC38441) DJ1033B10.5.1 (SAC2	CAC38441	90%	9	749
  				(suppressor of actin		33%	596	694
  						100%	727	1086
  HSRAO56	719816	2706	WUblastx.64	(Q9HBS7) HYPOTHETICAL 14.2 KDA	Q9HBS7	68%	1419	1324
  				PROTEIN.		72%	1592	1416
  HSRAV28	581102	2707	WUblastx.64	(Q9H387) PRO2550.	Q9H387	61%	655	524
  						61%	823	785
  						75%	788	654
  HSRDW57	562019	2709	WUblastx.64	(BAB55068) CDNA FLJ14466 fis,	BAB55068	100%	8	259
  				clone MAMMA1000416.		91%	262	495
  						83%	485	520
  HSREC72	601368	2710	WUblastx.64	(Q9H387) PRO2550.	Q9H387	88%	838	812
  						69%	783	625
  HSREG42	839481	2711	WUblastx.64	(Q9VZZ4) PXN PROTEIN.	Q9VZZ4	47%	6	1484
  HSRFD18	840771	2712	WUblastx.64	(Q9H941) CDNA FLJ13033 FIS,	Q9H941	100%	437	559
  				CLONE NT2RP3001126.
  HSRGZ11	801929	2713	WUblastx.64	(Q9BYN8) DJ534B8.3 (NOVEL	Q9BYN8	100%	253	384
  				PROTEIN).
  HSRHB59	840384	2714	WUblastx.64	(Q9NWT0) HYPOTHETICAL 17.7 KDA	Q9NWT0	100%	8	121
  				PROTEIN.		100%	123	308
  HSSCC66	559402	2716	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	65%	548	667
  				PRODUCT.
  HSSEL28	838618	2721	WUblastx.64	(Q9P195) PRO1722.	Q9P195	68%	755	1042
  HSSFP88	658726	2722	HMMER	PFAM: Zinc finger, C3HC4 type	PF00097	36.1	828	953
  			2.1.1	(RING finger)
  			WUblastx.64	(Q9H6Y7) CDNA: FLJ21676 FIS,	Q9H6Y7	90%	204	1133
  				CLONE COL09164.
  HSSGS62	741162	2723	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	60%	899	762
  				PROTEIN.		60%	200	141
  HSSJA23	847089	2724	WUblastx.64	(Q9BTH7) UNKNOWN (PROTEIN	Q9BTH7	94%	272	685
  				FOR MGC: 5601).		96%	1379	1561
  HSSJF26	567431	2725	WUblastx.64	(Q9HBS7) HYPOTHETICAL 14.2 KDA	Q9HBS7	76%	600	689
  				PROTEIN.		46%	401	580
  HSSJM47	796096	2727	WUblastx.64	(Q9UJ98) STROMAL ANTIGEN 3,	Q9UJ98	77%	129	194
  				(STAG3).		75%	527	637
  						96%	899	994
  HSSJW30	779822	2728	WUblastx.64	(BAB55147) CDNA FLJ14580 fis,	BAB55147	86%	2	46
  				clone NT2RM4001204.		92%	45	488
  HSSJW30	850566	2729	WUblastx.64	(BAB55147) CDNA FLJ14580 fis,	BAB55147	67%	14	703
  				clone NT2RM4001204.
  HSSJW30	867721	2730	WUblastx.64	(BAB55147) CDNA FLJ14580 fis,	BAB55147	82%	672	842
  				clone NT2RM4001204.		100%	625	675
  						89%	76	186
  						46%	131	220
  						60%	24	83
  HSSMY35	740765	2731	WUblastx.64	(Q9H7J9) FLJ00075 PROTEIN	Q9H7J9	96%	20	217
  				(FRAGMENT).
  HSTAL93	841863	2732	WUblastx.64	(AAK52433) Low density lipoprotein	AAK52433	98%	287	799
  				receptor-related		35%	284	787
  						35%	299	787
  						35%	287	637
  HSUAF06	863206	2734	WUblastx.64	pol polyprotein - Cas-Br-E murine	pir|A26103|A26103	47%	630	944
  				leukemia virus (fragment)		50%	1194	1307
  HSUBX67	751266	2735	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	61%	624	499
  				PRODUCT.		64%	766	608
  HSUSB73	603912	2736	WUblastx.64	(Q9GMU5) HYPOTHETICAL 14.1 KDA	Q9GMU5	57%	1158	1117
  				PROTEIN.		80%	130	56
  HSVAC05	836051	2737	WUblastx.64	(Q9H6G8) CDNA: FLJ22294 FIS,	Q9H6G8	64%	688	539
  				CLONE HRC04426.
  HSVBA83	812059	2742	WUblastx.64	(O60593) ARG/ABL-INTERACTING	O60593	98%	313	516
  				PROTEIN ARGBP2B (FRAGMENT).
  HSVBY62	637113	2745	WUblastx.64	(Q9Y2Q7) HSPC005 PROTEIN	Q9Y2Q7	100%	35	217
  				(C11ORF10) (CHROMOSOME 11
  				OPEN READING FRAME
  HSXAI44	590743	2750	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	90%	1024	992
  				CLONE COL04765.		73%	1007	828
  HSXAS59	838072	2752	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	85%	1515	1474
  				CLONE COL04765.		81%	1303	1256
  						62%	1478	1305
  HSXAY60	737753	2754	WUblastx.64	(Q9N032) UNNAMED PROTEIN	Q9N032	59%	1742	1602
  				PRODUCT.
  HSXCA83	830046	2756	WUblastx.64	(Q9UHD2) TANK BINDING	Q9UHD2	39%	1121	1219
  				KINASE TBK1 (NF-KB-		98%	1215	1382
  				ACTIVATING KINASE NAK).		93%	109	1197
  HSXCX20	658728	2757	WUblastx.64	(Q9BXA5) G-PROTEIN COUPLED	Q9BXA5	100%	891	1049
  				RECEPTOR 91.		86%	61	942
  HSXFG21	805972	2758	WUblastx.64	(Q9CQQ2) 1700066F09RIK	Q9CQQ2	47%	499	933
  				PROTEIN (FRAGMENT).
  HSXFH82	699863	2759	WUblastx.64	(Q9MZZ7) HYPOTHETICAL 16.3 KDA	Q9MZZ7	69%	1447	1611
  				PROTEIN.		92%	1124	1162
  						65%	1146	1598
  HSYBR79	873848	2761	WUblastx.64	vesicle-associated membrane protein-	pir|JG0186|JG0186	100%	193	861
  				associated protein B - human
  HSYBV44	753253	2762	WUblastx.64	(Q9B2U5) ATP SYNTHASE 6.	Q9B2U5	66%	84	761
  HSYBZ94	799543	2763	WUblastx.64	(Q9BZ73) NIR2.	Q9BZ73	98%	2168	2392
  						99%	2388	2924
  						33%	1313	1399
  						33%	1895	1999
  						79%	21	2174
  HT3AB13	841680	2764	WUblastx.64	(Q9UP93) SHORT FORM	Q9UP93	53%	663	980
  				TRANSCRIPTION FACTOR C-MAF.		74%	271	612
  HT4SB02	837688	2765	HMMER	PFAM: emp24/gp25L/p24 family	PF01105	231.4	78	521
  			2.1.1
  			WUblastx.64	protein trafficking protein tmp21-I -	pir|G01159|G01159	100%	84	524
  				human		100%	21	74
  HT4SB81	756723	2767	WUblastx.64	(O95621) TIC.	O95621	95%	3	194
  						77%	580	789
  						62%	185	631
  						41%	19	54
  HT4SB81	844512	2768	WUblastx.64	(O95621) TIC.	O95621	72%	185	790
  						95%	3	194
  						41%	19	54
  						57%	872	913
  HT4SB81	858154	2769	WUblastx.64	(O95621) TIC.	O95621	71%	185	790
  						41%	19	54
  						95%	3	194
  						57%	872	913
  HTABF81	610040	2771	WUblastx.64	(Q9NRR3) NON-KINASE CDC42	Q9NRR3	100%	48	299
  				EFFECTOR PROTEIN SPEC2.
  HTACX63	602694	2772	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	65%	919	860
  				CLONE COL04765.		78%	825	784
  						53%	1067	921
  HTADC63	842131	2773	WUblastx.64	(Q9D7U1) 2210407P13RIK	Q9D7U1	40%	557	793
  				PROTEIN.		46%	162	557
  HTADO61	695760	2774	WUblastx.64	hypothetical protein	pir|T42648|T42648	100%	9	95
  				DKFZp434C1415.1 - human
  HTAEC59	846728	2776	WUblastx.64	ubiquitin-conjugating enzyme	pir|S53358|S53358	100%	409	591
  				E2.17 kB - rat		100%	156	383
  HTAED89	801931	2777	HMMER	PFAM: 7 transmembrane receptor	PF00003	78.6	1240	1578
  			2.1.1	(metabotropic glutamate family)
  			WUblastx.64	(O35363) CALCIUM SENSING	O35363	51%	1590	1718
  				RECEPTOR, RELATED SEQUENCE		38%	858	920
  				2 (CALCIUM-SENSIN		38%	908	1171
  						53%	1216	1611
  HTAEO35	732379	2780	WUblastx.64	(Q9H387) PRO2550.	Q9H387	73%	1476	1195
  HTDAF68	637685	2781	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	72%	892	1041
  				CLONE COL04765.		50%	2	91
  						70%	711	890
  HTDAI38	878930	2782	WUblastx.64	(Q9NX73) CDNA FLJ20400 FIS,	Q9NX73	88%	954	1268
  				CLONE KAT00587 (FRAGMENT).		30%	361	669
  						34%	1146	1874
  						37%	1314	1898
  						100%	343	678
  						32%	930	1628
  HTECE87	702025	2786	WUblastx.64	(Q9NVC3) CDNA FLJ10815 FIS,	Q9NVC3	100%	53	133
  				CLONE NT2RP4000989, WEAKLY		100%	133	279
  				SIMILAR TO UNC
  HTEDF78	564215	2787	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	75%	1545	1423
  				PROTEIN.
  HTEDX05	862064	2789	WUblastx.64	(Q9D4H6) 4932415A06RIK	Q9D4H6	79%	16	441
  				PROTEIN.		86%	429	1649
  HTEEC19	862065	2790	WUblastx.64	translation initiation factor IF-2	pir|T43483|T43483	100%	11	1321
  				homolog [similarity] - 1
  HTEGH03	815562	2791	WUblastx.64	(Q9UHB5) EPITHELIAL PROTEIN	Q9UHB5	93%	32	928
  				LOST IN NEOPLASM ALPHA.
  HTEGH03	839477	2792	WUblastx.64	(Q9UHB5) EPITHELIAL PROTEIN	Q9UHB5	82%	678	845
  				LOST IN NEOPLASM ALPHA.		88%	1	732
  HTEGY81	637689	2794	WUblastx.64	(Q9CYN5) 5730405I09RIK	Q9CYN5	74%	11	544
  				PROTEIN.
  HTEHB49	823145	2796	WUblastx.64	(Q9H2T7) RANBP17.	Q9H2T7	65%	126	254
  						99%	203	1537
  HTEHV60	637690	2798	WUblastx.64	(Q9D2H5) 4930486B16RIK	Q9D2H5	88%	72	1028
  				PROTEIN.		33%	153	281
  						89%	1027	1113
  HTEHW80	862068	2799	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	71%	297	578
  				CLONE HEMBA1006036.
  HTEID25	737852	2800	WUblastx.64	(Q9Y4V7) DJ1178H5.3 (NOVEL	Q9Y4V7	100%	259	384
  				PROTEIN) (FRAGMENT).
  HTEIJ23	784268	2801	WUblastx.64	(Q9NX98) CDNA FLJ20363 FIS,	Q9NX98	93%	107	1303
  				CLONE HEP17001.
  HTEIM62	806472	2802	WUblastx.64	(Q9H3C1) PRO0872.	Q9H3C1	81%	62	15
  						76%	123	61
  HTEIV33	603393	2803	WUblastx.64	hypothetical protein	pir|T47135|T47135	53%	259	11
  				DKFZp761L0812.1 - human
  				(fragment)
  HTEJD61	828178	2807	WUblastx.64	(BAB49394) Ml12208 protein.	BAB49394	37%	437	682
  						27%	21	380
  						30%	437	682
  						27%	9	383
  						24%	18	392
  						29%	21	386
  						32%	401	682
  						30%	138	395
  						24%	452	682
  						27%	90	380
  						30%	395	679
  						28%	437	682
  						26%	392	592
  						30%	12	386
  HTEJL16	603409	2810	WUblastx.64	(Q9CPU8) 4921511D23RIK	Q9CPU8	71%	453	412
  				PROTEIN.		34%	1042	722
  						34%	1027	698
  						80%	412	257
  						32%	952	728
  						64%	1030	596
  HTEKD35	604979	2813	WUblastx.64	(Q9D6N1) CARBONIC	Q9D6N1	89%	40	594
  				ANHYDRASE (EC 4.2.1.1)
  				(CARBONATE DEHYDRATASE).
  HTEKP82	694648	2814	WUblastx.64	(Q9D9W1) 1700027A23RIK	Q9D9W1	68%	147	689
  				PROTEIN.
  HTEKV69	877673	2815	WUblastx.64	(Q9D400) 4933425K02RIK	Q9D400	61%	157	1005
  				PROTEIN.		66%	1052	1096
  						35%	52	210
  HTFOB75	900824	2818	WUblastx.64	(Q9BX86) HP95.	Q9BX86	96%	162	2366
  						50%	2469	2675
  						21%	1857	2354
  HTGAA35	737945	2819	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	64%	1474	1349
  				CLONE KAIA0536.		78%	1632	1465
  HTGAD74	834464	2820	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	66%	665	621
  						67%	868	668
  HTGAP05	637715	2821	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	65%	871	749
  				CLONE KAIA0536.		73%	990	868
  HTGAR21	838159	2823	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	80%	864	820
  				FOR MGC: 5149).		67%	1081	878
  HTGAS70	827320	2824	WUblastx.64	(AAH00037) DNA fragmentation	AAH00037	95%	290	1216
  				factor, 45 kD, alpha p
  HTGAT65	688864	2825	WUblastx.64	(Q9P0D8) HSPC098 (FRAGMENT).	Q9P0D8	42%	1074	1130
  						60%	1469	1573
  HTGAU17	605125	2826	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	58%	621	692
  				PROTEIN.		44%	703	876
  HTGBK95	834490	2828	WUblastx.64	(Q9GMX5) HYPOTHETICAL 12.9 KDA	Q9GMX5	66%	126	55
  				PROTEIN.		70%	235	116
  HTGCC01	598903	2829	WUblastx.64	(Q9H387) PRO2550.	Q9H387	73%	1099	962
  						68%	963	784
  HTGCK43	828867	2830	WUblastx.64	(P82914) 28S RIBOSOMAL	RT15_HUMAN	91%	862	92
  				PROTEIN S15, MITOCHONDRIAL
  				PRECURSOR
  HTGDS43	605094	2831	WUblastx.64	(O60921) HUS1+-LIKE PROTEIN.	O60921	97%	706	831
  HTGDS92	839478	2832	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	69%	1300	1001
  				CLONE COL04765.
  HTGEX34	877490	2833	WUblastx.64	(Q9H387) PRO2550.	Q9H387	77%	2277	1966
  HTGGM37	827310	2835	WUblastx.64	(Q9NUM6) CDNA FLJ11267 FIS,	Q9NUM6	76%	1781	1909
  				CLONE PLACE1009174.
  HTGGN22	782853	2836	WUblastx.64	(Q9H5R3) CDNA: FLJ23147 FIS,	Q9H5R3	57%	741	664
  				CLONE LNG09295.		66%	682	524
  HTHBC58	839916	2838	WUblastx.64	(Q9H387) PRO2550.	Q9H387	80%	1267	1208
  						71%	1452	1294
  HTHBQ29	561547	2840	WUblastx.64	(Q9BGV8) HYPOTHETICAL 10.0 KDA	Q9BGV8	78%	786	1010
  				PROTEIN.
  HTHBZ91	637697	2842	WUblastx.64	(O60448) NEURONAL THREAD	O60448	53%	823	668
  				PROTEIN AD7C-NTP.		61%	948	802
  						69%	743	705
  						44%	458	273
  						52%	947	747
  						59%	594	466
  						61%	963	748
  						50%	360	313
  						36%	891	802
  						39%	948	880
  						57%	569	324
  						50%	493	425
  						25%	848	561
  						48%	408	289
  						26%	425	249
  						34%	454	272
  						30%	513	301
  						52%	588	367
  HTHCA30	637124	2843	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	67%	627	466
  				FOR MGC: 5149).
  HTHDB20	669032	2845	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	79%	426	355
  				CLONE COL04765.		60%	629	429
  HTHDF45	621313	2846	WUblastx.64	(Q9H387) PRO2550.	Q9H387	42%	987	691
  HTHDF86	815686	2847	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	63%	1184	1249
  				CLONE KAIA0536.		83%	1029	1193
  						50%	1495	1247
  HTHDP65	637699	2849	WUblastx.64	(Q9H387) PRO2550.	Q9H387	100%	42	16
  						66%	205	35
  HTHDV50	789402	2851	WUblastx.64	(Q9GMI7) HYPOTHETICAL 9.0 KDA	Q9GMI7	47%	299	78
  				PROTEIN.
  HTJMA64	775181	2852	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	75%	1065	1211
  				CLONE COL04765.		75%	1369	1500
  						52%	1209	1391
  						44%	1187	1267
  						64%	893	1087
  HTLAD74	638978	2854	WUblastx.64	(P78525) MYB PROTO-ONCOGENE	P78525	48%	929	633
  				PROTEIN (C-MYB).
  HTLAF81	855970	2855	WUblastx.64	(Q9BUS3) UNKNOWN (PROTEIN	Q9BUS3	72%	306	1073
  				FOR IMAGE: 3461487)
  				(FRAGMENT).
  HTLBF46	839774	2856	HMMER	PFAM: Papain family cysteine	PF00112	255	176	751
  			2.1.1	protease
  			WUblastx.64	(Q9UBX1) CATHEPSIN F	CATF_HUMAN	99%	56	757
  				PRECURSOR (EC 3.4.22.41)
  				(CATSF).
  HTLBF63	762847	2857	HMMER	PFAM: MYND finger	PF01753	53.8	652	762
  			2.1.1
  			WUblastx.64	(O75800) BLU PROTEIN.	O75800	94%	10	792
  HTLCX82	847091	2858	WUblastx.64	(Q9BTZ4) SIMILAR TO	Q9BTZ4	100%	127	258
  				EXPRESSED SEQUENCE 2		72%	237	629
  				EMBRYONIC LETHAL
  				(FRAGMENT).
  HTLDN34	866199	2860	WUblastx.64	(O94993) SOX30 PROTEIN.	O94993	82%	242	751
  						89%	726	842
  						69%	9	137
  HTLDP19	885353	2861	WUblastx.64	(Q9D5P4) 4930403J07RIK PROTEIN.	Q9D5P4	62%	530	751
  						84%	8	121
  						61%	159	581
  HTLEJ24	608317	2863	WUblastx.64	(Q9D7G6) 2310009N05RIK	Q9D7G6	84%	2	619
  				PROTEIN.
  HTLEJ75	815631	2864	WUblastx.64	(AAK52668) MMS19.	AAK52668	92%	31	2133
  HTLEJ75	762849	2865	WUblastx.64	(BAB55315) CDNA FLJ14804 fis,	BAB55315	85%	7	798
  				clone NT2RP4001638, w
  HTLEP55	637704	2866	WUblastx.64	(AAL37611) Carboxypeptidase A5.	AAL37611	97%	1242	1352
  						92%	1171	1251
  						53%	561	656
  						100%	178	561
  						87%	608	1168
  HTLEV80	866200	2867	WUblastx.64	(O15020) BETA-SPECTRIN III	O15020	100%	9	452
  				(FNTA III SPECTRIN).
  HTLEZ57	634874	2868	WUblastx.64	(Q9P195) PRO1722.	Q9P195	57%	662	531
  						59%	121	26
  						62%	481	401
  HTLFA90	740770	2869	WUblastx.64	(Q9BRY0) UNKNOWN (PROTEIN	Q9BRY0	100%	2	1072
  				FOR IMAGE: 2966557)
  				(FRAGMENT).
  HTLGL33	835020	2870	WUblastx.64	N-type calcium channel alpha-1 chain, 1	pir|T45115|T45115	22%	364	1092
  HTLGQ25	898114	2871	HMMER	PFAM: Immunoglobulin domain	PF00047	26.6	153	377
  			2.1.1
  			WUblastx.64	(Q9H106) DJ576H24.4 (NOVEL	Q9H106	100%	114	443
  				PROTEIN MEMBER OF THE PTPNS
  				(PROTEIN TYROS
  HTLGS72	897278	2872	WUblastx.64	(Q9JJC0) BRAIN CDNA, CLONE	Q9JJC0	34%	6	563
  				MNCB-2717.
  HTLGY50	839479	2873	WUblastx.64	(O73884) PUTATIVE	O73884	39%	987	1100
  				PHOSPHATASE.		65%	589	1002
  HTLHN86	896930	2874	WUblastx.64	(BAB55144) CDNA FLJ14576 fis,	BAB55144	95%	318	938
  				clone NT2RM4001092, w
  HTLHN86	838287	2875	WUblastx.64	(BAB55144) CDNA FLJ14576 fis,	BAB55144	95%	318	938
  				clone NT2RM4001092, w
  HTLHN86	843766	2876	WUblastx.64	(BAB55144) CDNA FLJ14576 fis,	BAB55144	95%	318	938
  				clone NT2RM4001092, w
  HTLHN86	883351	2877	WUblastx.64	(BAB55144) CDNA FLJ14576 fis,	BAB55144	95%	318	938
  				clone NT2RM4001092, w
  HTLIW29	899417	2878	HMMER	PFAM: Trypsin	PF00089	226.4	133	852
  			2.1.1
  			WUblastx.64	(Q9Y6M0) TESTISIN PRECURSOR	TEST_HUMAN	100%	67	885
  				(EC 3.4.21.—) (EOSINOPHIL SERIN
  HTLJC15	898235	2879	WUblastx.64	(Q9D123) 1110032O16RIK	Q9D123	71%	996	1715
  				PROTEIN.
  HTNAL14	886203	2880	WUblastx.64	(Q9NXU2) CDNA FLJ20054 FIS,	Q9NXU2	100%	580	687
  				CLONE COL00849.
  HTNBJ15	834872	2882	WUblastx.64	(Q9H055) HYPOTHETICAL 13.8 KDA	Q9H055	99%	1536	1889
  				PROTEIN.
  HTNBJ15	845783	2883	WUblastx.64	(Q9H055) HYPOTHETICAL 13.8 KDA	Q9H055	99%	1536	1889
  				PROTEIN.
  HTNBJ15	853916	2884	WUblastx.64	(Q9H055) HYPOTHETICAL 13.8 KDA	Q9H055	99%	1536	1889
  				PROTEIN.
  HTNBJ15	884024	2885	WUblastx.64	(Q9H055) HYPOTHETICAL 13.8 KDA	Q9H055	99%	1536	1889
  				PROTEIN.
  HTOAO58	855972	2889	WUblastx.64	(Q9GZW5) SCAN DOMAIN-	Q9GZW5	49%	974	1321
  				CONTAINING PROTEIN 2
  				(SCAND2).
  HTOAT56	702026	2890	WUblastx.64	(AAH00407) Synaptogyrin 2.	AAH00407	82%	425	673
  						83%	10	495
  HTOBG07	566862	2891	WUblastx.64	(Q9H288) SEROLOGICALLY	Q9H288	100%	225	1343
  				DEFINED BREAST CANCER		41%	498	1343
  				ANTIGEN NY-BR-16.		37%	438	1343
  						37%	276	1187
  						33%	258	1319
  						34%	462	1304
  						30%	462	1286
  						50%	798	953
  						44%	1182	1343
  						66%	941	967
  						25%	261	839
  						39%	1098	1247
  						100%	42	71
  HTOBG62	566830	2892	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	66%	1378	1112
  				CLONE COL04765.
  HTODO45	823125	2895	WUblastx.64	(AAH08373) Similar to hypothetical	AAH08373	67%	1408	1226
  				protein PRO1722.
  HTOET03	845230	2899	WUblastx.64	(Q9BQC3) SIMILAR TO	Q9BQC3	83%	4	1041
  				DIPTHERIA TOXIN RESISTANCE
  				PROTEIN REQUIRED FOR D
  HTOET03	837213	2900	WUblastx.64	(Q9BQC3) SIMILAR TO	Q9BQC3	83%	4	1041
  				DIPTHERIA TOXIN RESISTANCE
  				PROTEIN REQUIRED FOR D
  HTOFA11	637719	2902	WUblastx.64	(Q30086) MHC CLASS II HLA-DQ-	Q30086	96%	1348	1635
  				ALPHA (DR2-DQW1/DR4 DQW3)		80%	1962	2069
  				(FRAGMENT).		97%	694	942
  						68%	1882	1968
  HTOFC33	824604	2903	WUblastx.64	(Q9H387) PRO2550.	Q9H387	65%	900	754
  						73%	1069	878
  HTOGB79	762835	2904	WUblastx.64	(O60448) NEURONAL THREAD	O60448	52%	1639	1917
  				PROTEIN AD7C-NTP.		60%	1642	1806
  						50%	1661	1825
  						31%	1465	1530
  						26%	1238	1528
  						27%	69	227
  						60%	1854	1928
  						43%	1797	1865
  						32%	1420	1542
  						75%	2727	2596
  						52%	2606	2472
  						42%	1214	1137
  						77%	2727	2551
  						49%	2661	2500
  						42%	2663	2553
  						56%	1213	1166
  						45%	2715	2596
  						68%	1284	1219
  						41%	1302	1141
  						55%	1332	1273
  						48%	1332	1264
  						52%	2730	2674
  						55%	1333	1274
  						55%	1208	1155
  						60%	1339	1157
  						62%	2756	2478
  HTOHE22	821698	2905	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	75%	1008	1217
  				CLONE KAIA0536.
  HTOHG63	834832	2906	WUblastx.64	(Q9H8L6) CDNA FLJ13465 FIS,	Q9H8L6	98%	2	670
  				CLONE PLACE1003493, WEAKLY
  				SIMILAR TO END
  HTOHJ93	762836	2907	WUblastx.64	(Q9H743) CDNA: FLJ21394 FIS,	Q9H743	70%	1323	1048
  				CLONE COL03536.
  HTOHN40	843372	2910	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	64%	1889	1839
  				CLONE KAIA0536.		50%	1845	1654
  HTOHR59	762850	2911	WUblastx.64	(AAH07609) Similar to hypothetical	AAH07609	92%	1249	1127
  				protein PRO1722.
  HTOHS29	722256	2912	WUblastx.64	(AAH08373) Similar to hypothetical	AAH08373	80%	129	10
  				protein PRO1722.
  HTOID65	636069	2913	WUblastx.64	(Q9H387) PRO2550.	Q9H387	56%	630	761
  						50%	467	658
  						77%	1420	1316
  						78%	1314	1165
  HTOIE17	688061	2914	WUblastx.64	(Q9NWI4) CDNA FLJ20837 FIS,	Q9NWI4	66%	942	889
  				CLONE ADKA02602.		61%	1078	938
  HTOIG16	845999	2915	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	93%	1558	1511
  				CLONE COL04765.		63%	1793	1563
  HTOIH39	839245	2916	WUblastx.64	(O60448) NEURONAL THREAD	O60448	53%	750	1106
  				PROTEIN AD7C-NTP.		46%	53	130
  						50%	1095	1184
  						51%	840	1049
  						57%	64	126
  						56%	14	121
  						54%	987	1151
  						54%	29	133
  						40%	1039	1167
  						68%	19	93
  HTOJB02	600376	2918	WUblastx.64	(O60448) NEURONAL THREAD	O60448	54%	780	652
  				PROTEIN AD7C-NTP.		33%	1322	1143
  						50%	1275	1108
  						66%	919	770
  						47%	682	626
  						52%	1235	1161
  						40%	889	770
  						50%	787	632
  						58%	816	727
  						41%	1424	1338
  						62%	708	661
  						37%	784	614
  						56%	918	658
  						58%	1411	1265
  						56%	1274	1134
  						30%	1275	1093
  						57%	1403	1251
  						59%	1424	1098
  						60%	949	713
  HTOJJ26	821702	2919	WUblastx.64	(Q9UHT1) PRO1902 PROTEIN.	Q9UHT1	71%	2969	2844
  HTOJP25	853623	2920	WUblastx.64	hypothetical L1 protein (third intron of	pir|JU0033|JU0033	60%	1388	1519
  				gene TS) - human
  HTOJS23	737735	2921	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	57%	1564	1523
  				CLONE COL04765.		46%	1670	1575
  						59%	1833	1657
  HTOJY56	664504	2922	WUblastx.64	(Q9P1F2) PRO2032.	Q9P1F2	100%	88	240
  HTPCO75	853645	2925	WUblastx.64	(O00549) ORF2-LIKE PROTEIN	O00549	43%	325	26
  				(FRAGMENT).		36%	1318	1253
  HTPDD68	590530	2927	WUblastx.64	(Q9H0G2) HYPOTHETICAL 136.4 KDA	Q9H0G2	88%	10	2004
  				PROTEIN.
  HTSET62	833692	2930	WUblastx.64	(Q9HCS4) HMG-BOX	Q9HCS4	95%	59	124
  				TRANSCRIPTION FACTOR TCF-3.		66%	124	327
  HTSFV18	609939	2931	HMMER	PFAM: Low-density lipoprotein	PF00057	49.4	170	280
  			2.1.1	receptor domain class A
  			WUblastx.64	LDL receptor related protein 105 -	pir|T00204|T00204	82%	10	984
  				human
  HTSGO13	789723	2932	WUblastx.64	(Q9NWJ5) CDNA FLJ20807 FIS,	Q9NWJ5	99%	195	548
  				CLONE ADSE01784.		97%	489	845
  HTSGO88	634834	2933	WUblastx.64	(O60448) NEURONAL THREAD	O60448	76%	797	612
  				PROTEIN AD7C-NTP.		52%	640	521
  						66%	636	574
  						45%	560	492
  						69%	791	627
  						61%	137	75
  HTTAH05	772560	2934	WUblastx.64	(Q9H397) PRO2852.	Q9H397	60%	548	676
  						39%	684	926
  HTTBJ38	863131	2936	WUblastx.64	(Q9VST7) CG4911 PROTEIN.	Q9VST7	25%	497	1177
  HTTDB11	638132	2937	WUblastx.64	(Q99LX8) UNKNOWN (PROTEIN	Q99LX8	100%	130	240
  				FOR MGC: 7346).
  HTTDG27	608318	2938	WUblastx.64	(O62658) LINE-1 ELEMENT ORF2.	O62658	44%	454	380
  						35%	392	3
  HTTDN24	766485	2939	WUblastx.64	(Q9BVN5) HYPOTHETICAL 120.6 KDA	Q9BVN5	95%	628	1725
  				PROTEIN.		32%	937	1593
  						95%	3	629
  						32%	1114	1596
  HTTDO33	899418	2940	WUblastx.64	(Q96GQ9) Unknown (protein for	Q96GQ9	95%	22	753
  				MGC: 16648).
  HTTEO25	853403	2942	WUblastx.64	(Q9BWK9) UNKNOWN (PROTEIN	Q9BWK9	97%	1314	1424
  				FOR IMAGE: 2900813)
  				(FRAGMENT).
  HTTEP11	562023	2943	WUblastx.64	hypothetical protein	pir|T46441|T46441	70%	753	830
  				DKFZp434C0927.1 - human		74%	1006	1110
  						51%	812	928
  						100%	625	753
  HTTES77	844417	2944	WUblastx.64	(AAK40083) Inhibin binding protein	AAK40083	82%	697	2085
  				long isoform.		37%	3	725
  						31%	6	713
  						30%	93	704
  						36%	659	733
  						39%	697	1818
  						34%	697	1815
  						33%	982	1818
  						88%	3	719
  						33%	697	1827
  						31%	1096	1821
  						31%	736	1833
  						35%	45	719
  						28%	901	1761
  						35%	6	737
  						34%	9	680
  						32%	45	716
  						36%	1240	1818
  HTTFG15	600377	2946	WUblastx.64	(Q9GMK2) HYPOTHETICAL 10.0 KDA	Q9GMK2	66%	327	482
  				PROTEIN.
  HTWAM19	618318	2947	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	76%	1852	1763
  				CLONE COL04765.		64%	1764	1525
  HTWBO30	655371	2949	WUblastx.64	(Q9N083) UNNAMED PORTEIN	Q9N083	47%	560	453
  				PRODUCT.		55%	751	578
  HTWBZ57	828030	2950	WUblastx.64	(O95273) D-TYPE CYCLIN-	O95273	87%	218	1276
  				INTERACTING PROTEIN 1.
  HTWCC10	747687	2951	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	60%	1051	947
  				CLONE KAIA0536.		63%	1241	1095
  HTWCE14	762839	2952	WUblastx.64	(Q9BSZ7) UNKNOWN (PROTEIN	Q9BSZ7	100%	121	231
  				FOR MGC: 4322).
  HTWCT76	767758	2953	WUblastx.64	(Q9BGW3) HYPOTHETICAL 13.5 KDA	Q9BGW3	68%	1550	1368
  				PROTEIN.		58%	1361	1311
  HTWDJ17	784037	2954	WUblastx.64	(Q9CYV6) 2810439F02RIK	Q9CYV6	93%	17	1255
  				PROTEIN.
  HTWDM89	569787	2955	WUblastx.64	(Q9P195) PRO1722.	Q9P195	63%	1413	1267
  						61%	1277	1098
  HTWEQ36	823369	2958	WUblastx.64	(Q9NXK9) CDNA FLJ20187 FIS,	Q9NXK9	68%	748	891
  				CLONE COLF0433.
  HTWFA88	596942	2960	WUblastx.64	probable transposase - human	pir|S72481|S72481	58%	1286	1357
  				transposon MER37		70%	1350	1559
  						51%	709	1272
  HTWFO43	858741	2962	WUblastx.64	(O60448) NEURONAL THREAD	O60448	67%	890	627
  				PROTEIN AD7C-NTP.		62%	1241	1056
  						63%	754	689
  						62%	1108	980
  						65%	1242	1102
  						61%	905	744
  						53%	684	601
  						40%	790	665
  						58%	675	625
  						34%	1184	975
  						35%	863	744
  						61%	1257	1039
  						45%	1063	992
  						64%	657	607
  						40%	1215	1087
  						34%	778	659
  						100%	1016	996
  HTXAD75	745410	2965	WUblastx.64	(Q9GLG1) CALPAIN 2.	Q9GLG1	92%	345	917
  HTXAR92	656935	2966	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	77%	1578	1297
  				CLONE COL04765.
  HTXBU88	604985	2968	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	75%	771	736
  				CLONE KAIA0536.		65%	739	680
  						60%	701	453
  HTXCP27	892364	2969	WUblastx.64	(AAH00057) Protein phosphatase 1G	AAH00057	97%	521	628
  				(formerly 2C), ma		100%	946	1149
  						98%	285	524
  HTXCU30	839846	2970	WUblastx.64	(Q9H728) CDNA: FLJ21463 FIS,	Q9H728	81%	706	641
  				CLONE COL04765.		80%	639	577
  						59%	875	684
  HTXCV44	740773	2971	WUblastx.64	(Q9HA67) CDNA FLJ12155 FIS,	Q9HA67	52%	883	945
  				CLONE MAMMA1000472.		70%	809	898
  						63%	630	803
  						63%	1521	1366
  						68%	1370	1323
  HTXDJ75	562780	2973	WUblastx.64	(Q26195) PVA1 GENE.	Q26195	51%	1598	1335
  HTXDT72	778083	2977	WUblastx.64	(Q9BSV9) SIMILAR TO 95 KDA	Q9BSV9	100%	8	400
  				RETINOBLASTOMA PROTEIN
  				BINDING PROTEIN, KI
  HTXDZ68	824545	2979	WUblastx.64	(Q9P0D0) HSPC106 (FRAGMENT).	Q9P0D0	72%	80	277
  						54%	136	498
  HTXEN33	589556	2980	WUblastx.64	(Q99419) ICSAT TRANSCRIPTION	Q99419	78%	843	968
  				FACTOR (FRAGMENT).
  HTXJD08	566885	2985	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	59%	743	1111
  				PROTEIN (FRAGMENT).		47%	604	756
  						45%	395	583
  HTXJD85	840391	2986	WUblastx.64	(Q9HAD8) CDNA FLJ11786 FIS,	Q9HAD8	52%	1093	818
  				CLONE HEMBA1006036.
  HTXJI59	869905	2988	WUblastx.64	(O15290) P66SHC.	O15290	93%	1708	1571
  HTXJJ92	688062	2989	WUblastx.64	(Q9N044) UNNAMED PROTEIN	Q9N044	59%	709	614
  				PRODUCT.		66%	611	531
  HTXJM94	853413	2990	WUblastx.64	(Q9Y2S9) HSPC019.	Q9Y2S9	83%	44	262
  HTXJW06	789201	2992	WUblastx.64	(Q9UI28) ADRENAL GLAND	Q9UI28	100%	868	623
  				PROTEIN AD-003.
  HTXKB57	844329	2993	WUblastx.64	(Q9P073) HSPC309 (FRAGMENT).	Q9P073	94%	1072	1128
  						65%	1125	1523
  HTXKH40	600378	2995	WUblastx.64	(Q9Y380) CGI-71 PROTEIN.	Q9Y380	94%	9	290
  HTXKK76	858755	2996	WUblastx.64	pro-pol-dUTPase polyprotein - murine	pir|T29097|T29097	44%	466	2
  				endogenous retrovirus ERV-L
  				(fragment)
  HTXKL53	783141	2997	WUblastx.64	(Q9Y3F0) CGI-05 PROTEIN.	Q9Y3F0	100%	1315	1359
  						90%	3	89
  HTXLC05	843523	3000	WUblastx.64	(Q9H189) SPHINGOSINE-1-	Q9H189	25%	4	531
  				PHOSPHATASE.
  HTXLY94	844165	3003	WUblastx.64	hypothetical protein	pir|T14744|T14744	64%	77	643
  				DKFZp586F0424.1 - human
  				(fragment)
  HTXNV66	840597	3004	WUblastx.64	(Q9UJY4) ADP-RIBOSYLATION	GGA2_HUMAN	99%	18	431
  				FACTOR BINDING PROTEIN
  				GGA2 (GOLG
  HTXOW27	839280	3006	WUblastx.64	(Q9P2V7) PROTEIN CONTAINING	Q9P2V7	100%	1652	2086
  				CXXC DOMAIN 1		92%	1309	1656
  				(HYPOTHETICAL 75.7 KDA PROT		78%	229	1188
  HTXPD86	834772	3007	WUblastx.64	(Q9UGP9) WD-REPEAT PROTEIN 5	WDR5_HUMAN	95%	74	1132
  				(FRAGMENT).
  HTXPT57	897834	3008	WUblastx.64	(O60448) NEURONAL THREAD	O60448	60%	939	838
  				PROTEIN AD7C-NTP.		52%	693	544
  						42%	564	451
  						53%	471	427
  						26%	722	543
  						35%	725	474
  						37%	1123	938
  						45%	974	849
  						53%	705	526
  						40%	1054	911
  						53%	911	834
  						75%	1104	1069
  						64%	891	841
  						51%	551	432
  						57%	425	384
  						52%	1084	926
  HTYSJ88	598718	3009	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	64%	729	457
  				CLONE KAIA0536.
  HUFAB57	587275	3012	WUblastx.64	ISOFORM B OF Q9UNP9	sp_vs|Q9UNP9-	94%	1266	1316
  					01|Q9UNP9	53%	826	1035
  HUFAO92	608175	3014	WUblastx.64	(Q9D922) 1810010G06RIK	Q9D922	69%	232	522
  				PROTEIN.
  HUFAO94	668267	3015	WUblastx.64	(Q9UI14) PRENYLATED RAB	Q9UI14	100%	3	551
  				ACCEPTOR 1.
  HUFAP33	600379	3016	HMMER	PFAM: Zona pellucida-like domain	PF00100	223.2	282	1016
  			2.1.1
  			WUblastx.64	(Q9Y211) DMBT1 PROTEIN.	Q9Y211	93%	228	1016
  						100%	26	226
  						43%	29	217
  						41%	445	546
  						38%	445	546
  						38%	445	522
  						100%	1013	1117
  HUFBV62	742892	3020	WUblastx.64	(Q9CWU4) 2410004B18RIK	Q9CWU4	76%	76	264
  				PROTEIN.
  HUKAD46	604986	3024	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	46%	589	747
  				CLONE KAIA0536.		63%	377	598
  HUKCS86	826468	3027	WUblastx.64	(Q9NX09) CDNA FLJ20500 FIS,	Q9NX09	82%	201	896
  				CLONE KAT09159.
  HUKCS86	844245	3028	WUblastx.64	(Q9NX09) CDNA FLJ20500 FIS,	Q9NX09	82%	201	896
  				CLONE KAT09159.
  HUKEA22	702029	3029	WUblastx.64	(Q9PTD7) CINGULIN.	Q9PTD7	68%	575	12
  HUKFC71	1300740	3032	WUblastx.64	(P82914) 28S RIBOSOMAL	RT15_HUMAN	97%	292	414
  				PROTEIN S15, MITOCHONDRIAL		65%	408	929
  				PRECURSOR
  HUKFC71	845161	3111	blastx.2	MITOCHONDRIAL 28S	sp|P82914|P82914	100%	293	931
  				RIBOSOMAL PROTEIN S15 (MRP-
  				S15).
  HUKFV37	844644	3033	WUblastx.64	(AAH00407) Synaptogyrin 2.	AAH00407	91%	49	720
  HUNAL39	559448	3035	WUblastx.64	(Q14288) HYPOTHETICAL	Q14288	81%	1009	629
  				PROTEIN (FRAGMENT).
  HUSAO04	877889	3036	HMMER	PFAM: tRNA synthetases class I (W	PF00579	260.4	255	965
  			2.1.1	and Y)
  			WUblastx.64	(Q9H817) CDNA FLJ13995 FIS,	Q9H817	92%	30	1451
  				CLONE Y79AA1002209, WEAKLY
  				SIMILAR TO TYR
  HUSAO04	870791	3037	HMMER	PFAM: tRNA synthetases class I (W	PF00579	212.2	297	965
  			2.1.1	and Y)
  			WUblastx.64	(Q9H817) CDNA FLJ13995 FIS,	Q9H817	92%	30	1451
  				CLONE Y79AA1002209, WEAKLY
  				SIMILAR TO TYR
  HUSCA09	853417	3038	HMMER	PFAM: Leucine Rich Repeat	PF00560	69.9	1259	1327
  			2.1.1
  			WUblastx.64	(Q9H075) HYPOTHETICAL 81.8 KDA	Q9H075	52%	44	1615
  				PROTEIN.
  HUSCJ01	740775	3039	WUblastx.64	(Q9CWP6) 2410013I23RIK	Q9CWP6	70%	1	252
  				PROTEIN.
  HUSGB23	704094	3040	WUblastx.64	(Q9H0F5) HYPOTHETICAL 43.9 KDA	Q9H0F5	97%	143	247
  				PROTEIN.
  HUSGY15	759888	3043	WUblastx.64	(Q9JKN1) ZINC TRANSPORTER	Q9JKN1	76%	162	1289
  				LIKE 2 (1810059J10RIK PROTEIN).
  HUSHD41	866498	3044	WUblastx.64	(BAB55441) CDNA FLJ14993 fis,	BAB55441	86%	2575	1541
  				clone Y79AA1001874, w		50%	2569	2483
  HUSHK65	847188	3045	HMMER	PFAM: CUB domain	PF00431	292.1	887	1222
  			2.1.1
  			WUblastx.64	(AAG41897) Neuropilin-2a(17).	AAG41897	97%	806	2122
  						95%	2727	3023
  						82%	3046	3579
  						92%	2089	2721
  						32%	2086	2619
  						31%	1547	2086
  						92%	3008	3046
  HUSIK45	853932	3046	WUblastx.64	(Q96CN8) Hypothetical 30.9 kDa	Q96CN8	91%	23	454
  				protein (Fragment).
  HUSIO57	886206	3047	WUblastx.64	(Q9Y3E2) HYPOTHETICAL	YCE3_HUMAN	100%	170	565
  				PROTEIN CGI-143.
  HUSIR70	838163	3049	WUblastx.64	(Q9HDC9) BSCV PROTEIN	Q9HDC9	97%	1	345
  				(FRAGMENT).
  HUSXP50	561962	3050	WUblastx.64	(Q63777) HYPOTHETICAL 32.0 KDA	Q63777	26%	178	291
  				PROTEIN.		57%	27	68
  						55%	113	172
  HUVCQ68	561963	3053	WUblastx.64	(Q9BV40) SIMILAR TO VESICLE-	Q9BV40	82%	40	339
  				ASSOCIATED MEMBRANE
  				PROTEIN 8 (ENDOBREVIN
  HUVEG53	797556	3055	WUblastx.64	(O62658) LINE-1 ELEMENT ORF2.	O62658	35%	1843	1676
  						39%	2148	1939
  						60%	1391	1263
  HWAAH11	815548	3056	WUblastx.64	(Q9HAB3) CDNA FLJ11856 FIS,	Q9HAB3	74%	362	754
  				CLONE HEMBA1006789 (SIMILAR
  				TO HYPOTHETIC
  HWAAQ28	806588	3057	WUblastx.64	(Q9NZZ8) HSPC169	Q9NZZ8	96%	62	676
  				(HYPOTHETICAL 33.9 KDA		23%	649	801
  				PROTEIN).		100%	651	980
  HWAAY60	745917	3058	WUblastx.64	(Q9BQA1) BA552M11.2.2 (NOVEL	Q9BQA1	76%	504	722
  				PROTEIN (ISOFORM 2))
  				(UNKNOWN) (PROTEIN
  HWABR43	823367	3059	WUblastx.64	(Q9BVD9) UNKNOWN (PROTEIN	Q9BVD9	75%	2081	2034
  				FOR MGC: 5149).		79%	2266	2093
  HWACZ33	827311	3061	WUblastx.64	(Q9CTA6) 1110035E02RIK	Q9CTA6	62%	389	436
  				PROTEIN (FRAGMENT).		73%	234	392
  HWADV90	897732	3062	WUblastx.64	(Q9NX17) CDNA FLJ20489 FIS,	Q9NX17	78%	2375	2163
  				CLONE KAT08285.
  HWAEB52	873207	3063	WUblastx.64	(Q9H8U5) CDNA FLJ13219 FIS,	Q9H8U5	68%	878	1426
  				CLONE NT2RP4001849, WEAKLY		60%	389	685
  				SIMILAR TO SH3		23%	356	667
  						28%	874	1113
  						31%	1191	1286
  						40%	344	442
  						35%	299	466
  HWBAK71	853581	3064	WUblastx.64	(Q9T9V8) NADH	Q9T9V8	87%	77	175
  				DEHYDROGENASE SUBUNIT 3.		83%	305	340
  HWBBU75	780360	3065	WUblastx.64	(Q9R189) MUNC13-4 PROTEIN.	Q9R189	82%	1454	2362
  						73%	913	1434
  						80%	194	952
  						62%	2229	2729
  						31%	1586	1711
  						34%	401	532
  HWBCN81	853580	3066	WUblastx.64	(Q9H101) DJ776F14.2 (A NOVEL	Q9H101	94%	195	560
  				PROTEIN MEMBER OF THE PTPNS		33%	222	560
  				(PROTEIN TYR		100%	150	221
  HWBCX93	853418	3068	WUblastx.64	(Q9NX85) CDNA FLJ20378 FIS,	Q9NX85	80%	642	553
  				CLONE KAIA0536.		84%	796	641
  HWHGV77	788555	3072	WUblastx.64	(Q9D6W1) 2310050C09RIK	Q9D6W1	70%	136	621
  				PROTEIN.		51%	127	552
  						37%	127	588
  						47%	124	318
  						100%	721	756
  HWHGW09	702032	3073	HMMER	PFAM: Uncharacterized protein family	PF01027	68.3	271	444
  			2.1.1
  			WUblastx.64	(Q9Y3C2) CGI-119 PROTEIN.	Q9Y3C2	84%	256	450
  HWHPU44	778090	3075	WUblastx.64	(Q9H1C8) PUTATIVE	Q9H1C8	97%	311	547
  				CYTOPLASMATIC PROTEIN.
  HWLAT50	840753	3077	WUblastx.64	(Q99JR4) UNKNOWN (PROTEIN	Q99JR4	48%	1864	2106
  				FOR IMAGE: 3599558)
  				(FRAGMENT).
  HWLGP26	834770	3079	WUblastx.64	(Q9NP87) DNA POLYMERASE MU.	Q9NP87	93%	674	760
  						100%	269	298
  						94%	295	465
  						87%	432	623
  						100%	3	254
  HWLHO31	837480	3080	WUblastx.64	(Q9NQT8) KINESIN-LIKE PROTEIN	Q9NQT8	100%	41	205
  				GAKIN.		96%	190	1674
  HWLJN08	800626	3082	WUblastx.64	(Q9BX68) HIT-17 KDA.	Q9BX68	99%	434	844
  HWLRE03	831198	3083	WUblastx.64	(O60646) HYPOTHETICAL 53.8 KDA	O60646	100%	1008	1232
  				PROTEIN (FRAGMENT).		99%	1238	2431
  HWTBL86	791720	3089	WUblastx.64	hypothetical protein	pir|T17285|T17285	91%	1	1080
  				DKFZp434N0535.1 - human
  				(fragment)
  HWTBX66	732187	3090	WUblastx.64	(Q9BRI8) UNKNOWN (PROTEIN	Q9BRI8	71%	63	1073
  				FOR MGC: 11332).
  HYAAD61	897736	3092	WUblastx.64	(O21101) ATPASE 6/8	O21101	56%	2009	2179
  				(FRAGMENT).
  HYBAP75	853561	3094	WUblastx.64	(Q9N032) UNNAMED PROTEIN	Q9N032	64%	909	1025
  				PRODUCT.

  
  RACE Protocol for Recovery of Full-Length Genes
• Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNA is reverse transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI, SalI and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.
• Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from Clontech which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
• An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
• RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes
• Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′ RACE. While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant gene.
• The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC (e.g., as described in columns 2 and 3 of Table 1A, and/or as set forth in Table 1B, Table 6, or Table 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Table 1A and Table 7. These deposits are referred to as “the deposits” herein. The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A and/or 1B (ATCC Deposit No: Z). A clone which is isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC Deposits by use of a sequence (or portion thereof) described in, for example Tables 1A and/or 1B or 2, by procedures hereinafter further described, and others apparent to those skilled in the art.
• Also provided in Table 1A and 7 is the name of the vector which contains the cDNA clone. Each vector is routinely used in the art. The following additional information is provided for convenience.
• Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from Stratagene.
• Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. See, for instance, Gruber, C. E., et al., Focus 15:59-(1993). Vector lafinid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).
• The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or the deposited clone (ATCC Deposit No: Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
• Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in ATCC Deposit No: Z, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
• The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
• The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
• The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.
• The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA sequence contained in ATCC Deposit No: Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in ATCC Deposit No: Z.
• Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in Table 1C column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in Table 1C column 6, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
• Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
• Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
• Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, wherein sequentially delineated sequences in the table (i.e. corresponding to those exons located closest to each other) are directly contiguous in a 5′ to 3′ orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1C. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.
• In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same Clone ID (see Table 1C, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• Table 3
• Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fifth column of Table 1A and/or the fourth column of Table 1B.1, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a +14. More specifically, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone). In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.

  LENGTHY TABLE REFERENCED HERE
  US20070048297A1-20070301-T00006
  Please refer to the end of the specification for access instructions.

  
  Description of Table 4
• Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 of Table 4 provides the tissue/cell source identifier code disclosed in Table 1B.2, Column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.

  TABLE 4
  Code	Description	Tissue	Organ	Cell Line	Disease	Vector
  AR022	a_Heart	a_Heart
  AR023	a_Liver	a_Liver
  AR024	a_mammary gland	a_mammary gland
  AR025	a_Prostate	a_Prostate
  AR026	a_small intestine	a_small intestine
  AR027	a_Stomach	a_Stomach
  AR028	Blood B cells	Blood B cells
  AR029	Blood B cells activated	Blood B cells activated
  AR030	Blood B cells resting	Blood B cells resting
  AR031	Blood T cells activated	Blood T cells activated
  AR032	Blood T cells resting	Blood T cells resting
  AR033	brain	brain
  AR034	breast	breast
  AR035	breast cancer	breast cancer
  AR036	Cell Line CAOV3	Cell Line CAOV3
  AR037	cell line PA-1	cell line PA-1
  AR038	cell line transformed	cell line transformed
  AR039	colon	colon
  AR040	colon (9808co65R)	colon (9808co65R)
  AR041	colon (9809co15)	colon (9809co15)
  AR042	colon cancer	colon cancer
  AR043	colon cancer (9808co64R)	colon cancer (9808co64R)
  AR044	colon cancer 9809co14	colon cancer 9809co14
  AR050	Donor II B Cells 24 hrs	Donor II B Cells 24 hrs
  AR051	Donor II B Cells 72 hrs	Donor II B Cells 72 hrs
  AR052	Donor II B-Cells 24 hrs.	Donor II B-Cells 24 hrs.
  AR053	Donor II B-Cells 72 hrs	Donor II B-Cells 72 hrs
  AR054	Donor II Resting B Cells	Donor II Resting B Cells
  AR055	Heart	Heart
  AR056	Human Lung (clonetech)	Human Lung (clonetech)
  AR057	Human Mammary (clontech)	Human Mammary (clontech)
  AR058	Human Thymus (clonetech)	Human Thymus (clonetech)
  AR059	Jurkat (unstimulated)	Jurkat (unstimulated)
  AR060	Kidney	Kidney
  AR061	Liver	Liver
  AR062	Liver (Clontech)	Liver (Clontech)
  AR063	Lymphocytes chronic lymphocytic leukaemia	Lymphocytes chronic
  		lymphocytic leukaemia
  AR064	Lymphocytes diffuse large B cell lymphoma	Lymphocytes diffuse large B
  		cell lymphoma
  AR065	Lymphocytes follicular lymphoma	Lymphocytes follicular
  		lymphoma
  AR066	normal breast	normal breast
  AR067	Normal Ovarian (4004901)	Normal Ovarian (4004901)
  AR068	Normal Ovary 9508G045	Normal Ovary 9508G045
  AR069	Normal Ovary 9701G208	Normal Ovary 9701G208
  AR070	Normal Ovary 9806G005	Normal Ovary 9806G005
  AR071	Ovarian Cancer	Ovarian Cancer
  AR072	Ovarian Cancer (9702G001)	Ovarian Cancer (9702G001)
  AR073	Ovarian Cancer (9707G029)	Ovarian Cancer (9707G029)
  AR074	Ovarian Cancer (9804G011)	Ovarian Cancer (9804G011)
  AR075	Ovarian Cancer (9806G019)	Ovarian Cancer (9806G019)
  AR076	Ovarian Cancer (9807G017)	Ovarian Cancer (9807G017)
  AR077	Ovarian Cancer (9809G001)	Ovarian Cancer (9809G001)
  AR078	ovarian cancer 15799	ovarian cancer 15799
  AR079	Ovarian Cancer 17717AID	Ovarian Cancer 17717AID
  AR080	Ovarian Cancer 4004664B1	Ovarian Cancer 4004664B1
  AR081	Ovarian Cancer 4005315A1	Ovarian Cancer 4005315A1
  AR082	ovarian cancer 94127303	ovarian cancer 94127303
  AR083	Ovarian Cancer 96069304	Ovarian Cancer 96069304
  AR084	Ovarian Cancer 9707G029	Ovarian Cancer 9707G029
  AR085	Ovarian Cancer 9807G045	Ovarian Cancer 9807G045
  AR086	ovarian cancer 9809G001	ovarian cancer 9809G001
  AR087	Ovarian Cancer 9905C032RC	Ovarian Cancer
  		9905C032RC
  AR088	Ovarian cancer 9907 C00 3rd	Ovarian cancer 9907 C00 3rd
  AR089	Prostate	Prostate
  AR090	Prostate (clonetech)	Prostate (clonetech)
  AR091	prostate cancer	prostate cancer
  AR092	prostate cancer #15176	prostate cancer #15176
  AR093	prostate cancer #15509	prostate cancer #15509
  AR094	prostate cancer #15673	prostate cancer #15673
  AR095	Small Intestine (Clontech)	Small Intestine (Clontech)
  AR096	Spleen	Spleen
  AR097	Thymus T cells activated	Thymus T cells activated
  AR098	Thymus T cells resting	Thymus T cells resting
  AR099	Tonsil	Tonsil
  AR100	Tonsil geminal center centroblast	Tonsil geminal center
  		centroblast
  AR101	Tonsil germinal center B cell	Tonsil germinal center B cell
  AR102	Tonsil lymph node	Tonsil lymph node
  AR103	Tonsil memory B cell	Tonsil memory B cell
  AR104	Whole Brain	Whole Brain
  AR105	Xenograft ES-2	Xenograft ES-2
  AR106	Xenograft SW626	Xenograft SW626
  AR119	001: IL-2	001: IL-2
  AR120	001: IL-2.1	001: IL-2.1
  AR121	001: IL-2_b	001: IL-2_b
  AR124	002: Monocytes untreated (1 hr)	002: Monocytes untreated
  		(1 hr)
  AR125	002: Monocytes untreated (5 hrs)	002: Monocytes untreated
  		(5 hrs)
  AR126	002: Control.1C	002: Control.1C
  AR127	002: IL2.1C	002: IL2.1C
  AR130	003: Placebo-treated Rat Lacrimal Gland	003: Placebo-treated Rat
  		Lacrimal Gland
  AR131	003: Placebo-treated Rat Submandibular	003: Placebo-treated Rat
  	Gland	Submandibular Gland
  AR135	004: Monocytes untreated (5 hrs)	004: Monocytes untreated
  		(5 hrs)
  AR136	004: Monocytes untreated 1 hr	004: Monocytes untreated
  		1 hr
  AR139	005: Placebo (48 hrs)	005: Placebo (48 hrs)
  AR140	006: pC4 (24 hrs)	006: pC4 (24 hrs)
  AR141	006: pC4 (48 hrs)	006: pC4 (48 hrs)
  AR152	007: PHA(1 hr)	007: PHA(1 hr)
  AR153	007: PHA(6 HRS)	007: PHA(6 HRS)
  AR154	007: PMA(6 hrs)	007: PMA(6 hrs)
  AR155	008: 1449_#2	008: 1449_#2
  AR161	01: A - max 24	01: A - max 24
  AR162	01: A - max 26	01: A - max 26
  AR163	01: A - max 30	01: A - max 30
  AR164	01: B - max 24	01: B - max 24
  AR165	01: B - max 26	01: B - max 26
  AR166	01: B - max 30	01: B - max 30
  AR167	1449 Sample	1449 Sample
  AR168	3T3P10 1.0 uM insulin	3T3P10 1.0 uM insulin
  AR169	3T3P10 10 nM Insulin	3T3P10 10 nM Insulin
  AR170	3T3P10 10 uM insulin	3T3P10 10 uM insulin
  AR171	3T3P10 No Insulin	3T3P10 No Insulin
  AR172	3T3P4	3T3P4
  AR173	Adipose (41892)	Adipose (41892)
  AR174	Adipose Diabetic (41611)	Adipose Diabetic (41611)
  AR175	Adipose Diabetic (41661)	Adipose Diabetic (41661)
  AR176	Adipose Diabetic (41689)	Adipose Diabetic (41689)
  AR177	Adipose Diabetic (41706)	Adipose Diabetic (41706)
  AR178	Adipose Diabetic (42352)	Adipose Diabetic (42352)
  AR179	Adipose Diabetic (42366)	Adipose Diabetic (42366)
  AR180	Adipose Diabetic (42452)	Adipose Diabetic (42452)
  AR181	Adipose Diabetic (42491)	Adipose Diabetic (42491)
  AR182	Adipose Normal (41843)	Adipose Normal (41843)
  AR183	Adipose Normal (41893)	Adipose Normal (41893)
  AR184	Adipose Normal (42452)	Adipose Normal (42452)
  AR185	Adrenal Gland	Adrenal Gland
  AR186	Adrenal Gland + Whole Brain	Adrenal Gland + Whole
  		Brain
  AR187	B7(1 hr) + (inverted)	B7(1 hr) + (inverted)
  AR188	Breast (18275A2B)	Breast (18275A2B)
  AR189	Breast (4004199)	Breast (4004199)
  AR190	Breast (4004399)	Breast (4004399)
  AR191	Breast (4004943B7)	Breast (4004943B7)
  AR192	Breast (4005570B1)	Breast (4005570B1)
  AR193	Breast Cancer (4004127A30)	Breast Cancer (4004127A30)
  AR194	Breast Cancer (400443A21)	Breast Cancer (400443A21)
  AR195	Breast Cancer (4004643A2)	Breast Cancer (4004643A2)
  AR196	Breast Cancer (4004710A7)	Breast Cancer (4004710A7)
  AR197	Breast Cancer (4004943A21)	Breast Cancer (4004943A21)
  AR198	Breast Cancer (400553A2)	Breast Cancer (400553A2)
  AR199	Breast Cancer (9805C046R)	Breast Cancer (9805C046R)
  AR200	Breast Cancer (9806C012R)	Breast Cancer (9806C012R)
  AR201	Breast Cancer (ODQ 45913)	Breast Cancer (ODQ 45913)
  AR202	Breast Cancer (ODQ45913)	Breast Cancer (ODQ45913)
  AR203	Breast Cancer (ODQ4591B)	Breast Cancer (ODQ4591B)
  AR204	Colon Cancer (15663)	Colon Cancer (15663)
  AR205	Colon Cancer (4005144A4)	Colon Cancer (4005144A4)
  AR206	Colon Cancer (4005413A4)	Colon Cancer (4005413A4)
  AR207	Colon Cancer (4005570B1)	Colon Cancer (4005570B1)
  AR208	Control RNA #1	Control RNA #1
  AR209	Control RNA #2	Control RNA #2
  AR210	Cultured Preadipocyte (blue)	Cultured Preadipocyte (blue)
  AR211	Cultured Preadipocyte (Red)	Cultured Preadipocyte (Red)
  AR212	Donor II B-Cells 24 hrs	Donor II B-Cells 24 hrs
  AR213	Donor II Resting B-Cells	Donor II Resting B-Cells
  AR214	H114EP12 10 nM Insulin	H114EP12 10 nM Insulin
  AR215	H114EP12 (10 nM insulin)	H114EP12 (10 nM insulin)
  AR216	H114EP12 (2.6 ug/ul)	H114EP12 (2.6 ug/ul)
  AR217	H114EP12 (3.6 ug/ul)	H114EP12 (3.6 ug/ul)
  AR218	HUVEC #1	HUVEC #1
  AR219	HUVEC #2	HUVEC #2
  AR221	L6 undiff.	L6 undiff.
  AR222	L6 Undifferentiated	L6 Undifferentiated
  AR223	L6P8 + 10 nM Insulin	L6P8 + 10 nM Insulin
  AR224	L6P8 + HS	L6P8 + HS
  AR225	L6P8 10 nM Insulin	L6P8 10 nM Insulin
  AR226	Liver (00-06-A007B)	Liver (00-06-A007B)
  AR227	Liver (96-02-A075)	Liver (96-02-A075)
  AR228	Liver (96-03-A144)	Liver (96-03-A144)
  AR229	Liver (96-04-A138)	Liver (96-04-A138)
  AR230	Liver (97-10-A074B)	Liver (97-10-A074B)
  AR231	Liver (98-09-A242A)	Liver (98-09-A242A)
  AR232	Liver Diabetic (1042)	Liver Diabetic (1042)
  AR233	Liver Diabetic (41616)	Liver Diabetic (41616)
  AR234	Liver Diabetic (41955)	Liver Diabetic (41955)
  AR235	Liver Diabetic (42352R)	Liver Diabetic (42352R)
  AR236	Liver Diabetic (42366)	Liver Diabetic (42366)
  AR237	Liver Diabetic (42483)	Liver Diabetic (42483)
  AR238	Liver Diabetic (42491)	Liver Diabetic (42491)
  AR239	Liver Diabetic (99-09-A281A)	Liver Diabetic (99-09-
  		A281A)
  AR240	Lung	Lung
  AR241	Lung (27270)	Lung (27270)
  AR242	Lung (2727Q)	Lung (2727Q)
  AR243	Lung Cancer (4005116A1)	Lung Cancer (4005116A1)
  AR244	Lung Cancer (4005121A5)	Lung Cancer (4005121A5)
  AR245	Lung Cancer (4005121A5))	Lung Cancer (4005121A5))
  AR246	Lung Cancer (4005340A4)	Lung Cancer (4005340A4)
  AR247	Mammary Gland	Mammary Gland
  AR248	Monocyte (CT)	Monocyte (CT)
  AR249	Monocyte (OCT)	Monocyte (OCT)
  AR250	Monocytes (CT)	Monocytes (CT)
  AR251	Monocytes (INFG 18 hr)	Monocytes (INFG 18 hr)
  AR252	Monocytes (INFG 18 hr)	Monocytes (INFG 18 hr)
  AR253	Monocytes (INFG 8-11)	Monocytes (INFG 8-11)
  AR254	Monocytes (OCT)	Monocytes (OCT)
  AR255	Muscle (91-01-A105)	Muscle (91-01-A105)
  AR256	Muscle (92-04-A059)	Muscle (92-04-A059)
  AR257	Muscle (97-11-A056d)	Muscle (97-11-A056d)
  AR258	Muscle (99-06-A210A)	Muscle (99-06-A210A)
  AR259	Muscle (99-07-A203B)	Muscle (99-07-A203B)
  AR260	Muscle (99-7-A203B)	Muscle (99-7-A203B)
  AR261	Muscle Diabetic (42352R)	Muscle Diabetic (42352R)
  AR262	Muscle Diabetic (42366)	Muscle Diabetic (42366)
  AR263	NK-19 Control	NK-19 Control
  AR264	NK-19 IL Treated 72 hrs	NK-19 IL Treated 72 hrs
  AR265	NK-19 UK Treated 72 hrs.	NK-19 UK Treated 72 hrs.
  AR266	Omentum Normal (94-08-B009)	Omentum Normal (94-08-
  		B009)
  AR267	Omentum Normal (97-01-A039A)	Omentum Normal (97-01-
  		A039A)
  AR268	Omentum Normal (97-04-A114C)	Omentum Normal (97-04-
  		A114C)
  AR269	Omentum Normal (97-06-A117C)	Omentum Normal (97-06-
  		A117C)
  AR270	Omentum Normal (97-09-B004C)	Omentum Normal (97-09-
  		B004C)
  AR271	Ovarian Cancer (17717AID)	Ovarian Cancer (17717AID)
  AR272	Ovarian Cancer (9905C023RC)	Ovarian Cancer
  		(9905C023RC)
  AR273	Ovarian Cancer (9905C032RC)	Ovarian Cancer
  		(9905C032RC)
  AR274	Ovary (9508G045)	Ovary (9508G045)
  AR275	Ovary (9701G208)	Ovary (9701G208)
  AR276	Ovary 9806G005	Ovary 9806G005
  AR277	Pancreas	Pancreas
  AR278	Placebo	Placebo
  AR279	rIL2 Control	rIL2 Control
  AR280	RSS288L	RSS288L
  AR281	RSS288LC	RSS288LC
  AR282	Salivary Gland	Salivary Gland
  AR283	Skeletal Muscle	Skeletal Muscle
  AR284	Skeletal Muscle (91-01-A105)	Skeletal Muscle (91-01-
  		A105)
  AR285	Skeletal Muscle (42180)	Skeletal Muscle (42180)
  AR286	Skeletal Muscle (42386)	Skeletal Muscle (42386)
  AR287	Skeletal Muscle (42461)	Skeletal Muscle (42461)
  AR288	Skeletal Muscle (91-01-A105)	Skeletal Muscle (91-01-
  		A105)
  AR289	Skeletal Muscle (92-04-A059)	Skeletal Muscle (92-04-
  		A059)
  AR290	Skeletal Muscle (96-08-A171)	Skeletal Muscle (96-08-
  		A171)
  AR291	Skeletal Muscle (97-07-A190A)	Skeletal Muscle (97-07-
  		A190A)
  AR292	Skeletal Muscle Diabetic (42352)	Skeletal Muscle Diabetic
  		(42352)
  AR293	Skeletal Muscle Diabetic (42366)	Skeletal Muscle Diabetic
  		(42366)
  AR294	Skeletal Muscle Diabetic (42395)	Skeletal Muscle Diabetic
  		(42395)
  AR295	Skeletal Muscle Diabetic (42483)	Skeletal Muscle Diabetic
  		(42483)
  AR296	Skeletal Muscle Diabetic (42491)	Skeletal Muscle Diabetic
  		(42491)
  AR297	Skeletal Muscle Diabetic 42352	Skeletal Muscle Diabetic
  		42352
  AR298	Skeletal Musle (42461)	Skeletal Musle (42461)
  AR299	Small Intestine	Small Intestine
  AR300	Stomach	Stomach
  AR301	T-Cell + HDPBQ71.fc 1449 16 hrs	T-Cell + HDPBQ71.fc 1449
  		16 hrs
  AR302	T-Cell + HDPBQ71.fc 1449 6 hrs	T-Cell + HDPBQ71.fc 1449
  		6 hrs
  AR303	T-Cell + IL2 16 hrs	T-Cell + IL2 16 hrs
  AR304	T-Cell + IL2 6 hrs	T-Cell + IL2 6 hrs
  AR306	T-Cell Untreated 16 hrs	T-Cell Untreated 16 hrs
  AR307	T-Cell Untreated 6 hrs	T-Cell Untreated 6 hrs
  AR308	T-Cells 24 hours	T-Cells 24 hours
  AR309	T-Cells 24 hrs	T-Cells 24 hrs
  AR310	T-Cells 24 hrs.	T-Cells 24 hrs.
  AR311	T-Cells 24 hrs	T-Cells 24 hrs
  AR312	T-Cells 4 days	T-Cells 4 days
  AR313	Thymus	Thymus
  AR314	TRE	TRE
  AR315	TREC	TREC
  AR316	Virtual Mixture	Virtual Mixture
  AR317	B lymphocyte,	B lymphocyte,
  AR318	(non-T; non-B)	(non-T; non-B)
  AR326	001-293 RNA (Vector Control)	001-293 RNA (Vector
  		Control)
  AR327	001: Control	001: Control
  AR328	001: Control.1	001: Control.1
  AR355	Acute Lymphocyte Leukemia	Acute Lymphocyte
  		Leukemia
  AR356	AML Patient #11	AML Patient #11
  AR357	AML Patient #2	AML Patient #2
  AR358	AML Patient #2 SGAH	AML Patient #2 SGAH
  AR359	AML Patient#2	AML Patient#2
  AR360	Aorta	Aorta
  AR361	B Cell	B Cell
  AR362	B lymphoblast	B lymphoblast
  AR363	B lymphocyte	B lymphocyte
  AR364	B lymphocytes	B lymphocytes
  AR365	B-cell	B-cell
  AR366	B-Cells	B-Cells
  AR367	B-Lymphoblast	B-Lymphoblast
  AR368	B-Lymphocytes	B-Lymphocytes
  AR369	Bladder	Bladder
  AR370	Bone Marrow	Bone Marrow
  AR371	Bronchial Epithelial Cell	Bronchial Epithelial Cell
  AR372	Bronchial Epithelial Cells	Bronchial Epithelial Cells
  AR373	Caco-2A	Caco-2A
  AR374	Caco-2B	Caco-2B
  AR375	Caco-2C	Caco-2C
  AR376	Cardiac #1	Cardiac #1
  AR377	Cardiac #2	Cardiac #2
  AR378	Chest Muscle	Chest Muscle
  AR381	Dendritic Cell	Dendritic Cell
  AR382	Dendritic cells	Dendritic cells
  AR383	E. coli	E. coli
  AR384	Epithelial Cells	Epithelial Cells
  AR385	Esophagus	Esophagus
  AR386	FPPS	FPPS
  AR387	FPPSC	FPPSC
  AR388	HepG2 Cell Line	HepG2 Cell Line
  AR389	HepG2 Cell line Buffer 1 hr.	HepG2 Cell line Buffer 1 hr.
  AR390	HepG2 Cell line Buffer 06 hr	HepG2 Cell line Buffer 06 hr
  AR391	HepG2 Cell line Buffer 24 hr.	HepG2 Cell line Buffer 24 hr.
  AR392	HepG2 Cell line Insulin 01 hr.	HepG2 Cell line Insulin 01 hr.
  AR393	HepG2 Cell line Insulin 06 hr.	HepG2 Cell line Insulin 06 hr.
  AR394	HepG2 Cell line Insulin 24 hr.	HepG2 Cell line insulin 24 hr.
  AR398	HMC-1	HMC-1
  AR399	HMCS	HMCS
  AR400	HMSC	HMSC
  AR401	HUVEC #3	HUVEC #3
  AR402	HUVEC #4	HUVEC #4
  AR404	KIDNEY NORMAL	KIDNEY NORMAL
  AR405	KIDNEY TUMOR	KIDNEY TUMOR
  AR406	KIDNEY TUMOR
  AR407	Lymph Node	Lymph Node
  AR408	Macrophage	Macrophage
  AR409	Megakarioblast	Megakarioblast
  AR410	Monocyte	Monocyte
  AR411	Monocytes	Monocytes
  AR412	Myocardium	Myocardium
  AR413	Myocardium #3	Myocardium #3
  AR414	Myocardium #4	Myocardium #4
  AR415	Myocardium #5	Myocardium #5
  AR416	NK	NK
  AR417	NK cell	NK cell
  AR418	NK cells	NK cells
  AR419	NKYa	NKYa
  AR420	NKYa019	NKYa019
  AR421	Ovary	Ovary
  AR422	Patient #11	Patient #11
  AR423	Peripheral blood	Peripheral blood
  AR424	Primary Adipocytes	Primary Adipocytes
  AR425	Promyeloblast	Promyeloblast
  AR427	RSSWT	RSSWT
  AR428	RSSWTC	RSSWTC
  AR429	SW 480(G1)	SW 480(G1)
  AR430	SW 480(G2)	SW 480(G2)
  AR431	SW 480(G3)	SW 480(G3)
  AR432	SW 480(G4)	SW 480(G4)
  AR433	SW 480(G5)	SW 480(G5)
  AR434	T Lymphoblast	T Lymphoblast
  AR435	T Lymphocyte	T Lymphocyte
  AR436	T-Cell	T-Cell
  AR438	T-Cell,	T-Cell,
  AR439	T-Cells	T-Cells
  AR440	T-lymphoblast	T-lymphoblast
  AR441	Th 1	Th 1
  AR442	Th 2	Th 2
  AR443	Th1	Th1
  AR444	Th2	Th2
  H0002	Human Adult Heart	Human Adult Heart	Heart			Uni-ZAP XR
  H0003	Human Adult Liver	Human Adult Liver	Liver			Uni-ZAP XR
  H0004	Human Adult Spleen	Human Adult Spleen	Spleen			Uni-ZAP XR
  H0007	Human Cerebellum	Human Cerebellum	Brain			Uni-ZAP XR
  H0008	Whole 6 Week Old Embryo					Uni-ZAP XR
  H0009	Human Fetal Brain					Uni-ZAP XR
  H0010	Human Fetal Hepatic	Human Fetal Liver	Liver			Uni-ZAP XR
  H0011	Human Fetal Kidney	Human Fetal Kidney	Kidney			Uni-ZAP XR
  H0012	Human Fetal Kidney	Human Fetal Kidney	Kidney			Uni-ZAP XR
  H0013	Human 8 Week Whole Embryo	Human 8 Week Old Embryo	Embryo			Uni-ZAP XR
  H0014	Human Gall Bladder	Human Gall Bladder	Gall Bladder			Uni-ZAP XR
  H0015	Human Gall Bladder, fraction II	Human Gall Bladder	Gall Bladder			Uni-ZAP XR
  H0018	Human Greater Omentum, fII remake	Human Greater Omentum	peritoneum			Uni-ZAP XR
  H0019	Human Fetal Heart	Human Fetal Heart	Heart			pBluescript
  H0020	Human Hippocampus	Human Hippocampus	Brain			Uni-ZAP XR
  H0021	Human Infant Adrenal Gland	Human Infant Adrenal Gland	Adrenal gland			Uni-ZAP XR
  H0022	Jurkat Cells	Jurkat T-Cell Line				Lambda ZAP II
  H0023	Human Fetal Lung					Uni-ZAP XR
  H0024	Human Fetal Lung III	Human Fetal Lung	Lung			Uni-ZAP XR
  H0025	Human Adult Lymph Node	Human Adult Lymph Node	Lymph Node			Lambda ZAP II
  H0026	Namalwa Cells	Namalwa B-Cell Line, EBV				Lambda ZAP II
  		immortalized
  H0028	Human Old Ovary	Human Old Ovary	Ovary			pBluescript
  H0029	Human Pancreas	Human Pancreas	Pancreas			Uni-ZAP XR
  H0030	Human Placenta					Uni-ZAP XR
  H0031	Human Placenta	Human Placenta	Placenta			Uni-ZAP XR
  H0032	Human Prostate	Human Prostate	Prostate			Uni-ZAP XR
  H0033	Human Pituitary	Human Pituitary				Uni-ZAP XR
  H0035	Human Salivary Gland	Human Salivary Gland	Salivary gland			Uni-ZAP XR
  H0036	Human Adult Small Intestine	Human Adult Small Intestine	Small Int.			Uni-ZAP XR
  H0037	Human Adult Small Intestine	Human Adult Small Intestine	Small Int.			pBluescript
  H0038	Human Testes	Human Testes	Testis			Uni-ZAP XR
  H0039	Human Pancreas Tumor	Human Pancreas Tumor	Pancreas		disease	Uni-ZAP XR
  H0040	Human Testes Tumor	Human Testes Tumor	Testis		disease	Uni-ZAP XR
  H0041	Human Fetal Bone	Human Fetal Bone	Bone			Uni-ZAP XR
  H0042	Human Adult Pulmonary	Human Adult Pulmonary	Lung			Uni-ZAP XR
  H0044	Human Cornea	Human Cornea	eye			Uni-ZAP XR
  H0045	Human Esophagus, Cancer	Human Esophagus, cancer	Esophagus		disease	Uni-ZAP XR
  H0046	Human Endometrial Tumor	Human Endometrial Tumor	Uterus		disease	Uni-ZAP XR
  H0047	Human Fetal Liver	Human Fetal Liver	Liver			Uni-ZAP XR
  H0048	Human Pineal Gland	Human Pineal Gland				Uni-ZAP XR
  H0049	Human Fetal Kidney	Human Fetal Kidney	Kidney			Uni-ZAP XR
  H0050	Human Fetal Heart	Human Fetal Heart	Heart			Uni-ZAP XR
  H0051	Human Hippocampus	Human Hippocampus	Brain			Uni-ZAP XR
  H0052	Human Cerebellum	Human Cerebellum	Brain			Uni-ZAP XR
  H0053	Human Adult Kidney	Human Adult Kidney	Kidney			Uni-ZAP XR
  H0056	Human Umbilical Vein, Endo. remake	Human Umbilical Vein	Umbilical vein			Uni-ZAP XR
  		Endothelial Cells
  H0057	Human Fetal Spleen					Uni-ZAP XR
  H0058	Human Thymus Tumor	Human Thymus Tumor	Thymus		disease	Lambda ZAP II
  H0059	Human Uterine Cancer	Human Uterine Cancer	Uterus		disease	Lambda ZAP II
  H0060	Human Macrophage	Human Macrophage	Blood	Cell Line		pBluescript
  H0061	Human Macrophage	Human Macrophage	Blood	Cell Line		pBluescript
  H0062	Human Thymus	Human Thymus	Thymus			Uni-ZAP XR
  H0063	Human Thymus	Human Thymus	Thymus			Uni-ZAP XR
  H0064	Human Right Hemisphere of Brain	Human Brain, right	Brain			Uni-ZAP XR
  		hemisphere
  H0067	Human left hemisphere, adult	Human Left Hemisphere,	Brain			Lambda ZAP II
  		Adult
  H0068	Human Skin Tumor	Human Skin Tumor	Skin		disease	Uni-ZAP XR
  H0069	Human Activated T-Cells	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0070	Human Pancreas	Human Pancreas	Pancreas			Uni-ZAP XR
  H0071	Human Infant Adrenal Gland	Human Infant Adrenal Gland	Adrenal gland			Uni-ZAP XR
  H0073	Human Leiomyeloid Carcinoma	Human Leiomyeloid	Muscle		disease	Uni-ZAP XR
  		Carcinoma
  H0074	Human Platelets	Human Platelets	Blood	Cell Line		Uni-ZAP XR
  H0075	Human Activated T-Cells (II)	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0077	Human Thymus Tumor	Human Thymus Tumor	Thymus		disease	Lambda ZAP II
  H0078	Human Lung Cancer	Human Lung Cancer	Lung		disease	Lambda ZAP II
  H0079	Human Whole 7 Week Old Embryo (II)	Human Whole 7 Week Old	Embryo			Uni-ZAP XR
  		Embryo
  H0080	Human Whole 6 Week Old Embryo (II)	Human Whole Six Week Old	Embryo			Lambda ZAP II
  		Embryo
  H0081	Human Fetal Epithelium (Skin)	Human Fetal Skin	Skin			Uni-ZAP XR
  H0082	Human Fetal Muscle	Human Fetal Muscle	Sk Muscle			Uni-ZAP XR
  H0083	HUMAN JURKAT MEMBRANE BOUND	Jurkat Cells				Uni-ZAP XR
  	POLYSOMES
  H0085	Human Colon	Human Colon				Lambda ZAP II
  H0086	Human epithelioid sarcoma	Epithelioid Sarcoma, muscle	Sk Muscle		disease	Uni-ZAP XR
  H0087	Human Thymus	Human Thymus				pBluescript
  H0090	Human T-Cell Lymphoma	T-Cell Lymphoma	T-Cell		disease	Uni-ZAP XR
  H0092	Human Pancreas Tumor	Human Pancreas Tumor	Pancreas		disease	Uni-ZAP XR
  H0093	Human Greater Omentum Tumor	Human Greater Omentum	peritoneum		disease	Uni-ZAP XR
  H0095	Human Greater Omentum, RNA Remake	Human Greater Omentum	peritoneum			Uni-ZAP XR
  H0097	Human Adult Heart, subtracted	Human Adult Heart	Heart			pBluescript
  H0098	Human Adult Liver, subtracted	Human Adult Liver	Liver			Uni-ZAP XR
  H0099	Human Lung Cancer, subtracted	Human Lung Cancer	Lung			pBluescript
  H0100	Human Whole Six Week Old Embryo	Human Whole Six Week Old	Embryo			Uni-ZAP XR
  		Embryo
  H0101	Human 7 Weeks Old Embryo, subtracted	Human Whole 7 Week Old	Embryo			Lambda ZAP II
  		Embryo
  H0102	Human Whole 6 Week Old Embryo (II), subt	Human Whole Six Week Old	Embryo			pBluescript
  		Embryo
  H0103	Human Fetal Brain, subtracted	Human Fetal Brain	Brain			Uni-ZAP XR
  H0105	Human Fetal Heart, subtracted	Human Fetal Heart	Heart			pBluescript
  H0107	Human Infant Adrenal Gland, subtracted	Human Infant Adrenal Gland	Adrenal gland			pBluescript
  H0108	Human Adult Lymph Node, subtracted	Human Adult Lymph Node	Lymph Node			Uni-ZAP XR
  H0109	Human Macrophage, subtracted	Macrophage	Blood	Cell Line		pBluescript
  H0110	Human Old Ovary, subtracted	Human Old Ovary	Ovary			pBluescript
  H0111	Human Placenta, subtracted	Human Placenta	Placenta			pBluescript
  H0113	Human skin Tumor, subtracted	Human Skin Tumor	Skin			Uni-ZAP XR
  H0116	Human Thymus Tumor, subtracted	Human Thymus Tumor	Thymus			pBluescript
  H0117	Human Uterine Cancer, subtracted	Human Uterine Cancer	Uterus			pBluescript
  H0119	Human Pediatric Kidney	Human Pediatric Kidney	Kidney			Uni-ZAP XR
  H0120	Human Adult Spleen, subtracted	Human Adult Spleen	Spleen			Uni-ZAP XR
  H0121	Human Cornea, subtracted	Human Cornea	eye			Uni-ZAP XR
  H0122	Human Adult Skeletal Muscle	Human Skeletal Muscle	Sk Muscle			Uni-ZAP XR
  H0123	Human Fetal Dura Mater	Human Fetal Dura Mater	Brain			Uni-ZAP XR
  H0124	Human Rhabdomyosarcoma	Human Rhabdomyosarcoma	Sk Muscle		disease	Uni-ZAP XR
  H0125	Cem cells cyclohexamide treated	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  		Cem, Jurkat, Raji, and Supt
  H0128	Jurkat cells, thiouridine activated	Jurkat Cells				Uni-ZAP XR
  H0129	Jurkat cells, thiouridine activated, fract II	Jurkat Cells				Uni-ZAP XR
  H0130	LNCAP untreated	LNCAP Cell Line	Prostate	Cell Line		Uni-ZAP XR
  H0131	LNCAP + o.3 nM R1881	LNCAP Cell Line	Prostate	Cell Line		Uni-ZAP XR
  H0132	LNCAP + 30 nM R1881	LNCAP Cell Line	Prostate	Cell Line		Uni-ZAP XR
  H0134	Raji Cells, cyclohexamide treated	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  		Cem, Jurkat, Raji, and Supt
  H0135	Human Synovial Sarcoma	Human Synovial Sarcoma	Synovium			Uni-ZAP XR
  H0136	Supt Cells, cyclohexamide treated	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  		Cem, Jurkat, Raji, and Supt
  H0140	Activated T-Cells, 8 hrs.	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0141	Activated T-Cells, 12 hrs.	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0142	MCF7 Cell Line	MCF7 Cell line	Breast	Cell Line		Uni-ZAP XR
  H0144	Nine Week Old Early Stage Human	9 Wk Old Early Stage	Embryo			Uni-ZAP XR
  		Human
  H0147	Human Adult Liver	Human Adult Liver	Liver			Uni-ZAP XR
  H0149	7 Week Old Early Stage Human, subtracted	Human Whole 7 Week Old	Embryo			Uni-ZAP XR
  		Embryo
  H0150	Human Epididymus	Epididymis	Testis			Uni-ZAP XR
  H0151	Early Stage Human Liver	Human Fetal Liver	Liver			Uni-ZAP XR
  H0152	Early Stage Human Liver, fract (II)	Human Fetal Liver	Liver			Uni-ZAP XR
  H0154	Human Fibrosarcoma	Human Skin Fibrosarcoma	Skin		disease	Uni-ZAP XR
  H0155	Human Thymus, subtracted	Human Thymus Tumor	Thymus			pBluescript
  H0156	Human Adrenal Gland Tumor	Human Adrenal Gland	Adrenal Gland		disease	Uni-ZAP XR
  		Tumor
  H0157	Activated T-Cells, 0 hrs, ligation 2	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0158	Activated T-Cells, 4 hrs., ligation 2	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0159	Activated T-Cells, 8 hrs., ligation 2	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0163	Human Synovium	Human Synovium	Synovium			Uni-ZAP XR
  H0164	Human Trachea Tumor	Human Trachea Tumor	Trachea		disease	Uni-ZAP XR
  H0165	Human Prostate Cancer, Stage B2	Human Prostate Cancer,	Prostate		disease	Uni-ZAP XR
  		stage B2
  H0166	Human Prostate Cancer, Stage B2 fraction	Human Prostate Cancer,	Prostate		disease	Uni-ZAP XR
  		stage B2
  H0167	Activated T-Cells, 24 hrs.	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0168	Human Prostate Cancer, Stage C	Human Prostate Cancer,	Prostate		disease	Uni-ZAP XR
  		stage C
  H0169	Human Prostate Cancer, Stage C fraction	Human Prostate Cancer,	Prostate		disease	Uni-ZAP XR
  		stage C
  H0170	12 Week Old Early Stage Human	Twelve Week Old Early	Embryo			Uni-ZAP XR
  		Stage Human
  H0171	12 Week Old Early Stage Human, II	Twelve Week Old Early	Embryo			Uni-ZAP XR
  		Stage Human
  H0172	Human Fetal Brain, random primed	Human Fetal Brain	Brain			Lambda ZAP II
  H0173	Human Cardiomyopathy, RNA remake	Human Cardiomyopathy	Heart		disease	Uni-ZAP XR
  H0175	H. Adult Spleen, ziplox					pSport1
  H0176	CAMA1Ee Cell Line	CAMA1Ee Cell Line	Breast	Cell Line		Uni-ZAP XR
  H0177	CAMA1Ee Cell Line	CAMA1Ee Cell Line	Breast	Cell Line		Uni-ZAP XR
  H0178	Human Fetal Brain	Human Fetal Brain	Brain			Uni-ZAP XR
  H0179	Human Neutrophil	Human Neutrophil	Blood	Cell Line		Uni-ZAP XR
  H0181	Human Primary Breast Cancer	Human Primary Breast	Breast		disease	Uni-ZAP XR
  		Cancer
  H0182	Human Primary Breast Cancer	Human Primary Breast	Breast		disease	Uni-ZAP XR
  		Cancer
  H0183	Human Colon Cancer	Human Colon Cancer	Colon		disease	Uni-ZAP XR
  H0184	Human Colon Cancer, metasticized to live	Human Colon Cancer,	Liver		disease	Lambda ZAP II
  		metasticized to liver
  H0185	Activated T-Cell labeled with 4-thioluri	T-Cells	Blood	Cell Line		Lambda ZAP II
  H0186	Activated T-Cell	T-Cells	Blood	Cell Line		Lambda ZAP II
  H0187	Resting T-Cell	T-Cells	Blood	Cell Line		Lambda ZAP II
  H0188	Human Normal Breast	Human Normal Breast	Breast			Uni-ZAP XR
  H0189	Human Resting Macrophage	Human	Blood	Cell Line		Uni-ZAP XR
  		Macrophage/Monocytes
  H0190	Human Activated Macrophage (LPS)	Human	Blood	Cell Line		Uni-ZAP XR
  		Macrophage/Monocytes
  H0191	Human Activated Macrophage (LPS), thiour	Human	Blood	Cell Line		Uni-ZAP XR
  		Macrophage/Monocytes
  H0192	Cem Cells, cyclohexamide treated, subtra	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  		Cem, Jurkat, Raji, and Supt
  H0193	Cem Cells, cyclohexamide treated, differ	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  		Cem, Jurkat, Raji, and Supt
  H0194	Human Cerebellum, subtracted	Human Cerebellum	Brain			pBluescript
  H0196	Human Cardiomyopathy, subtracted	Human Cardiomyopathy	Heart			Uni-ZAP XR
  H0197	Human Fetal Liver, subtracted	Human Fetal Liver	Liver			Uni-ZAP XR
  H0198	Human Fetal Liver, subtracted, pos. clon	Human Fetal Liver	Liver			Uni-ZAP XR
  H0199	Human Fetal Liver, subtracted, neg clone	Human Fetal Liver	Liver			Uni-ZAP XR
  H0200	Human Greater Omentum, fract II remake,	Human Greater Omentum	peritoneum			Uni-ZAP XR
  H0201	Human Hippocampus, subtracted	Human Hippocampus	Brain			pBluescript
  H0202	Jurkat Cells, cyclohexamide treated,	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  	subtraction	Cem, Jurkat, Raji, and Supt
  H0203	Jurkat Cells, cyclohexamide treated, dif	Cyclohexamide Treated	Blood	Cell Line		Uni-ZAP XR
  		Cem, Jurkat, Raji, and Supt
  H0204	Human Colon Cancer, subtracted	Human Colon Cancer	Colon			pBluescript
  H0205	Human Colon Cancer, differential	Human Colon Cancer	Colon			pBluescript
  H0207	LNCAP, differential expression	LNCAP Cell Line	Prostate	Cell Line		pBluescript
  H0208	Early Stage Human Lung, subtracted	Human Fetal Lung	Lung			pBluescript
  H0209	Human Cerebellum, differentially expressed	Human Cerebellum	Brain			Uni-ZAP XR
  H0211	Human Prostate, differential expression	Human Prostate	Prostate			pBluescript
  H0212	Human Prostate, subtracted	Human Prostate	Prostate			pBluescript
  H0213	Human Pituitary, subtracted	Human Pituitary				Uni-ZAP XR
  H0214	Raji cells, cyclohexamide treated, subtracted	Cyclohexamide Treated	Blood	Cell Line		pBluescript
  		Cem, Jurkat, Raji, and Supt
  H0215	Raji cells, cyclohexamide treated,	Cyclohexamide Treated	Blood	Cell Line		pBluescript
  	differentially expressed	Cem, Jurkat, Raji, and Supt
  H0216	Supt cells, cyclohexamide treated, subtracted	Cyclohexamide Treated	Blood	Cell Line		pBluescript
  		Cem, Jurkat, Raji, and Supt
  H0217	Supt cells, cyclohexamide treated,	Cyclohexamide Treated	Blood	Cell Line		pBluescript
  	differentially expressed	Cem, Jurkat, Raji, and Supt
  H0218	Activated T-Cells, 0 hrs, subtracted	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0219	Activated T-Cells, 0 hrs, differentially	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  	expressed
  H0220	Activated T-Cells, 4 hrs, subtracted	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0222	Activated T-Cells, 8 hrs, subtracted	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0224	Activated T-Cells, 12 hrs, subtracted	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0225	Activated T-Cells, 12 hrs, differentially	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  	expressed
  H0228	C7MCF7 cell line, estrogen treated	C7MCF7 Cell Line, estrogen	Breast	Cell Line		Uni-ZAP XR
  		treated
  H0229	Early Stage Human Brain, random primed	Early Stage Human Brain	Brain			Lambda ZAP II
  H0230	Human Cardiomyopathy, diff exp	Human Cardiomyopathy	Heart		disease	Uni-ZAP XR
  H0231	Human Colon, subtraction	Human Colon				pBluescript
  H0232	Human Colon, differential expression	Human Colon				pBluescript
  H0233	Human Fetal Heart, Differential (Adult-	Human Fetal Heart	Heart			pBluescript
  	Specific)
  H0234	human colon cancer, metastatic to liver,	Human Colon Cancer,	Liver			pBluescript
  	differentially expressed	metasticized to liver
  H0235	Human colon cancer, metaticized to liver,	Human Colon Cancer,	Liver			pBluescript
  	subtraction	metasticized to liver
  H0239	Human Kidney Tumor	Human Kidney Tumor	Kidney		disease	Uni-ZAP XR
  H0241	C7MCF7 cell line, estrogen treated,	C7MCF7 Cell Line, estrogen	Breast	Cell Line		Uni-ZAP XR
  	subtraction	treated
  H0242	Human Fetal Heart, Differential (Fetal-	Human Fetal Heart	Heart			pBluescript
  	Specific)
  H0244	Human 8 Week Whole Embryo, subtracted	Human 8 Week Old Embryo	Embryo			Uni-ZAP XR
  H0246	Human Fetal Liver- Enzyme subtraction	Human Fetal Liver	Liver			Uni-ZAP XR
  H0247	Human Membrane Bound Polysomes-	Human Membrane Bound	Blood	Cell Line		Uni-ZAP XR
  	Enzyme Subtraction	Polysomes
  H0249	HE7, subtracted by hybridization with E7	Human Whole 7 Week Old	Embryo			Uni-ZAP XR
  	cDNA	Embryo
  H0250	Human Activated Monocytes	Human Monocytes				Uni-ZAP XR
  H0251	Human Chondrosarcoma	Human Chondrosarcoma	Cartilage		disease	Uni-ZAP XR
  H0252	Human Osteosarcoma	Human Osteosarcoma	Bone		disease	Uni-ZAP XR
  H0253	Human adult testis, large inserts	Human Adult Testis	Testis			Uni-ZAP XR
  H0254	Breast Lymph node cDNA library	Breast Lymph Node	Lymph Node			Uni-ZAP XR
  H0255	breast lymph node CDNA library	Breast Lymph Node	Lymph Node			Lambda ZAP II
  H0256	HL-60, unstimulated	Human HL-60 Cells,	Blood	Cell Line		Uni-ZAP XR
  		unstimulated
  H0257	HL-60, PMA 4 H	HL-60 Cells, PMA	Blood	Cell Line		Uni-ZAP XR
  		stimulated 4 H
  H0261	H. cerebellum, Enzyme subtracted	Human Cerebellum	Brain			Uni-ZAP XR
  H0263	human colon cancer	Human Colon Cancer	Colon		disease	Lambda ZAP II
  H0264	human tonsils	Human Tonsil	Tonsil			Uni-ZAP XR
  H0265	Activated T-Cell (12 hs)/Thiouridine	T-Cells	Blood	Cell Line		Uni-ZAP XR
  	labelledEco
  H0266	Human Microvascular Endothelial Cells,	HMEC	Vein	Cell Line		Lambda ZAP II
  	fract. A
  H0267	Human Microvascular Endothelial Cells,	HMEC	Vein	Cell Line		Lambda ZAP II
  	fract. B
  H0268	Human Umbilical Vein Endothelial Cells,	HUVE Cells	Umbilical vein	Cell Line		Lambda ZAP II
  	fract. A
  H0269	Human Umbilical Vein Endothelial Cells,	HUVE Cells	Umbilical vein	Cell Line		Lambda ZAP II
  	fract. B
  H0270	HPAS (human pancreas, subtracted)	Human Pancreas	Pancreas			Uni-ZAP XR
  H0271	Human Neutrophil, Activated	Human Neutrophil -	Blood	Cell Line		Uni-ZAP XR
  		Activated
  H0272	HUMAN TONSILS, FRACTION 2	Human Tonsil	Tonsil			Uni-ZAP XR
  H0274	Human Adult Spleen, fractionII	Human Adult Spleen	Spleen			Uni-ZAP XR
  H0275	Human Infant Adrenal Gland, Subtracted	Human Infant Adrenal Gland	Adrenal gland			pBluescript
  H0279	K562 cells	K562 Cell line	cell line	Cell Line		ZAP Express
  H0280	K562 + PMA (36 hrs)	K562 Cell line	cell line	Cell Line		ZAP Express
  H0281	Lymph node, abnorm. cell line (ATCC	Lymph Node, abnormal cell	Lymph Node	Cell Line		ZAP Express
  	#7225)	line
  H0282	HBGB''s differential consolidation	Human Primary Breast	Breast			Uni-ZAP XR
  		Cancer
  H0284	Human OB MG63 control fraction I	Human Osteoblastoma	Bone	Cell Line		Uni-ZAP XR
  		MG63 cell line
  H0286	Human OB MG63 treated (10 nM E2)	Human Osteoblastoma	Bone	Cell Line		Uni-ZAP XR
  	fraction I	MG63 cell line
  H0288	Human OB HOS control fraction I	Human Osteoblastoma HOS	Bone	Cell Line		Uni-ZAP XR
  		cell line
  H0290	Human OB HOS treated (1 nM E2) fraction I	Human Osteoblastoma HOS	Bone	Cell Line		Uni-ZAP XR
  		cell line
  H0292	Human OB HOS treated (10 nM E2)	Human Osteoblastoma HOS	Bone	Cell Line		Uni-ZAP XR
  	fraction I	cell line
  H0293	WI 38 cells					Uni-ZAP XR
  H0294	Amniotic Cells - TNF induced	Amniotic Cells - TNF	Placenta	Cell Line		Uni-ZAP XR
  		induced
  H0295	Amniotic Cells - Primary Culture	Amniotic Cells - Primary	Placenta	Cell Line		Uni-ZAP XR
  		Culture
  H0298	HCBB''s differential consolidation	CAMA1Ee Cell Line	Breast	Cell Line		Uni-ZAP XR
  H0299	HCBA''s differential consolidation	CAMA1Ee Cell Line	Breast	Cell Line		Uni-ZAP XR
  H0300	CD34 positive cells (Cord Blood)	CD34 Positive Cells	Cord Blood			ZAP Express
  H0305	CD34 positive cells (Cord Blood)	CD34 Positive Cells	Cord Blood			ZAP Express
  H0306	CD34 depleted Buffy Coat (Cord Blood)	CD34 Depleted Buffy Coat	Cord Blood			ZAP Express
  		(Cord Blood)
  H0309	Human Chronic Synovitis	Synovium, Chronic	Synovium		disease	Uni-ZAP XR
  		Synovitis/Osteoarthritis
  H0310	human caudate nucleus	Brain	Brain			Uni-ZAP XR
  H0313	human pleural cancer	pleural cancer			disease	pBluescript
  H0316	HUMAN STOMACH	Human Stomach	Stomach			Uni-ZAP XR
  H0318	HUMAN B CELL LYMPHOMA	Human B Cell Lymphoma	Lymph Node		disease	Uni-ZAP XR
  H0320	Human frontal cortex	Human Frontal Cortex	Brain			Uni-ZAP XR
  H0321	HUMAN SCHWANOMA	Schwanoma	Nerve		disease	Uni-ZAP XR
  H0327	human corpus colosum	Human Corpus Callosum	Brain			Uni-ZAP XR
  H0328	human ovarian cancer	Ovarian Cancer	Ovary		disease	Uni-ZAP XR
  H0329	Dermatofibrosarcoma Protuberance	Dermatofibrosarcoma	Skin		disease	Uni-ZAP XR
  		Protuberans
  H0330	HCBB''s Subtractive (-mito genes)	CAMA1Ee Cell Line	Breast	Cell Line		Uni-ZAP XR
  H0331	Hepatocellular Tumor	Hepatocellular Tumor	Liver		disease	Lambda ZAP II
  H0333	Hemangiopericytoma	Hemangiopericytoma	Blood vessel		disease	Lambda ZAP II
  H0334	Kidney cancer	Kidney Cancer	Kidney		disease	Uni-ZAP XR
  H0339	Duodenum	Duodenum				Uni-ZAP XR
  H0340	Corpus Callosum	Corpus Collosum-93052				Uni-ZAP XR
  H0341	Bone Marrow Cell Line (RS4; 11)	Bone Marrow Cell Line	Bone Marrow	Cell Line		Uni-ZAP XR
  		RS4; 11
  H0342	Lingual Gyrus	Lingual Gyrus	Brain			Uni-Zap XR
  H0343	stomach cancer (human)	Stomach Cancer - 5383A			disease	Uni-ZAP XR
  		(human)
  H0344	Adipose tissue (human)	Adipose - 6825A (human)				Uni-ZAP XR
  H0345	SKIN	Skin - 4000868H	Skin			Uni-ZAP XR
  H0346	Brain-medulloblastoma	Brain (Medulloblastoma)-	Brain		disease	Uni-ZAP XR
  		9405C006R
  H0349	human adult liver cDNA library	Human Adult Liver	Liver			pCMVSport 1
  H0350	Human Fetal Liver, mixed 10 & 14 week	Human Fetal Liver, mixed	Liver			Uni-ZAP XR
  		10&14 Week
  H0351	Glioblastoma	Glioblastoma	Brain		disease	Uni-ZAP XR
  H0352	wilm''s tumor	Wilm''s Tumor			disease	Uni-ZAP XR
  H0354	Human Leukocytes	Human Leukocytes	Blood	Cell Line		pCMVSport 1
  H0355	Human Liver	Human Liver, normal Adult				pCMVSport 1
  H0356	Human Kidney	Human Kidney	Kidney			pCMVSport 1
  H0357	H. Normalized Fetal Liver, II	Human Fetal Liver	Liver			Uni-ZAP XR
  H0359	KMH2 cell line	KMH2				ZAP Express
  H0360	Hemangiopericytoma	Hemangiopericytoma			disease
  H0361	Human rejected kidney	Human Rejected Kidney			disease	pBluescript
  H0362	HeLa cell line	HELA CELL LINE				pSport1
  H0363	Human Brain Medulla, subtracted	Human Brain Medulla				pBluescript
  H0364	Human Osteoclastoma, excised	Human Osteoclastoma			disease	pBluescript
  H0365	Osteoclastoma-normalized B	Human Osteoclastoma			disease	Uni-ZAP XR
  H0366	L428 cell line	L428				ZAP Express
  H0369	H. Atrophic Endometrium	Atrophic Endometrium and				Uni-ZAP XR
  		myometrium
  H0370	H. Lymph node breast Cancer	Lymph node with Met.			disease	Uni-ZAP XR
  		Breast Cancer
  H0371	Eosinophils-Hypereosinophilia patient	Eosinophils-			disease	Uni-ZAP XR
  		Hypereosinophilia patient
  H0372	Human Testes	Human Testes	Testis			pCMVSport 1
  H0373	Human Heart	Human Adult Heart	Heart			pCMVSport 1
  H0374	Human Brain	Human Brain				pCMVSport 1
  H0375	Human Lung	Human Lung				pCMVSport 1
  H0376	Human Spleen	Human Adult Spleen	Spleen			pCMVSport 1
  H0379	Human Tongue, frac 1	Human Tongue				pSport1
  H0380	Human Tongue, frac 2	Human Tongue				pSport1
  H0381	Bone Cancer	Bone Cancer			disease	Uni-ZAP XR
  H0383	Human Prostate BPH, re-excision	Human Prostate BPH				Uni-ZAP XR
  H0384	Brain, Kozak	Human Brain				pCMVSport 1
  H0385	H. Leukocytes, Kozak	Human Leukocytes	Blood	Cell Line		pCMVSport 1
  H0386	Leukocyte and Lung; 4 screens	Human Leukocytes	Blood	Cell Line		pCMVSport 1
  H0388	Human Rejected Kidney, 704 re-excision	Human Rejected Kidney			disease	pBluescript
  H0389	H. Brain, X-Chromosome hybridization	Human Brain				pCMVSport 1
  H0390	Human Amygdala Depression, re-excision	Human Amygdala			disease	pBluescript
  		Depression
  H0391	H. Meniingima, M6	Human Meningima	brain			pSport1
  H0392	H. Meningima, M1	Human Meningima	brain			pSport1
  H0393	Fetal Liver, subtraction II	Human Fetal Liver	Liver			pBluescript
  H0394	A-14 cell line	Redd-Sternberg cell				ZAP Express
  H0395	A1-CELL LINE	Redd-Sternberg cell				ZAP Express
  H0396	L1 Cell line	Redd-Sternberg cell				ZAP Express
  H0398	Human Newborn Bladder	Human Newborn Bladder				pBluescript
  H0399	Human Kidney Cortex, re-rescue	Human Kidney Cortex				Lambda ZAP II
  H0400	Human Striatum Depression, re-rescue	Human Brain, Striatum	Brain			Lambda ZAP II
  		Depression
  H0401	Human Pituitary, subtracted V	Human Pituitary				pBluescript
  H0402	CD34 depleted Buffy Coat (Cord Blood), re-	CD34 Depleted Buffy Coat	Cord Blood			ZAP Express
  	excision	(Cord Blood)
  H0403	H. Umbilical Vein Endothelial Cells, IL4	HUVE Cells	Umbilical vein	Cell Line		Uni-ZAP XR
  	induced
  H0404	H. Umbilical Vein endothelial cells,	HUVE Cells	Umbilical vein	Cell Line		Uni-ZAP XR
  	uninduced
  H0405	Human Pituitary, subtracted VI	Human Pituitary				pBluescript
  H0406	H Amygdala Depression, subtracted	Human Amygdala				Uni-ZAP XR
  		Depression
  H0408	Human kidney Cortex, subtracted	Human Kidney Cortex				pBluescript
  H0409	H. Striatum Depression, subtracted	Human Brain, Striatum	Brain			pBluescript
  		Depression
  H0410	H. Male bladder, adult	H Male Bladder, Adult	Bladder			pSport1
  H0411	H Female Bladder, Adult	Human Female Adult	Bladder			pSport1
  		Bladder
  H0412	Human umbilical vein endothelial cells, IL-4	HUVE Cells	Umbilical vein	Cell Line		pSport1
  	induced
  H0413	Human Umbilical Vein Endothelial Cells,	HUVE Cells	Umbilical vein	Cell Line		pSport1
  	uninduced
  H0414	Ovarian Tumor I, OV5232	Ovarian Tumor, OV5232	Ovary		disease	pSport1
  H0415	H. Ovarian Tumor, II, OV5232	Ovarian Tumor, OV5232	Ovary		disease	pCMVSport 2.0
  H0416	Human Neutrophils, Activated, re-excision	Human Neutrophil -	Blood	Cell Line		pBluescript
  		Activated
  H0417	Human Pituitary, subtracted VIII	Human Pituitary				pBluescript
  H0418	Human Pituitary, subtracted VII	Human Pituitary				pBluescript
  H0419	Bone Cancer, re-excision	Bone Cancer				Uni-ZAP XR
  H0421	Human Bone Marrow, re-excision	Bone Marrow				pBluescript
  H0422	T-Cell PHA 16 hrs	T-Cells	Blood	Cell Line		pSport1
  H0423	T-Cell PHA 24 hrs	T-Cells	Blood	Cell Line		pSport1
  H0424	Human Pituitary, subt IX	Human Pituitary				pBluescript
  H0427	Human Adipose	Human Adipose, left				pSport1
  		hiplipoma
  H0428	Human Ovary	Human Ovary Tumor	Ovary			pSport1
  H0429	K562 + PMA (36 hrs), re-excision	K562 Cell line	cell line	Cell Line		ZAP Express
  H0431	H. Kidney Medulla, re-excision	Kidney medulla	Kidney			pBluescript
  H0432	H. Kidney Pyramid	Kidney pyramids	Kidney			pBluescript
  H0433	Human Umbilical Vein	HUVE Cells	Umbilical vein	Cell Line		pBluescript
  	Endothelial cells, frac B, re-excision
  H0434	Human Brain, striatum, re-excision	Human Brain, Striatum				pBluescript
  H0435	Ovarian Tumor 10-3-95	Ovarian Tumor, OV350721	Ovary			pCMVSport 2.0
  H0436	Resting T-Cell Library, II	T-Cells	Blood	Cell Line		pSport1
  H0437	H Umbilical Vein Endothelial Cells, frac A,	HUVE Cells	Umbilical vein	Cell Line		Lambda ZAP II
  	re-excision
  H0438	H. Whole Brain #2, re-excision	Human Whole Brain #2				ZAP Express
  H0439	Human Eosinophils	Eosinophils				pBluescript
  H0440	FGF enriched mixed library	Mixed libraries				pCMVSport 1
  H0441	H. Kidney Cortex, subtracted	Kidney cortex	Kidney			pBluescript
  H0442	H. Striatum Depression, subt II	Human Brain, Striatum	Brain			pBluescript
  		Depression
  H0443	H. Adipose, subtracted	Human Adipose, left				pSport1
  		hiplipoma
  H0444	Spleen metastic melanoma	Spleen, Metastic malignant	Spleen		disease	pSport1
  		melanoma
  H0445	Spleen, Chronic lymphocytic leukemia	Human Spleen, CLL	Spleen		disease	pSport1
  H0447	Salivary gland, re-excision	Human Salivary Gland	Salivary gland			Uni-ZAP XR
  H0448	Salivary gland, subtracted	Human Salivary Gland	Salivary gland			Lambda ZAP II
  H0449	CD34 + cell, I	CD34 positive cells				pSport1
  H0450	CD34 +cells, II	CD34 positive cells				pCMVSport 2.0
  H0453	H. Kidney Pyramid, subtracted	Kidney pyramids	Kidney			pBluescript
  H0455	H. Striatum Depression, subt	Human Brain, Striatum	Brain			pBluescript
  		Depression
  H0456	H Kidney Cortex, subtracted III	Human Kidney Cortex				pBluescript
  H0457	Human Eosinophils	Human Eosinophils				pSport1
  H0458	CD34 + cell, I, frac II	CD34 positive cells				pSport1
  H0459	CD34+cells, II, FRACTION 2	CD34 positive cells				pCMVSport 2.0
  H0461	H. Kidney Medulla, subtracted	Kidney medulla	Kidney			pBluescript
  H0462	H. Amygdala Depression, subtracted		Brain			pBluescript
  H0477	Human Tonsil, Lib 3	Human Tonsil	Tonsil			pSport1
  H0478	Salivary Gland, Lib 2	Human Salivary Gland	Salivary gland			pSport1
  H0479	Salivary Gland, Lib 3	Human Salivary Gland	Salivary gland			pSport1
  H0480	L8 cell line	L8 cell line				ZAP Express
  H0483	Breast Cancer cell line, MDA 36	Breast Cancer Cell line,				pSport1
  		MDA 36
  H0484	Breast Cancer Cell line, angiogenic	Breast Cancer Cell line,				pSport1
  		Angiogenic, 36T3
  H0485	Hodgkin''s Lymphoma I	Hodgkin''s Lymphoma I			disease	pCMVSport 2.0
  H0486	Hodgkin''s Lymphoma II	Hodgkin''s Lymphoma II			disease	pCMVSport 2.0
  H0487	Human Tonsils, lib I	Human Tonsils				pCMVSport 2.0
  H0488	Human Tonsils, Lib 2	Human Tonsils				pCMVSport 2.0
  H0489	Crohn''s Disease	Ileum	Intestine		disease	pSport1
  H0490	HI-60, untreated, subtracted	Human HL-60 Cells,	Blood	Cell Line		Uni-ZAP XR
  		unstimulated
  H0491	HL-60, PMA 4 H, subtracted	HL-60 Cells, PMA	Blood	Cell Line		Uni-ZAP XR
  		stimulated 4 H
  H0492	HL-60, RA 4 h, Subtracted	HL-60 Cells, RA stimulated	Blood	Cell Line		Uni-ZAP XR
  		for 4 H
  H0493	HL-60, PMA 1 d, subtracted	HL-60 Cells, PMA	Blood	Cell Line		Uni-ZAP XR
  		stimulated for 1 day
  H0494	Keratinocyte	Keratinocyte				pCMVSport 2.0
  H0497	HEL cell line	HEL cell line		HEL 92.1.7		pSport1
  H0505	Human Astrocyte	Human Astrocyte				pSport1
  H0506	Ulcerative Colitis	Colon	Colon			pSport1
  H0509	Liver, Hepatoma	Human Liver, Hepatoma,	Liver		disease	pCMVSport 3.0
  		patient 8
  H0510	Human Liver, normal	Human Liver, normal,	Liver			pCMVSport 3.0
  		Patient # 8
  H0512	Keratinocyte, lib 3	Keratinocyte				pCMVSport 2.0
  H0517	Nasal polyps	Nasal polyps				pCMVSport 2.0
  H0518	pBMC stimulated w/ poly I/C	pBMC stimulated with poly				pCMVSport 3.0
  		I/C
  H0519	NTERA2, control	NTERA2, Teratocarcinoma				pCMVSport 3.0
  		cell line
  H0520	NTERA2 + retinoic acid, 14 days	NTERA2, Teratocarcinoma				pSport1
  		cell line
  H0521	Primary Dendritic Cells, lib 1	Primary Dendritic cells				pCMVSport 3.0
  H0522	Primary Dendritic cells, frac 2	Primary Dendritic cells				pCMVSport 3.0
  H0523	Primary Dendritic cells, CapFinder2, frac 1	Primary Dendritic cells				pSport1
  H0524	Primary Dendritic Cells, CapFinder, frac 2	Primary Dendritic cells				pSport1
  H0525	PCR, pBMC I/C treated	pBMC stimulated with poly				PCRII
  		I/C
  H0528	Poly[I]/Poly[C] Normal Lung Fibroblasts	Poly[I]/Poly[C] Normal				pCMVSport 3.0
  		Lung Fibroblasts
  H0529	Myoloid Progenitor Cell Line	TF-1 Cell Line; Myoloid				pCMVSport 3.0
  		progenitor cell line
  H0530	Human Dermal Endothelial Cells, untreated	Human Dermal Endothelial				pSport1
  		Cells; untreated
  H0535	Human ovary tumor cell OV350721	Ovarian Tumor, OV350721	Ovary		disease	pSport1
  H0537	H. Primary Dendritic Cells, lib 3	Primary Dendritic cells				pCMVSport 2.0
  H0538	Merkel Cells	Merkel cells	Lymph node			pSport1
  H0539	Pancreas Islet Cell Tumor	Pancreas Islet Cell Tumour	Pancreas		disease	pSport1
  H0540	Skin, burned	Skin, leg burned	Skin			pSport1
  H0542	T Cell helper I	Helper T cell				pCMVSport 3.0
  H0543	T cell helper II	Helper T cell				pCMVSport 3.0
  H0544	Human endometrial stromal cells	Human endometrial stromal				pCMVSport 3.0
  		cells
  H0545	Human endometrial stromal	Human endometrial stromal				pCMVSport 3.0
  	cells-treated with progesterone	cells-treated with proge
  H0546	Human endometrial stromal	Human endometrial stromal				pCMVSport 3.0
  	cells-treated with
  	estradiol	cells-treated with estra
  H0547	NTERA2 teratocarcinoma cell line + retinoic	NTERA2, Teratocarcinoma				pSport1
  	acid (14 days)	cell line
  H0549	H. Epididiymus, caput & corpus	Human Epididiymus, caput				Uni-ZAP XR
  		and corpus
  H0550	H. Epididiymus, cauda	Human Epididiymus, cauda				Uni-ZAP XR
  H0551	Human Thymus Stromal Cells	Human Thymus Stromal				pCMVSport 3.0
  		Cells
  H0552	Signal trap, Femur Bone Marrow, pooled	Femur Bone marrow, pooled				Other
  		from 8 male/female
  H0553	Human Placenta	Human Placenta				pCMVSport 3.0
  H0555	Rejected Kidney, lib 4	Human Rejected Kidney	Kidney		disease	pCMVSport 3.0
  H0556	Activated T-cell(12 h)/Thiouridine-re-	T-Cells	Blood	Cell Line		Uni-ZAP XR
  	excision
  H0559	HL-60, PMA 4 H, re-excision	HL-60 Cells, PMA	Blood	Cell Line		Uni-ZAP XR
  		stimulated 4 H
  H0560	KMH2	KMH2				pCMVSport 3.0
  H0561	L428	L428				pCMVSport 3.0
  H0562	Human Fetal Brain, normalized c5-11-26	Human Fetal Brain				pCMVSport 2.0
  H0563	Human Fetal Brain, normalized 50021F	Human Fetal Brain				pCMVSport 2.0
  H0564	Human Fetal Brain, normalized C5001F	Human Fetal Brain				pCMVSport 2.0
  H0565	HUman Fetal Brain, normalized 100024F	Human Fetal Brain				pCMVSport 2.0
  H0566	Human Fetal Brain, normalized c50F	Human Fetal Brain				pCMVSport 2.0
  H0567	Human Fetal Brain, normalized A5002F	Human Fetal Brain				pCMVSport 2.0
  H0569	Human Fetal Brain, normalized CO	Human Fetal Brain				pCMVSport 2.0
  H0570	Human Fetal Brain, normalized C500H	Human Fetal Brain				pCMVSport 2.0
  H0571	Human Fetal Brain, normalized C500HE	Human Fetal Brain				pCMVSport 2.0
  H0572	Human Fetal Brain, normalized AC5002	Human Fetal Brain				pCMVSport 2.0
  H0574	Hepatocellular Tumor; re-excision	Hepatocellular Tumor	Liver		disease	Lambda ZAP II
  H0575	Human Adult Pulmonary; re-excision	Human Adult Pulmonary	Lung			Uni-ZAP XR
  H0576	Resting T-Cell; re-excision	T-Cells	Blood	Cell Line		Lambda ZAP II
  H0578	Human Fetal Thymus	Fetal Thymus	Thymus			pSport1
  H0579	Pericardium	Pericardium	Heart			pSport1
  H0580	Dendritic cells, pooled	Pooled dendritic cells				pCMVSport 3.0
  H0581	Human Bone Marrow, treated	Human Bone Marrow	Bone Marrow			pCMVSport 3.0
  H0583	B Cell lymphoma	B Cell Lymphoma	B Cell		disease	pCMVSport 3.0
  H0584	Activated T-cells, 24 hrs, re-excision	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0585	Activated T-Cells, 12 hrs, re-excision	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0586	Healing groin wound, 6.5 hours post incision	healing groin wound, 6.5	groin		disease	pCMVSport 3.0
  		hours post incision - 2/
  H0587	Healing groin wound; 7.5 hours post incision	Groin-2/19/97	groin		disease	pCMVSport 3.0
  H0589	CD34 positive cells (cord blood), re-ex	CD34 Positive Cells	Cord Blood			ZAP Express
  H0590	Human adult small intestine, re-excision	Human Adult Small Intestine	Small Int.			Uni-ZAP XR
  H0591	Human T-cell lymphoma; re-excision	T-Cell Lymphoma	T-Cell		disease	Uni-ZAP XR
  H0592	Healing groin wound - zero hr post-incision	HGS wound healing project;			disease	pCMVSport 3.0
  	(control)	abdomen
  H0593	Olfactory epithelium; nasalcavity	Olfactory epithelium from				pCMVSport 3.0
  		roof of left nasal cacit
  H0594	Human Lung Cancer; re-excision	Human Lung Cancer	Lung		disease	Lambda ZAP II
  H0595	Stomach cancer (human); re-excision	Stomach Cancer - 5383A			disease	Uni-ZAP XR
  		(human)
  H0596	Human Colon Cancer; re-excision	Human Colon Cancer	Colon			Lambda ZAP II
  H0597	Human Colon; re-excision	Human Colon				Lambda ZAP II
  H0598	Human Stomach; re-excision	Human Stomach	Stomach			Uni-ZAP XR
  H0599	Human Adult Heart; re-excision	Human Adult Heart	Heart			Uni-ZAP XR
  H0600	Healing Abdomen wound; 70&90 min post	Abdomen			disease	pCMVSport 3.0
  	incision
  H0601	Healing Abdomen Wound; 15 days post	Abdomen			disease	pCMVSport 3.0
  	incision
  H0602	Healing Abdomen Wound; 21&29 days post	Abdomen			disease	pCMVSport 3.0
  	incision
  H0604	Human Pituitary, re-excision	Human Pituitary				pBluescript
  H0606	Human Primary Breast Cancer; re-excision	Human Primary Breast	Breast		disease	Uni-ZAP XR
  		Cancer
  H0607	H. Leukocytes, normalized cot 50A3	H. Leukocytes				pCMVSport 1
  H0608	H. Leukocytes, control	H. Leukocytes				pCMVSport 1
  H0609	H. Leukocytes, normalized cot >500A	H. Leukocytes				pCMVSport 1
  H0610	H. Leukocytes, normalized cot 5A	H. Leukocytes				pCMVSport 1
  H0611	H. Leukocytes, normalized cot 500 B	H. Leukocytes				pCMVSport 1
  H0612	H. Leukocytes, normalized cot 50 B	H. Leukocytes				pCMVSport 1
  H0613	H. Leukocytes, normalized cot 5B	H. Leukocytes				pCMVSport 1
  H0614	H. Leukocytes, normalized cot 500 A	H. Leukocytes				pCMVSport 1
  H0615	Human Ovarian Cancer Reexcision	Ovarian Cancer	Ovary		disease	Uni-ZAP XR
  H0616	Human Testes, Reexcision	Human Testes	Testis			Uni-ZAP XR
  H0617	Human Primary Breast Cancer Reexcision	Human Primary Breast	Breast		disease	Uni-ZAP XR
  		Cancer
  H0618	Human Adult Testes, Large Inserts,	Human Adult Testis	Testis			Uni-ZAP XR
  	Reexcision
  H0619	Fetal Heart	Human Fetal Heart	Heart			Uni-ZAP XR
  H0620	Human Fetal Kidney; Reexcision	Human Fetal Kidney	Kidney			Uni-ZAP XR
  H0622	Human Pancreas Tumor; Reexcision	Human Pancreas Tumor	Pancreas		disease	Uni-ZAP XR
  H0623	Human Umbilical Vein; Reexcision	Human Umbilical Vein	Umbilical vein			Uni-ZAP XR
  		Endothelial Cells
  H0624	12 Week Early Stage Human II; Reexcision	Twelve Week Old Early	Embryo			Uni-ZAP XR
  		Stage Human
  H0625	Ku 812F Basophils Line	Ku812F Basophils				pSport1
  H0626	Saos2 Cells; Untreated	Saos2 Cell Line; Untreated				pSport1
  H0627	Saos2 Cells; Vitamin D3 Treated	Saos2 Cell Line; Vitamin D3				pSport1
  		Treated
  H0628	Human Pre-Differentiated Adipocytes	Human Pre-Differentiated				Uni-ZAP XR
  		Adipocytes
  H0629	Human Leukocyte, control #2	Human Normalized				pCMVSport 1
  		leukocyte
  H0630	Human Leukocytes, normalized control #4	Human Normalized				pCMVSport 1
  		leukocyte
  H0631	Saos2, Dexamethosome Treated	Saos2 Cell Line;				pSport1
  		Dexamethosome Treated
  H0632	Hepatocellular Tumor; re-excision	Hepatocellular Tumor	Liver			Lambda ZAP II
  H0633	Lung Carcinoma A549 TNFalpha activated	TNFalpha activated A549-			disease	pSport1
  		Lung Carcinoma
  H0634	Human Testes Tumor, re-excision	Human Testes Tumor	Testis		disease	Uni-ZAP XR
  H0635	Human Activated T-Cells, re-excision	Activated T-Cells	Blood	Cell Line		Uni-ZAP XR
  H0637	Dendritic Cells From CD34 Cells	Dentritic cells from CD34				pSport1
  		cells
  H0638	CD40 activated monocyte dendridic cells	CD40 activated monocyte				pSport1
  		dendridic cells
  H0639	Ficolled Human Stromal Cells, 5Fu treated	Ficolled Human Stromal				Other
  		Cells, 5Fu treated
  H0640	Ficolled Human Stromal Cells, Untreated	Ficolled Human Stromal				Other
  		Cells, Untreated
  H0641	LPS activated derived dendritic cells	LPS activated monocyte				pSport1
  		derived dendritic cells
  H0642	Hep G2 Cells, lambda library	Hep G2 Cells				Other
  H0643	Hep G2 Cells, PCR library	Hep G2 Cells				Other
  H0644	Human Placenta (re-excision)	Human Placenta	Placenta			Uni-ZAP XR
  H0645	Fetal Heart, re-excision	Human Fetal Heart	Heart			Uni-ZAP XR
  H0646	Lung, Cancer (4005313 A3): Invasive	Metastatic squamous cell				pSport1
  	Poorly Differentiated Lung Adenocarcinoma,	lung carcinoma, poorly di
  H0647	Lung, Cancer (4005163 B7): Invasive,	Invasive poorly			disease	pSport1
  	Poorly Diff. Adenocarcinoma, Metastatic	differentiated lung
  		adenocarcinoma
  H0648	Ovary, Cancer: (4004562 B6) Papillary	Papillary Cstic neoplasm of			disease	pSport1
  	Serous Cystic Neoplasm, Low Malignant Pot	low malignant potentia
  H0649	Lung, Normal: (4005313 B1)	Normal Lung				pSport1
  H0650	B-Cells	B-Cells				pCMVSport 3.0
  H0651	Ovary, Normal: (9805C040R)	Normal Ovary				pSport1
  H0652	Lung, Normal: (4005313 B1)	Normal Lung				pSport1
  H0653	Stromal Cells	Stromal Cells				pSport1
  H0654	Lung, Cancer: (4005313 A3) Invasive	Metastatic Squamous cell				Other
  	Poorly-differentiated Metastatic lung adenoc	lung Carcinoma poorly dif
  H0656	B-cells (unstimulated)	B-cells (unstimulated)				pSport1
  H0657	B-cells (stimulated)	B-cells (stimulated)				pSport1
  H0658	Ovary, Cancer (9809C332): Poorly	9809C332- Poorly	Ovary &		disease	pSport1
  	differentiated adenocarcinoma	differentiate	Fallopian Tubes
  H0659	Ovary, Cancer (15395A1F): Grade II	Grade II Papillary	Ovary		disease	pSport1
  	Papillary Carcinoma	Carcinoma, Ovary
  H0660	Ovary, Cancer: (15799A1F) Poorly	Poorly differentiated			disease	pSport1
  	differentiated carcinoma	carcinoma, ovary
  H0661	Breast, Cancer: (4004943 A5)	Breast cancer			disease	pSport1
  H0662	Breast, Normal: (4005522B2)	Normal Breast -	Breast			pSport1
  		#4005522(B2)
  H0663	Breast, Cancer: (4005522 A2)	Breast Cancer -	Breast		disease	pSport1
  		#4005522(A2)
  H0664	Breast, Cancer: (9806C012R)	Breast Cancer	Breast		disease	pSport1
  H0665	Stromal cells 3.88	Stromal cells 3.88				pSport1
  H0666	Ovary, Cancer: (4004332 A2)	Ovarian Cancer, Sample			disease	pSport1
  		#4004332A2
  H0667	Stromal cells(HBM3.18)	Stromal cell(HBM 3.18)				pSport1
  H0668	stromal cell clone 2.5	stromal cell clone 2.5				pSport1
  H0669	Breast, Cancer: (4005385 A2)	Breast Cancer (4005385A2)	Breast			pSport1
  H0670	Ovary, Cancer(4004650 A3): Well-	Ovarian Cancer -				pSport1
  	Differentiated Micropapillary Serous	4004650A3
  	Carcinoma
  H0671	Breast, Cancer: (9802C02OE)	Breast Cancer- Sample #				pSport1
  		9802C02OE
  H0672	Ovary, Cancer: (4004576 A8)	Ovarian Cancer(4004576A8)	Ovary			pSport1
  H0673	Human Prostate Cancer, Stage B2; re-	Human Prostate Cancer,	Prostate			Uni-ZAP XR
  	excision	stage B2
  H0674	Human Prostate Cancer, Stage C; re-	Human Prostate Cancer,	Prostate			Uni-ZAP XR
  	excission	stage C
  H0675	Colon, Cancer: (9808C064R)	Colon Cancer 9808C064R				pCMVSport 3.0
  H0676	Colon, Cancer: (9808C064R)-total RNA	Colon Cancer 9808C064R				pCMVSport 3.0
  H0677	TNFR degenerate oligo	B-Cells				PCRII
  H0678	screened clones from placental library	Placenta	Placenta			Other
  H0679	screened clones from Tonsil library	Human Tonsils				Other
  H0682	Serous Papillary Adenocarcinoma	serous papillary				pCMVSport 3.0
  		adenocarcinoma
  		(9606G304SPA3B)
  H0683	Ovarian Serous Papillary Adenocarcinoma	Serous papillary				pCMVSport 3.0
  		adenocarcinoma, stage 3C
  		(9804G01
  H0684	Serous Papillary Adenocarcinoma	Ovarian Cancer-9810G606	Ovaries			pCMVSport 3.0
  H0685	Adenocarcinoma of Ovary, Human Cell Line	Adenocarcinoma of Ovary,				pCMVSport 3.0
  	# OVCAR-3	Human Cell Line, #
  		OVCAR-
  H0686	Adenocarcinoma of Ovary, Human Cell Line	Adenocarcinoma of Ovary,				pCMVSport 3.0
  		Human Cell Line, # SW-626
  H0687	Human normal ovary(#9610G215)	Human normal	Ovary			pCMVSport 3.0
  		ovary(#9610G215)
  H0688	Human Ovarian Cancer(#9807G017)	Human Ovarian				pCMVSport 3.0
  		cancer(#9807G017), mRNA
  		from Maura Ru
  H0689	Ovarian Cancer	Ovarian Cancer, #9806G019				pCMVSport 3.0
  H0690	Ovarian Cancer, # 9702G001	Ovarian Cancer, #9702G001				pCMVSport 3.0
  H0691	Normal Ovary, #9710G208	normal ovary, #9710G208				pCMVSport 3.0
  H0692	BLyS Receptor from Expression Cloning	B Cell Lymphoma	B Cell			pCMVSport 3.0
  H0693	Normal Prostate #ODQ3958EN	Normal Prostate Tissue #				pCMVSport 3.0
  		ODQ3958EN
  H0694	Prostate gland adenocarcinoma	Prostate gland,	prostate gland			pCMVSport 3.0
  		adenocarcinoma, mod/diff,
  		gleason
  H0695	mononucleocytes from patient	mononucleocytes from				pCMVSport 3.0
  		patient at Shady Grove
  		Hospit
  N0006	Human Fetal Brain	Human Fetal Brain
  N0007	Human Hippocampus	Human Hippocampus
  N0009	Human Hippocampus, prescreened	Human Hippocampus
  N0011	Human Brain	Human Brain
  S0001	Brain frontal cortex	Brain frontal cortex	Brain			Lambda ZAP II
  S0002	Monocyte activated	Monocyte-activated	blood	Cell Line		Uni-ZAP XR
  S0003	Human Osteoclastoma	Osteoclastoma	bone		disease	Uni-ZAP XR
  S0004	Prostate	Prostate BPH	Prostate			Lambda ZAP II
  S0005	Heart	Heart-left ventricle	Heart			pCDNA
  S0006	Neuroblastoma	Human Neural Blastoma			disease	pCDNA
  S0007	Early Stage Human Brain	Human Fetal Brain				Uni-ZAP XR
  S0010	Human Amygdala	Amygdala				Uni-ZAP XR
  S0011	STROMAL - OSTEOCLASTOMA	Osteoclastoma	bone		disease	Uni-ZAP XR
  S0013	Prostate	Prostate	prostate			Uni-ZAP XR
  S0014	Kidney Cortex	Kidney cortex	Kidney			Uni-ZAP XR
  S0015	Kidney medulla	Kidney medulla	Kidney			Uni-ZAP XR
  S0016	Kidney Pyramids	Kidney pyramids	Kidney			Uni-ZAP XR
  S0021	Whole brain	Whole brain	Brain			ZAP Express
  S0022	Human Osteoclastoma Stromal Cells -	Osteoclastoma Stromal Cells				Uni-ZAP XR
  	unamplified
  S0024	Human Kidney Medulla - unamplified	Human Kidney Medulla
  S0025	Human Kidney Pyramids - unamplified	Human Kidney Pyramids
  S0026	Stromal cell TF274	stromal cell	Bone marrow	Cell Line		Uni-ZAP XR
  S0027	Smooth muscle, serum treated	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0028	Smooth muscle, control	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0029	brain stem	Brain stem	brain			Uni-ZAP XR
  S0030	Brain pons	Brain Pons	Brain			Uni-ZAP XR
  S0031	Spinal cord	Spinal cord	spinal cord			Uni-ZAP XR
  S0032	Smooth muscle-ILb induced	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0035	Brain medulla oblongata	Brain medulla oblongata	Brain			Uni-ZAP XR
  S0036	Human Substantia Nigra	Human Substantia Nigra				Uni-ZAP XR
  S0037	Smooth muscle, IL1b induced	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0038	Human Whole Brain #2 - Oligo dT >1.5 Kb	Human Whole Brain #2				ZAP Express
  S0039	Hypothalamus	Hypothalamus	Brain			Uni-ZAP XR
  S0040	Adipocytes	Human Adipocytes from				Uni-ZAP XR
  		Osteoclastoma
  S0041	Thalamus	Human Thalamus				Uni-ZAP XR
  S0042	Testes	Human Testes				ZAP Express
  S0044	Prostate BPH	prostate BPH	Prostate		disease	Uni-ZAP XR
  S0045	Endothelial cells-control	Endothelial cell	endothelial cell-	Cell Line		Uni-ZAP XR
  			lung
  S0046	Endothelial-induced	Endothelial cell	endothelial cell-	Cell Line		Uni-ZAP XR
  			lung
  S0048	Human Hypothalamus, Alzheimer''s	Human Hypothalamus,			disease	Uni-ZAP XR
  		Alzheimer''s
  S0049	Human Brain, Striatum	Human Brain, Striatum				Uni-ZAP XR
  S0050	Human Frontal Cortex, Schizophrenia	Human Frontal Cortex,			disease	Uni-ZAP XR
  		Schizophrenia
  S0051	Human Hypothalmus, Schizophrenia	Human Hypothalamus,			disease	Uni-ZAP XR
  		Schizophrenia
  S0052	neutrophils control	human neutrophils	blood	Cell Line		Uni-ZAP XR
  S0053	Neutrophils IL-1 and LPS induced	human neutrophil induced	blood	Cell Line		Uni-ZAP XR
  S0106	STRIATUM DEPRESSION		BRAIN		disease	Uni-ZAP XR
  S0110	Brain Amygdala Depression		Brain		disease	Uni-ZAP XR
  S0112	Hypothalamus		Brain			Uni-ZAP XR
  S0114	Anergic T-cell	Anergic T-cell		Cell Line		Uni-ZAP XR
  S0116	Bone marrow	Bone marrow	Bone marrow			Uni-ZAP XR
  S0118	Smooth muscle control 2	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0122	Osteoclastoma-normalized A	Osteoclastoma	bone		disease	pBluescript
  S0124	Smooth muscle-edited A	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0126	Osteoblasts	Osteoblasts	Knee	Cell Line		Uni-ZAP XR
  S0132	Epithelial-TNFa and INF induced	Airway Epithelial				Uni-ZAP XR
  S0134	Apoptotic T-cell	apoptotic cells		Cell Line		Uni-ZAP XR
  S0136	PERM TF274	stromal cell	Bone marrow	Cell Line		Lambda ZAP II
  S0140	eosinophil-IL5 induced	eosinophil	lung	Cell Line		Uni-ZAP XR
  S0142	Macrophage-oxLDL	macrophage-oxidized LDL	blood	Cell Line		Uni-ZAP XR
  		treated
  S0144	Macrophage (GM-CSF treated)	Macrophage (GM-CSF				Uni-ZAP XR
  		treated)
  S0146	prostate-edited	prostate BPH	Prostate			Uni-ZAP XR
  S0148	Normal Prostate	Prostate	prostate			Uni-ZAP XR
  S0150	LNCAP prostate cell line	LNCAP Cell Line	Prostate	Cell Line		Uni-ZAP XR
  S0152	PC3 Prostate cell line	PC3 prostate cell line				Uni-ZAP XR
  S0168	Prostate/LNCAP, subtraction I	PC3 prostate cell line				pBluescript
  S0174	Prostate-BPH subtracted II	Human Prostate BPH				pBluescript
  S0176	Prostate, normal, subtraction I	Prostate	prostate			Uni-ZAP XR
  S0180	Bone Marrow Stroma, TNF&LPS ind	Bone Marrow Stroma, TNF			disease	Uni-ZAP XR
  		& LPS induced
  S0182	Human B Cell 8866	Human B-Cell 8866				Uni-ZAP XR
  S0184	7TM Receptor enriched, lib II	PBLS, 7TM receptor				Other
  		enriched
  S0188	Prostate, BPH, Lib 2	Human Prostate BPH			disease	pSport1
  S0190	Prostate BPH, Lib 2, subtracted	Human Prostate BPH				pSport1
  S0192	Synovial Fibroblasts (control)	Synovial Fibroblasts				pSport1
  S0194	Synovial hypoxia	Synovial Fibroblasts				pSport1
  S0196	Synovial IL-1/TNF stimulated	Synovial Fibroblasts				pSport1
  S0202	7TM-pbdd	PBLS, 7TM receptor				PCRII
  		enriched
  S0206	Smooth Muscle- HASTE normalized	Smooth muscle	Pulmanary	Cell Line		pBluescript
  			artery
  S0208	Messangial cell, frac 1	Messangial cell				pSport1
  S0210	Messangial cell, frac 2	Messangial cell				pSport1
  S0212	Bone Marrow Stromal Cell, untreated	Bone Marrow Stromal				pSport1
  		Cell, untreated
  S0214	Human Osteoclastoma, re-excision	Osteoclastoma	bone		disease	Uni-ZAP XR
  S0216	Neutrophils IL-1 and LPS induced	human neutrophil induced	blood	Cell Line		Uni-ZAP XR
  S0218	Apoptotic T-cell, re-excision	apoptotic cells		Cell Line		Uni-ZAP XR
  S0220	H. hypothalamus, frac A; re-excision	Hypothalamus	Brain			ZAP Express
  S0222	H. Frontal cortex, epileptic; re-excision	H. Brain, Frontal Cortex,	Brain		disease	Uni-ZAP XR
  		Epileptic
  S0242	Synovial Fibroblasts (Il1/TNF), subt	Synovial Fibroblasts				pSport1
  S0250	Human Osteoblasts II	Human Osteoblasts	Femur		disease	pCMVSport 2.0
  S0252	7TM-PIMIX	PBLS, 7TM receptor				PCRII
  		enriched
  S0256	7TM-PHMIX	PBLS, 7TM receptor				PCRII
  		enriched
  S0260	Spinal Cord, re-excision	Spinal cord	spinal cord			Uni-ZAP XR
  S0264	PPMIX	PPMIX (Human Pituitary)	Pituitary			PCRII
  S0266	PLMIX	PLMIX (Human Lung)	Lung			PCRII
  S0268	PRMIX	PRMIX (Human Prostate)	prostate			PCRII
  S0270	PTMIX	PTMIX (Human Thymus)	Thymus			PCRII
  S0276	Synovial hypoxia-RSF subtracted	Synovial fobroblasts	Synovial tissue			pSport1
  		(rheumatoid)
  S0278	H Macrophage (GM-CSF treated), re-	Macrophage (GM-CSF				Uni-ZAP XR
  	excision	treated)
  S0280	Human Adipose Tissue, re-excision	Human Adipose Tissue				Uni-ZAP XR
  S0282	Brain Frontal Cortex, re-excision	Brain frontal cortex	Brain			Lambda ZAP II
  S0292	Osteoarthritis (OA-4)	Human Osteoarthritic	Bone		disease	pSport1
  		Cartilage
  S0294	Larynx tumor	Larynx tumor	Larynx, vocal		disease	pSport1
  			cord
  S0296	Normal lung	Normal lung	Lung			pSport1
  S0298	Bone marrow stroma, treated	Bone marrow	Bone marrow			pSport1
  		stroma, treatedSB
  S0300	Frontal lobe, dementia; re-excision	Frontal Lobe	Brain			Uni-ZAP XR
  		dementia/Alzheimer''s
  S0306	Larynx normal #10 261-273	Larynx normal				pSport1
  S0308	Spleen/normal	Spleen normal				pSport1
  S0310	Normal trachea	Normal trachea				pSport1
  S0312	Human osteoarthritic; fraction II	Human osteoarthritic			disease	pSport1
  		cartilage
  S0314	Human osteoarthritis; fraction I	Human osteoarthritic			disease	pSport1
  		cartilage
  S0316	Human Normal Cartilage, Fraction I	Human Normal Cartilage				pSport1
  S0318	Human Normal Cartilage Fraction II	Human Normal Cartilage				pSport1
  S0320	Human Larynx	Larynx	Epiglottis			pSport1
  S0322	Siebben Polyposis	Siebben Polyposis				pSport1
  S0324	Human Brain	Brain	Cerebellum			pSport1
  S0328	Palate carcinoma	Palate carcinoma	Uvula		disease	pSport1
  S0330	Palate normal	Palate normal	Uvula			pSport1
  S0332	Pharynx carcinoma	Pharynx carcinoma	Hypopharynx			pSport1
  S0334	Human Normal Cartilage Fraction III	Human Normal Cartilage				pSport1
  S0336	Human Normal Cartilage Fraction IV	Human Normal Cartilage				pSport1
  S0338	Human Osteoarthritic Cartilage Fraction III	Human osteoarthritic			disease	pSport1
  		cartilage
  S0340	Human Osteoarthritic Cartilage Fraction IV	Human osteoarthritic			disease	pSport1
  		cartilage
  S0342	Adipocytees; re-excision	Human Adipocytes from				Uni-ZAP XR
  		Osteoclastoma
  S0344	Macrophage-oxLDL; re-excision	macrophage-oxidized LDL	blood	Cell Line		Uni-ZAP XR
  		treated
  S0346	Human Amygdala; re-excision	Amygdala				Uni-ZAP XR
  S0348	Cheek Carcinoma	Cheek Carcinoma			disease	pSport1
  S0350	Pharynx Carcinoma	Pharynx carcinoma	Hypopharynx		disease	pSport1
  S0352	Larynx Carcinoma	Larynx carcinoma			disease	pSport1
  S0354	Colon Normal II	Colon Normal	Colon			pSport1
  S0356	Colon Carcinoma	Colon Carcinoma	Colon		disease	pSport1
  S0358	Colon Normal III	Colon Normal	Colon			pSport1
  S0360	Colon Tumor II	Colon Tumor	Colon		disease	pSport1
  S0362	Human Gastrocnemius	Gastrocnemius muscle				pSport1
  S0364	Human Quadriceps	Quadriceps muscle				pSport1
  S0366	Human Soleus	Soleus Muscle				pSport1
  S0368	Human Pancreatic Langerhans	Islets of Langerhans				pSport1
  S0370	Larynx carcinoma II	Larynx carcinoma			disease	pSport1
  S0372	Larynx carcinoma III	Larynx carcinoma			disease	pSport1
  S0374	Normal colon	Normal colon				pSport1
  S0376	Colon Tumor	Colon Tumor			disease	pSport1
  S0378	Pancreas normal PCA4 No	Pancreas Normal PCA4 No				pSport1
  S0380	Pancreas Tumor PCA4 Tu	Pancreas Tumor PCA4 Tu			disease	pSport1
  S0382	Larynx carcinoma IV	Larynx carcinoma			disease	pSport1
  S0384	Tongue carcinoma	Tongue carcinoma			disease	pSport1
  S0386	Human Whole Brain, re-excision	Whole brain	Brain			ZAP Express
  S0388	Human Hypothalamus, schizophrenia, re-	Human Hypothalamus,			disease	Uni-ZAP XR
  	excision	Schizophrenia
  S0390	Smooth muscle, control; re-excision	Smooth muscle	Pulmanary	Cell Line		Uni-ZAP XR
  			artery
  S0392	Salivary Gland	Salivary gland; normal				pSport1
  S0394	Stomach; normal	Stomach; normal				pSport1
  S0396	Uterus; normal	Uterus; normal				pSport1
  S0398	Testis; normal	Testis; normal				pSport1
  S0400	Brain; normal	Brain; normal				pSport1
  S0402	Adrenal Gland, normal	Adrenal gland; normal				pSport1
  S0404	Rectum normal	Rectum, normal				pSport1
  S0406	Rectum tumour	Rectum tumour				pSport1
  S0408	Colon, normal	Colon, normal				pSport1
  S0410	Colon, tumour	Colon, tumour				pSport1
  S0412	Temporal cortex-Alzheizmer; subtracted	Temporal cortex, alzheimer			disease	Other
  S0414	Hippocampus, Alzheimer Subtracted	Hippocampus, Alzheimer				Other
  		Subtracted
  S0418	CHME Cell Line; treated 5 hrs	CHME Cell Line; treated				pCMVSport 3.0
  S0420	CHME Cell Line, untreated	CHME Cell line, untreatetd				pSport1
  S0422	Mo7e Cell Line GM-CSF treated (1 ng/ml)	Mo7e Cell Line GM-CSF				pCMVSport 3.0
  		treated (1 ng/ml)
  S0424	TF-1 Cell Line GM-CSF Treated	TF-1 Cell Line GM-CSF				pSport1
  		Treated
  S0426	Monocyte activated; re-excision	Monocyte-activated	blood	Cell Line		Uni-ZAP XR
  S0428	Neutrophils control; re-excision	human neutrophils	blood	Cell Line		Uni-ZAP XR
  S0430	Aryepiglottis Normal	Aryepiglottis Normal				pSport1
  S0432	Sinus piniformis Tumour	Sinus piniformis Tumour				pSport1
  S0434	Stomach Normal	Stomach Normal			disease	pSport1
  S0436	Stomach Tumour	Stomach Tumour			disease	pSport1
  S0438	Liver Normal Met5No	Liver Normal Met5No				pSport1
  S0440	Liver Tumour Met 5 Tu	Liver Tumour				pSport1
  S0442	Colon Normal	Colon Normal				pSport1
  S0444	Colon Tumor	Colon Tumour			disease	pSport1
  S0446	Tongue Tumour	Tongue Tumour				pSport1
  S0448	Larynx Normal	Larynx Normal				pSport1
  S0450	Larynx Tumour	Larynx Tumour				pSport1
  S0452	Thymus	Thymus				pSport1
  S0454	Placenta	Placenta	Placenta			pSport1
  S0456	Tongue Normal	Tongue Normal				pSport1
  S0458	Thyroid Normal (SDCA2 No)	Thyroid normal				pSport1
  S0460	Thyroid Tumour	Thyroid Tumour				pSport1
  S0462	Thyroid Thyroiditis	Thyroid Thyroiditis				pSport1
  S0464	Larynx Normal	Larynx Normal				pSport1
  S0466	Larynx Tumor	Larynx Tumor			disease	pSport1
  S0468	Ea.hy.926 cell line	Ea.hy.926 cell line				pSport1
  S0470	Adenocarcinoma	PYFD			disease	pSport1
  S0472	Lung Mesothelium	PYBT				pSport1
  S0474	Human blood platelets	Platelets	Blood platelets			Other
  S0665	Human Amygdala; re-excission	Amygdala				Uni-ZAP XR
  S3012	Smooth Muscle Serum Treated, Norm	Smooth muscle	Pulmanary	Cell Line		pBluescript
  			artery
  S3014	Smooth muscle, serum induced, re-exc	Smooth muscle	Pulmanary	Cell Line		pBluescript
  			artery
  S6014	H. hypothalamus, frac A	Hypothalamus	Brain			ZAP Express
  S6016	H. Frontal Cortex, Epileptic	H. Brain, Frontal Cortex,	Brain		disease	Uni-ZAP XR
  		Epileptic
  S6022	H. Adipose Tissue	Human Adipose Tissue				Uni-ZAP XR
  S6024	Alzheimers, spongy change	Alzheimer''s/Spongy change	Brain		disease	Uni-ZAP XR
  S6026	Frontal Lobe, Dementia	Frontal Lobe	Brain			Uni-ZAP XR
  		dementia/Alzheimer''s
  S6028	Human Manic Depression Tissue	Human Manic depression	Brain		disease	Uni-ZAP XR
  		tissue
  T0001	Human Brown Fat	Brown Fat				pBluescript SK−
  T0002	Activated T-cells	Activated T-Cell, PBL	Blood	Cell Line		pBluescript SK−
  		fraction
  T0003	Human Fetal Lung	Human Fetal Lung				pBluescript SK−
  T0004	Human White Fat	Human White Fat				pBluescript SK−
  T0006	Human Pineal Gland	Human Pinneal Gland				pBluescript SK−
  T0007	Colon Epithelium	Colon Epithelium				pBluescriptISK−
  T0008	Colorectal Tumor	Colorectal Tumor			disease	pBluescript SK−
  T0010	Human Infant Brain	Human Infant Brain				Other
  T0023	Human Pancreatic Carcinoma	Human Pancreatic			disease	pBluescript SK−
  		Carcinoma
  T0039	HSA 172 Cells	Human HSA 172 cell line				pBluescript SK−
  T0040	HSC172 cells	SA172 Cells				pBluescript SK−
  T0041	Jurkat T-cell G1 phase	Jurkat T-cell				pBluescript SK−
  T0042	Jurkat T-Cell, S phase	Jurkat T-Cell Line				pBluescript SK−
  T0047	T lymphocytes >70	T lymphocytes >70				pBluescript SK−
  T0048	Human Aortic Endothelium	Human Aortic Endothilium				pBluescript SK−
  T0049	Aorta endothelial cells + TNF-a	Aorta endothelial cells				pBluescript SK−
  T0060	Human White Adipose	Human White Fat				pBluescript SK−
  T0067	Human Thyroid	Human Thyroid				pBluescript SK−
  T0068	Normal Ovary, Premenopausal	Normal Ovary,				pBluescript SK−
  		Premenopausal
  T0069	Human Uterus, normal	Human Uterus, normal				pBluescript SK−
  T0070	Human Adrenal Gland	Human Adrenal Gland				pBluescript SK−
  T0071	Human Bone Marrow	Human Bone Marrow				pBluescript SK−
  T0078	Human Liver, normal adult	Human Liver, normal Adult				pBluescript SK−
  T0079	Human Kidney, normal Adult	Human Kidney, normal				pBluescript SK−
  		Adult
  T0082	Human Adult Retina	Human Adult Retina				pBluescript SK−
  T0090	Liver, normal					pBluescript SK−
  T0091	Liver, hepatocellular carcinoma					pBluescript SK−
  T0104	HCC cell line metastisis to liver					pBluescript SK−
  T0109	Human (HCC) cell line liver (mouse)					pBluescript SK−
  	metastasis, remake
  T0110	Human colon carcinoma (HCC) cell line,					pBluescript SK−
  	remake
  T0112	Human (Caco-2) cell line, adenocarcinoma,					pBluescript SK−
  	colon
  T0114	Human (Caco-2) cell line, adenocarcinoma,					pBluescript SK−
  	colon, remake
  T0115	Human Colon Carcinoma (HCC) cell line					pBluescript SK−
  L0002	Atrium cDNA library Human heart
  L0004	ClonTech HL 1065a
  L0005	Clontech human aorta polyA+ mRNA
  	(#6572)
  L0009	EST from 8p21.3-p22
  L0012	HDMEC cDNA library
  L0015	Human
  L0021	Human adult (K. Okubo)
  L0022	Human adult lung 3'' directed MboI cDNA
  L0024	Human brain ARSanders
  L0032	Human chromosome 12p cDNAs
  L0034	Human chromosome 14
  L0038	Human chromosome 6
  L0040	Human colon mucosa
  L0041	Human epidermal keratinocyte
  L0045	Human keratinocyte differential display
  	(B. Lin)
  L0051	Human mRNA (Tripodis and Ragoussis)
  L0052	Human normalized K562-cDNA
  L0055	Human promyclocyte
  L0060	Human thymus NSTH II
  L0065	Liver HepG2 cell line.
  L0070	Selected chromosome 21 cDNA library
  L0096	Subtracted human retina
  L0097	Subtracted human retinal pigment epithelium
  	(RPE)
  L0103	DKFZphamy1	amygdala
  L0105	Human aorta polyA+ (TFujiwara)	aorta
  L0109	Human brain cDNA	brain
  L0118	Human fetal brain S. Meier-Ewert	brain
  L0126	Human fibroblast cDNA	fibroblast
  L0138	Human normal gingiva	normal gingiva
  L0141	Human pancreatic islet cell	pancreatic islet
  L0142	Human placenta cDNA (TFujiwara)	placenta
  L0143	Human placenta polyA+ (TFujiwara)	placenta
  L0146	Human fovea cDNA	retinal fovea
  L0149	DKFZphsnu1	subthalamic nucleus
  L0151	Human testis (C. De Smet)	testis
  L0157	Human fetal brain (TFujiwara)		brain
  L0158	Human fetal brain QBoqin		brain
  L0162	Human brain frontal cortex	frontal cortex	brain
  L0163	Human heart cDNA (YNakamura)		heart
  L0171	Human lung adenocarcinoma A549	lung adenocarcinoma		A549
  L0175	Human retina cell line ARPE-19	retina		ARPE-19
  L0177	Human newborn melanocytes (T. Vogt)			Clonetics
  				Corp. (San
  				Diego, CA)
  				strain #68
  				and 2486
  L0182	Human HeLa (Y. Wang)			HeLa
  L0186	Human salivary gland cell line HSG	salivary gland		HSG
  L0194	Human pancreatic cancer cell line Patu 8988t	pancreatic cancer		Patu 8988t
  L0351	Infant brain, Bento Soares					BA, M13-derived
  L0352	Normalized infant brain, Bento Soares					BA, M13-derived
  L0353	21q Placenta, F. Tassone and K. Gardiner					Bluescript
  L0355	P, Human foetal Brain Whole tissue					Bluescript
  L0356	S, Human foetal Adrenals tissue					Bluescript
  L0361	Stratagene ovary (#937217)		ovary			Bluescript SK
  L0362	Stratagene ovarian cancer (#937219)					Bluescript SK−
  L0363	NCI_CGAP_GC2	germ cell tumor				Bluescript SK−
  L0364	NCI_CGAP_GC5	germ cell tumor				Bluescript SK−
  L0365	NCI_CGAP_Phe1	pheochromocytoma				Bluescript SK−
  L0366	Stratagene schizo brain S11	schizophrenic brain S-11				Bluescript SK−
  		frontal lobe
  L0367	NCI_CGAP_Sch1	Schwannoma tumor				Bluescript SK−
  L0368	NCI_CGAP_SS1	synovial sarcoma				Bluescript SK−
  L0369	NCI_CGAP_AA1	adrenal adenoma	adrenal gland			Bluescript SK−
  L0370	Johnston frontal cortex	pooled frontal lobe	brain			Bluescript SK−
  L0371	NCI_CGAP_Br3	breast tumor	breast			Bluescript SK−
  L0372	NCI_CGAP_Co12	colon tumor	colon			Bluescript SK−
  L0373	NCI_CGAP_Co11	tumor	colon			Bluescript SK−
  L0374	NCI_CGAP_Co2	tumor	colon			Bluescript SK−
  L0375	NCI_CGAP_Kid6	kidney tumor	kidney			Bluescript SK−
  L0376	NCI_CGAP_Lar1	larynx	larynx			Bluescript SK−
  L0377	NCI_CGAP_HN2	squamous cell carcinoma	larynx			Bluescript SK−
  		from vocal cord
  L0378	NCI_CGAP_Lu1	lung tumor	lung			Bluescript SK−
  L0379	NCI_CGAP_Lym3	lymphoma	lymph node			Bluescript SK−
  L0380	NCI_CGAP_HN1	squamous cell carcinoma	lymph node			Bluescript SK−
  L0381	NCI_CGAP_HN4	squamous cell carcinoma	pharynx			Bluescript SK−
  L0382	NCI_CGAP_Pr25	epithelium (cell line)	prostate			Bluescript SK−
  L0383	NCI_CGAP_Pr24	invasive tumor (cell line)	prostate			Bluescript SK−
  L0384	NCI_CGAP_Pr23	prostate tumor	prostate			Bluescript SK−
  L0385	NCI_CGAP_Gas1	gastric tumor	stomach			Bluescript SK−
  L0386	NCI_CGAP_HN3	squamous cell carcinoma	tongue			Bluescript SK−
  		from base of tongue
  L0387	NCI_CGAP_GCB0	germinal center B-cells	tonsil			Bluescript SK−
  L0388	NCI_CGAP_HN6	normal gingiva (cell line				Bluescript SK−
  		from immortalized kerati
  L0389	NCI_CGAP_HN5	normal gingiva (cell line				Bluescript SK−
  		from primary keratinocyt
  L0393	B, Human Liver tissue					gt11
  L0394	H, Human adult Brain Cortex tissue					gt11
  L0404	b4HB3MA Cot109 + 103 + 85-Bio					Lafmid A
  L0405	b4HB3MA Cot109 + 103-Bio					Lafmid A
  L0411	1-NIB					Lafmid BA
  L0414	b4HB3MA					Lafmid BA
  L0415	b4HB3MA Cot8-HAP-Ft					Lafmid BA
  L0416	b4HB3MA-Cot0.38-HAP-B					Lafmid BA
  L0417	b4HB3MA-Cot0.38-HAP-Ft-6					Lafmid BA
  L0422	b4HB3MA-Cot12-HAP-B					Lafmid BA
  L0424	b4HB3MA-Cot14.5					Lafmid BA
  L0425	b4HB3MA-Cot18-Bio					Lafmid BA
  L0426	b4HB3MA-Cot51.5-HAP-Ft					Lafmid BA
  L0427	b4HB3MA-FT20%-Biotin					Lafmid BA
  L0428	Cot1374Ft-4HB3MA					Lafmid BA
  L0430	Cot250Ft-b4HB3MA					Lafmid BA
  L0434	Infant brain library of Dr. M. Soares					lafmid BA
  L0435	Infant brain, LLNL array of Dr. M. Soares					lafmid BA
  	1NIB
  L0437	N-b4HB3MA-Cot109					Lafmid BA
  L0438	normalized infant brain cDNA	total brain	brain			lafmid BA
  L0439	Soares infant brain 1NIB		whole brain			Lafmid BA
  L0441	2HB3MK					Lafmid BK
  L0442	4HB3MK					Lafmid BK
  L0443	b4HB3MK					Lafmid BK
  L0446	N4HB3MK					Lafmid BK
  L0447	NHB3MK					Lafmid BK
  L0448	3HFLSK20					Lafmid K
  L0451	N3HFLSK20					Lafmid K
  L0453	BATM1					lambda gt10
  L0454	Clontech adult human fat cell library					lambda gt10
  	HL1108A
  L0455	Human retina cDNA randomly primed	retina	eye			lambda gt10
  	sublibrary
  L0456	Human retina cDNA Tsp509I-cleaved	retina	eye			lambda gt10
  	sublibrary
  L0457	multi-tissue normalized short-fragment	multi-tissue	pooled			lambda gt10
  L0459	Adult heart, Clontech					Lambda gt11
  L0460	Adult heart, Lambda gt11					Lambda gt11
  L0462	WATM1					lambda gt11
  L0463	fetal brain cDNA	brain	brain			lambda gt11
  L0465	TEST1, Human adult Testis tissue					lambda nm1149
  L0468	HE6W					lambda zap
  L0469	T, Human adult Rhabdomyosarcoma					Lambda Zap
  	cell-line
  L0470	BL29 Burkitt''s lymphoma, Pascalis Sideras					lambda ZAP 2
  L0471	Human fetal heart, Lambda ZAP Express					Lambda ZAP
  						Express
  L0475	KG1-a Lambda Zap Express cDNA library			KG1-a		Lambda Zap
  						Express
  						(Stratagene)
  L0476	Fetal brain, Stratagene					Lambda ZAP II
  L0477	HPLA CCLee	placenta				Lambda ZAP II
  L0480	Stratagene cat#937212 (1992)					Lambda ZAP,
  						pBluescript SK(−)
  L0481	CD34+DIRECTIONAL					Lambda ZAPII
  L0483	Human pancreatic islet					Lambda ZAPII
  L0485	STRATAGENE Human skeletal muscle	skeletal muscle	leg muscle			Lambda ZAPII
  	cDNA library, cat. #936215.
  L0487	Human peripheral blood (Steve Elledge)	whole peripheral blood				Lambda-Yes
  L0492	Human Genomic					pAMP
  L0493	NCI_CGAP_Ov26	papillary serous carcinoma	ovary			pAMP1
  L0497	NCI_CGAP_HSC4	CD34+, CD38− from normal	bone marrow			pAMP1
  		bone marrow donor
  L0498	NCI_CGAP_HSC3	CD34+, T negative, patient	bone marrow			pAMP1
  		with chronic myelogenou
  L0499	NCI_CGAP_HSC2	stem cell 34+/38+	bone marrow			pAMP1
  L0500	NCI_CGAP_Brn20	oligodendroglioma	brain			pAMP1
  L0501	NCI_CGAP_Brn21	oligodendroglioma	brain			pAMP1
  L0502	NCI_CGAP_Br15	adenocarcinoma	breast			pAMP1
  L0503	NCI_CGAP_Br17	adenocarcinoma	breast			pAMP1
  L0504	NCI_CGAP_Br13	breast carcinoma in situ	breast			pAMP1
  L0505	NCI_CGAP_Br12	invasive carcinoma	breast			pAMP1
  L0506	NCI_CGAP_Br16	lobullar carcinoma in situ	breast			pAMP1
  L0507	NCI_CGAP_Br14	normal epithelium	breast			pAMP1
  L0508	NCI_CGAP_Lu25	bronchioalveolar carcinoma	lung			pAMP1
  L0509	NCI_CGAP_Lu26	invasive adenocarcinoma	lung			pAMP1
  L0510	NCI_CGAP_Ov33	borderline ovarian carcinoma	ovary			pAMP1
  L0511	NCI_CGAP_Ov34	borderline ovarian carcinoma	ovary			pAMP1
  L0512	NCI_CGAP_Ov36	borderline ovarian carcinoma	ovary			pAMP1
  L0513	NCI_CGAP_Ov37	early stage papillary serous	ovary			pAMP1
  		carcinoma
  L0514	NCI_CGAP_Ov31	papillary serous carcinoma	ovary			pAMP1
  L0515	NCI_CGAP_Ov32	papillary serous carcinoma	ovary			pAMP1
  L0516	Chromosome 19p12-p13.1 exon					pAMP10
  L0517	NCI_CGAP_Pr1					pAMP10
  L0518	NCI_CGAP_Pr2					pAMP10
  L0519	NCI_CGAP_Pr3					pAMP10
  L0520	NCI_CGAP_Alv1	alveolar rhabdomyosarcoma				pAMP10
  L0521	NCI_CGAP_Ew1	Ewing''s sarcoma				pAMP10
  L0522	NCI_CGAP_Kid1	kidney				pAMP10
  L0523	NCI_CGAP_Lip2	liposarcoma				pAMP10
  L0524	NCI_CGAP_Li1	liver				pAMP10
  L0525	NCI_CGAP_Li2	liver				pAMP10
  L0526	NCI_CGAP_Pr12	metastatic prostate bone				pAMP10
  		lesion
  L0527	NCI_CGAP_Ov2	ovary				pAMP10
  L0528	NCI_CGAP_Pr5	prostate				pAMP10
  L0529	NCI_CGAP_Pr6	prostate				pAMP10
  L0530	NCI_CGAP_Pr8	prostate				pAMP10
  L0531	NCI_CGAP_Pr20	prostate metastasis, liver				pAMP10
  L0532	NCI_CGAP_Thy1	thyroid				pAMP10
  L0533	NCI_CGAP_HSC1	stem cells	bone marrow			pAMP10
  L0534	Chromosome 7 Fetal Brain cDNA Library	brain	brain			pAMP10
  L0535	NCI_CGAP_Br5	infiltrating ductal carcinoma	breast			pAMP10
  L0536	NCI_CGAP_Br4	normal ductal tissue	breast			pAMP10
  L0537	NCI_CGAP_Ov6	normal cortical stroma	ovary			pAMP10
  L0539	Chromosome 7 Placental cDNA Library		placenta			pAMP10
  L0540	NCI_CGAP_Pr10	invasive prostate tumor	prostate			pAMP10
  L0541	NCI_CGAP_Pr7	low-grade prostatic neoplasia	prostate			pAMP10
  L0542	NCI_CGAP_Pr11	normal prostatic epithelial	prostate			pAMP10
  		cells
  L0543	NCI_CGAP_Pr9	normal prostatic epithelial	prostate			pAMP10
  		cells
  L0544	NCI_CGAP_Pr4	prostatic intraepithelial	prostate			pAMP10
  		neoplasia —high grade
  L0545	NCI_CGAP_Pr4.1	prostatic intraepithelial	prostate			pAMP10
  		neoplasia —high grade
  L0546	NCI_CGAP_Pr18	stroma	prostate			pAMP10
  L0547	NCI_CGAP_Pr16	tumor	prostate			pAMP10
  L0548	Chromosome 7 Thymus cDNA Library	thymus	thymus			pAMP10
  L0549	NCI_CGAP_HN10	carcinoma in situ from				pAMP10
  		retromolar trigone
  L0550	NCI_CGAP_HN9	normal squamous epithelium				pAMP10
  		from retromolar trigone
  L0551	NCI_CGAP_HN7	normal squamous				pAMP10
  		epithelium, floor of mouth
  L0552	NCI_CGAP_HN8	well-differentiated invasive				pAMP10
  		carcinoma, floor of m
  L0553	NCI_CGAP_Co22	colonic adenocarcinoma	colon			pAMP10
  L0554	NCI_CGAP_Li8		liver			pAMP10
  L0555	NCI_CGAP_Lu34	large cell carcinoma	lung			pAMP10
  L0556	NCI_CGAP_Lu34.1	large cell carcinoma	lung			pAMP10
  L0558	NCI_CGAP_Ov40	endometrioid ovarian	ovary			pAMP10
  		metastasis
  L0559	NCI_CGAP_Ov39	papillary serous ovarian	ovary			pAMP10
  		metastasis
  L0560	NCI_CGAP_HN12	moderate to poorly	tongue			pAMP10
  		differentiated invasive
  		carcino
  L0561	NCI_CGAP_HN11	normal squamous epithelium	tongue			pAMP10
  L0562	Chromosome 7 HeLa cDNA Library			HeLa cell		pAMP10
  				line; ATCC
  L0563	Human Bone Marrow Stromal Fibroblast	bone marrow				pBluescript
  L0564	Jia bone marrow stroma	bone marrow stroma				pBluescript
  L0565	Normal Human Trabecular Bone Cells	Bone	Hip			pBluescript
  L0579	Human fetal brain QBoqin2	cerebrum and cerebellum				pBluescript SK
  L0581	Stratagene liver (#937224)		liver			pBluescript SK
  L0583	Stratagene cDNA library Human fibroblast,					pBluescript SK(+)
  	cat#937212
  L0584	Stratagene cDNA library Human heart,					pBluescript SK(+)
  	cat#936208
  L0586	HTCDLI					pBluescript SK(−)
  L0587	Stratagene colon HT29 (#937221)					pBluescript SK−
  L0588	Stratagene endothelial cell 937223					pBluescript SK−
  L0589	Stratagene fetal retina 937202					pBluescript SK−
  L0590	Stratagene fibroblast (#937212)					pBluescript SK−
  L0591	Stratagene HeLa cell s3 937216					pBluescript SK−
  L0592	Stratagene hNT neuron (#937233)					pBluescript SK−
  L0593	Stratagene neuroepithelium (#937231)					pBluescript SK−
  L0594	Stratagene neuroepithelium NT2RAMI					pBluescript SK−
  	937234
  L0595	Stratagene NT2 neuronal precursor 937230	neuroepithelial cells	brain			pBluescript SK−
  L0596	Stratagene colon (#937204)		colon			pBluescript SK−
  L0597	Stratagene corneal stroma (#937222)		cornea			pBluescript SK−
  L0598	Morton Fetal Cochlea	cochlea	ear			pBluescript SK−
  L0599	Stratagene lung (#937210)		lung			pBluescript SK−
  L0600	Weizmann Olfactory Epithelium	olfactory epithelium	nose			pBluescript SK−
  L0601	Stratagene pancreas (#937208)		pancreas			pBluescript SK−
  L0602	Pancreatic Islet	pancreatic islet	pancreas			pBluescript SK−
  L0603	Stratagene placenta (#937225)		placenta			pBluescript SK−
  L0604	Stratagene muscle 937209	muscle	skeletal muscle			pBluescript SK−
  L0605	Stratagene fetal spleen (#937205)	fetal spleen	spleen			pBluescript SK−
  L0606	NCI_CGAP_Lym5	follicular lymphoma	lymph node			pBluescript SK−
  L0607	NCI_CGAP_Lym6	mantle cell lymphoma	lymph node			pBluescript SK−
  L0608	Stratagene lung carcinoma 937218	lung carcinoma	lung	NCI-H69		pBluescript SK−
  L0609	Schiller astrocytoma	astrocytoma	brain			pBluescript SK−
  						(Stratagene)
  L0611	Schiller meningioma	meningioma	brain			pBluescript SK−
  						(Stratagene)
  L0612	Schiller oligodendroglioma	oligodendroglioma	brain			pBluescript SK−
  						(Stratagene)
  L0615	22 week old human fetal liver cDNA library					pBluescriptII SK(−)
  L0617	Chromosome 22 exon					pBluescriptIIKS+
  L0619	Chromosome 9 exon II					pBluescriptIIKS+
  L0622	HM1					pcDNAII
  						(Invitrogen)
  L0623	HM3	pectoral muscle (after				pcDNAII
  		mastectomy)				(Invitrogen)
  L0625	NCI_CGAP_AR1	bulk alveolar tumor				pCMV-SPORT2
  L0626	NCI_CGAP_GC1	bulk germ cell seminoma				pCMV-SPORT2
  L0627	NCI_CGAP_Col	bulk tumor	colon			pCMV-SPORT2
  L0628	NCI_CGAP_Ov1	ovary bulk tumor	ovary			pCMV-SPORT2
  L0629	NCI_CGAP_Mel3	metastatic melanoma to	bowel (skin			pCMV-SPORT4
  		bowel	primary)
  L0630	NCI_CGAP_CNS1	substantia nigra	brain			pCMV-SPORT4
  L0631	NCI_CGAP_Br7		breast			pCMV-SPORT4
  L0632	NCI_CGAP_Li5	hepatic adenoma	liver			pCMV-SPORT4
  L0633	NCI_CGAP_Lu6	small cell carcinoma	lung			pCMV-SPORT4
  L0634	NCI_CGAP_Ov8	serous adenocarcinoma	ovary			pCMV-SPORT4
  L0635	NCI_CGAP_PNS1	dorsal root ganglion	peripheral			pCMV-SPORT4
  			nervous system
  L0636	NCI_CGAP_Pit1	four pooled pituitary	brain			pCMV-SPORT6
  		adenomas
  L0637	NCI_CGAP_Brn53	three pooled meningiomas	brain			pCMV-SPORT6
  L0638	NCI_CGAP_Brn35	tumor, 5 pooled (see	brain			pCMV-SPORT6
  		description)
  L0639	NCI_CGAP_Brn52	tumor, 5 pooled (see	brain			pCMV-SPORT6
  		description)
  L0640	NCI_CGAP_Br18	four pooled high-grade	breast			pCMV-SPORT6
  		tumors, including two prima
  L0641	NCI_CGAP_Co17	juvenile granulosa tumor	colon			pCMV-SPORT6
  L0642	NCI_CGAP_Co18	moderately differentiated	colon			pCMV-SPORT6
  		adenocarcinoma
  L0643	NCI_CGAP_Co19	moderately differentiated	colon			pCMV-SPORT6
  		adenocarcinoma
  L0644	NCI_CGAP_Co20	moderately differentiated	colon			pCMV-SPORT6
  		adenocarcinoma
  L0645	NCI_CGAP_Co21	moderately differentiated	colon			pCMV-SPORT6
  		adenocarcinoma
  L0646	NCI_CGAP_Co14	moderately-differentiated	colon			pCMV-SPORT6
  		adenocarcinoma
  L0647	NCI_CGAP_Sar4	five pooled sarcomas,	connective tissue			pCMV-SPORT6
  		including myxoid
  		liposarcoma
  L0648	NCI_CGAP_Eso2	squamous cell carcinoma	esophagus			pCMV-SPORT6
  L0649	NCI_CGAP_GU1	2 pooled high-grade	genitourinary			pCMV-SPORT6
  		transitional cell tumors	tract
  L0650	NCI_CGAP_Kid13	2 pooled Wilms” tumors, one	kidney			pCMV-SPORT6
  		primary and one metast
  L0651	NCI_CGAP_Kid8	renal cell tumor	kidney			pCMV-SPORT6
  L0652	NCI_CGAP_Lu27	four pooled poorly-	lung			pCMV-SPORT6
  		differentiated
  		adenocarcinomas
  L0653	NCI_CGAP_Lu28	two pooled squamous cell	lung			pCMV-SPORT6
  		carcinomas
  L0654	NCI_CGAP_Lu31		lung, cell line			pCMV-SPORT6
  L0655	NCI_CGAP_Lym12	lymphoma, follicular mixed	lymph node			pCMV-SPORT6
  		small and large cell
  L0656	NCI_CGAP_Ov38	normal epithelium	ovary			pCMV-SPORT6
  L0657	NCI_CGAP_Ov23	tumor, 5 pooled (see	ovary			pCMV-SPORT6
  		description)
  L0658	NCI_CGAP_Ov35	tumor, 5 pooled (see	ovary			pCMV-SPORT6
  		description)
  L0659	NCI_CGAP_Pan1	adenocarcinoma	pancreas			pCMV-SPORT6
  L0661	NCI_CGAP_Mel15	malignant melanoma,	skin			pCMV-SPORT6
  		metastatic to lymph node
  L0662	NCI_CGAP_Gas4	poorly differentiated	stomach			pCMV-SPORT6
  		adenocarcinoma with signet r
  L0663	NCI_CGAP_Ut2	moderately-differentiated	uterus			pCMV-SPORT6
  		endometrial adenocarcino
  L0664	NCI_CGAP_Ut3	poorly-differentiated	uterus			pCMV-SPORT6
  		endometrial adenocarcinoma,
  L0665	NCI_CGAP_Ut4	serous papillary carcinoma,	uterus			pCMV-SPORT6
  		high grade, 2 pooled t
  L0666	NCI_CGAP_Ut1	well-differentiated	uterus			pCMV-SPORT6
  		endometrial
  		adenocarcinoma, 7
  L0667	NCI_CGAP_CML1	myeloid cells, 18 pooled	whole blood			pCMV-SPORT6
  		CML cases, BCR/ABL
  		rearra
  L0669	Human MCF7 cDNA subtracted with MDA-	breast adenocarcinoma	breast	MCF7		pCR II [Invitrogen]
  	MB-231 cDNA
  L0681	Stanley Frontal SN individual	frontal lobe (see description)	brain			pCR2.1
  						(Invitrogen)
  L0682	Stanley Frontal NB pool 2	frontal lobe (see description)	brain			pCR2.1-TOPO
  						(Invitrogen)
  L0683	Stanley Frontal NS pool 2	frontal lobe (see description)	brain			pCR2.1-TOPO
  						(Invitrogen)
  L0684	Stanley Frontal SB pool 1	frontal lobe (see description)	brain			pCR2.1-TOPO
  						(Invitrogen)
  L0685	Stanley Frontal SN pool 1	frontal lobe (see description)	brain			pCR2.1-TOPO
  						(Invitrogen)
  L0686	Stanley Frontal SN pool 2	frontal lobe (see description)	brain			pCR2.1-TOPO
  						(Invitrogen)
  L0687	Stanley Hippocampus NB pool 1	hippocampus (see	brain			pCR2.1-TOPO
  		description)				(Invitrogen)
  L0688	Stanley Hippocampus SB pool 1	hippocampus (see	brain			pCR2.1-TOPO
  		description)				(Invitrogen)
  L0689	Stanley Hippocampus SN pool 1	hippocampus (see	brain			pCR2.1-TOPO
  		description)				(Invitrogen)
  L0695	Human Glialblastoma Cell		Brain	BT-325		PCRII, Invitrogen
  L0697	Testis 1					PGEM 5zf(+)
  L0698	Testis 2					PGEM 5zf(+)
  L0700	Outward Alu-primed hncDNA library					pGEM-3Z
  L0709	NIH_MGC_21	choriocarcinoma	placenta			pOTB7
  L0710	NIH_MGC_7	small cell carcinoma	lung	MGC3		pOTB7
  L0716	PMA-induced HL60 cell subtraction library			PMA-		pSPORT 1
  				induced
  				HL60
  				human
  				leukemic
  				cell line
  L0717	Gessler Wilms tumor					pSPORT1
  L0718	Testis 5					pSPORT1
  L0719	human embryo cDNA library	Whole embryo				pSPORT1
  L0731	Soares_pregnant_uterus_NbHPU		uterus			pT7T3-Pac
  L0738	Human colorectal cancer					pT7T3D
  L0740	Soares melanocyte 2NbHM	melanocyte				pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0741	Soares adult brain N2b4HB55Y		brain			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0742	Soares adult brain N2b5HB55Y		brain			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0743	Soares breast 2NbHBst		breast			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0744	Soares breast 3NbHBst		breast			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0745	Soares retina N2b4HR	retina	eye			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0746	Soares retina N2b5HR	retina	eye			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0747	Soares_fetal_heart_NbHH19W		heart			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0748	Soares fetal liver spleen 1NFLS		Liver and Spleen			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0749	Soares_fetal_liver_spleen_1NFLS_S1		Liver and Spleen			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0750	Soares_fetal_lung_NbHL19W		lung			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0751	Soares ovary tumor NbHOT	ovarian tumor	ovary			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0752	Soares_parathyroid_tumor_NbHPA	parathyroid tumor	parathyroid			pT7T3D
  			gland			(Pharmacia) with a
  						modified polylinker
  L0753	Soares_pineal_gland_N3HPG		pineal gland			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0754	Soares placenta Nb2HP		placenta			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0755	Soares_placenta_8to9weeks_2NbHP8to9W		placenta			pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  L0756	Soares_multiple_sclerosis_2NbHMSP	multiple sclerosis lesions				pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  						V_TYPE
  L0757	Soares_senescent_fibroblasts_NbHSF	senescent fibroblast				pT7T3D
  						(Pharmacia) with a
  						modified polylinker
  						V_TYPE
  L0758	Soares_testis_NHT					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0759	Soares_total_fetus_Nb2HF8_9w					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0760	Barstead aorta HPLRB3	aorta				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0761	NCI_CGAP_CLL1	B-cell, chronic lymphotic				pT7T3D-Pac
  		leukemia				(Pharmacia) with a
  						modified polylinker
  L0762	NCI_CGAP_Br1.1	breast				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0763	NCI_CGAP_Br2	breast				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0764	NCI_CGAP_Co3	colon				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0765	NCI_CGAP_Co4	colon				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0766	NCI_CGAP_GCB1	germinal center B cell				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0767	NCI_CGAP_GC3	pooled germ cell tumors				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0768	NCI_CGAP_GC4	pooled germ cell tumors				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0769	NCI_CGAP_Brn25	anaplastic	brain			pT7T3D-Pac
  		oligodendroglioma				(Pharmacia) with a
  						modified polylinker
  L0770	NCI_CGAP_Brn23	glioblastoma (pooled)	brain			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0771	NCI_CGAP_Co8	adenocarcinoma	colon			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0772	NCI_CGAP_Co10	colon tumor RER+	colon			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0773	NCI_CGAP_Co9	colon tumor RER+	colon			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0774	NCI_CGAP_Kid3		kidney			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0775	NCI_CGAP_Kid5	2 pooled tumors (clear cell	kidney			pT7T3D-Pac
  		type)				(Pharmacia) with a
  						modified polylinker
  L0776	NCI_CGAP_Lu5	carcinoid	lung			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0777	Soares_NhHMPu_S1	Pooled human melanocyte,	mixed (see			pT7T3D-Pac
  		fetal heart, and pregnant	below)			(Pharmacia) with a
  						modified polylinker
  L0778	Barstead pancreas HPLRB1		pancreas			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0779	Soares_NFL_T_GBC_S1		pooled			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0780	Soares_NSF_F8_9W_OT_PA_P_S1		pooled			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0782	NCI_CGAP_Pr21	normal prostate	prostate			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0783	NCI_CGAP_Pr22	normal prostate	prostate			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0784	NCI_CGAP_Lei2	leiomyosarcoma	soft tissue			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0785	Barstead spleen HPLRB2		spleen			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0786	Soares_NbHFB		whole brain			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0787	NCI_CGAP_Sub1					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0788	NCI_CGAP_Sub2					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0789	NCI_CGAP_Sub3					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0790	NCI_CGAP_Sub4					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0791	NCI_CGAP_Sub5					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0792	NCI_CGAP_Sub6					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0793	NCI_CGAP_Sub7					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0794	NCI_CGAP_GC6	pooled germ cell tumors				pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0796	NCI_CGAP_Brn50	medulloblastoma	brain			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0800	NCI_CGAP_Co16	colon tumor, RER+	colon			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0803	NCI_CGAP_Kid11		kidney			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0804	NCI_CGAP_Kid12	2 pooled tumors (clear cell	kidney			pT7T3D-Pac
  		type)				(Pharmacia) with a
  						modified polylinker
  L0805	NCI_CGAP_Lu24	carcinoid	lung			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0806	NCI_CGAP_Lu19	squamous cell carcinoma,	lung			pT7T3D-Pac
  		poorly differentiated (4				(Pharmacia) with a
  						modified polylinker
  L0807	NCI_CGAP_Ov18	fibrotheoma	ovary			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0808	Barstead prostate BPH HPLRB4 1		prostate			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0809	NCI_CGAP_Pr28		prostate			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L0811	BATM2					PTZ18
  L0988	BT0387		breast			puc18
  L1430	CT0225		colon			puc18
  L1562	CN0027		colon_normal			puc18
  L1819	HT0268		head_neck			puc18
  L2138	ST0186		stomach			puc18
  L2245	NEM subtracted human fetal kidney cDNA					pUEX1
  L2250	Human cerebral cortex	cerebral cortex
  L2251	Human fetal lung	Fetal lung
  L2252	Human placenta	placenta
  L2255	GLC	corresponding non cancerous				pBluescript sk(−)
  		liver tissue
  L2257	NIH_MGC_65	adenocarcinoma	colon			pCMV-SPORT6
  L2258	NIH_MGC_67	retinoblastoma	eye			pCMV-SPORT6
  L2259	NIH_MGC_68	large cell carcinoma	lung			pCMV-SPORT6
  L2260	NIH_MGC_69	large cell carcinoma,	lung			pCMV-SPORT6
  		undifferentiated
  L2261	NIH_MGC_70	epithelioid carcinoma	pancreas			pCMV-SPORT6
  L2262	NIH_MGC_72	melanotic melanoma	skin			pCMV-SPORT6
  L2263	NIH_MGC_66	adenocarcinoma	ovary			pCMV-SPORT6
  L2264	NIH_MGC_71	leiomyosarcoma	uterus			pCMV-SPORT6
  L2265	NIH_MGC_39	adenocarcinoma	pancreas			pOTB7
  L2269	NCI_CGAP_Thy11	follicular carcinoma	thyroid			pAMP10
  L2270	Lupski_dorsal_root_ganglion	dorsal root ganglia				pCMV-SPORT6
  						(Life Technologies)
  L2285	BT0723		breast			puc18
  L2289	BT0757		breast			puc18
  L2293	BT0762		breast			puc18
  L2300	BT0789		breast			puc18
  L2323	CT0406		colon			puc18
  L2336	CT0428		colon			puc18
  L2352	UT0001		uterus_tumor			puc18
  L2357	UT0021		uterus_tumor			puc18
  L2359	UT0023		uterus_tumor			puc18
  L2368	UT0041		uterus_tumor			puc18
  L2380	NN0068		nervous_normal			puc18
  L2381	NN0070		nervous_normal			puc18
  L2402	NN0118		nervous_normal			puc18
  L2412	NN0136		nervous_normal			puc18
  L2439	NN1022		nervous_normal			puc18
  L2482	HT0497		head_neck			puc18
  L2486	HT0527		head_neck			puc18
  L2487	HT0542		head_neck			puc18
  L2491	HT0559		head_neck			puc18
  L2497	HT0618		head_neck			puc18
  L2499	HT0622		head_neck			puc18
  L2504	HT0636		head_neck			puc18
  L2519	HT0698		head_neck			puc18
  L2528	HT0713		head_neck			puc18
  L2543	HT0734		head_neck			puc18
  L2551	HT0744		head_neck			puc18
  L2630	HT0865		head_neck			puc18
  L2635	HT0875		head_neck			puc18
  L2647	HT0894		head_neck			puc18
  L2652	NIH_MGC_57	glioblastoma	brain			pDNR-LIB
  						(Clontech)
  L2653	NIH_MGC_58	hypernephroma	kidney			pDNR-LIB
  						(Clontech)
  L2654	NIH_MGC_9	adenocarcinoma cell line	ovary			pOTB7
  L2655	NIH_MGC_55	from acute myelogenous	bone marrow			pDNR-LIB
  		leukemia				(Clontech)
  L2657	NIH_MGC_54	from chronic myelogenous	bone marrow			pDNR-LIB
  		leukemia				(Clontech)
  L2669	NT0022		nervous_tumor			puc18
  L2670	NT0023		nervous_tumor			puc18
  L2673	NT0028		nervous_tumor			puc18
  L2675	NT0033		nervous_tumor			puc18
  L2706	NT0102		nervous_tumor			puc18
  L2744	FT0004		prostate_tumor			puc18
  L2758	FT0027		prostate_tumor			puc18
  L2759	FT0028		prostate_tumor			puc18
  L2767	FT0044		prostate_tumor			puc18
  L2771	FT0050		prostate_tumor			puc18
  L2777	FT0056		prostate_tumor			puc18
  L2791	FT0077		prostate_tumor			puc18
  L2800	FT0097		prostate_tumor			puc18
  L2842	UM0009		uterus			puc18
  L2877	AN0027		amnion_normal			puc18
  L2879	AN0032		amnion_normal			puc18
  L2903	BN0039		breast_normal			puc18
  L2904	BN0042		breast_normal			puc18
  L2906	BN0047		breast_normal			puc18
  L2909	BN0067		breast_normal			puc18
  L2910	BN0070		breast_normal			puc18
  L2915	BN0098		breast_normal			puc18
  L2985	BN0257		breast_normal			puc18
  L2991	BN0264		breast_normal			puc18
  L2999	BN0273		breast_normal			puc18
  L3001	BN0275		breast_normal			puc18
  L3012	BN0296		breast_normal			puc18
  L3019	BN0303		breast_normal			puc18
  L3092	ET0023		lung_tumor			puc18
  L3109	ET0046		lung_tumor			puc18
  L3117	ET0068		lung_tumor			puc18
  L3119	ET0072		lung_tumor			puc18
  L3180	MT0101		marrow			puc18
  L3181	MT0107		marrow			puc18
  L3210	OT0067		ovary			puc18
  L3215	OT0083		ovary			puc18
  L3271	FN0094		prostate_normal			puc18
  L3278	FN0104		prostate_normal			puc18
  L3280	FN0106		prostate_normal			puc18
  L3311	FN0180		prostate_normal			puc18
  L3327	SN0024		stomach_normal			puc18
  L3357	TN0034		testis_normal			puc18
  L3372	TN0068		testis_normal			puc18
  L3378	TN0080		testis_normal			puc18
  L3385	Homo sapiens HeLa			HeLa
  L3387	GKB	hepatocellular carcinoma				pBluescript sk(−)
  L3388	GKC	hepatocellular carcinoma				pBluescript sk(−)
  L3391	NIH_MGC_53	carcinoma, cell line	bladder			pDNR-LIB
  						(Clontech)
  L3450	CT0508		colon			puc18
  L3485	GN0070		placenta_normal			puc18
  L3499	HT0617		head_neck			puc18
  L3503	HT0870		head_neck			puc18
  L3516	HT0913		head_neck			puc18
  L3560	TN0023		testis_normal			puc18
  L3563	TN0037		testis_normal			puc18
  L3566	TN0046		testis_normal			puc18
  L3576	TN0086		testis_normal			puc18
  L3585	TN0119		testis_normal			puc18
  L3592	TN0129		testis_normal			puc18
  L3630	UT0071		uterus_tumor			puc18
  L3632	UT0074		uterus_tumor			puc18
  L3642	ADA	Adrenal gland				pBluescript sk(−)
  L3643	ADB	Adrenal gland				pBluescript sk(−)
  L3644	ADC	Adrenal gland				pBluescript sk(−)
  L3645	Cu	adrenal cortico adenoma for				pBluescript sk(−)
  		Cushing''s syndrome
  L3646	DCA					pTriplEx2
  L3647	Human HO-1 melanoma cells
  L3649	DCB					pTriplEx2
  L3651	FHTA	hypothalamus				pTriplEx2
  L3652	FHTB	hypothalamus				pTriplEx2
  L3653	HTB	Hypothalamus				pBluescript sk(−)
  L3655	HTC	Hypothalamus				pBluescript sk(−)
  L3657	HTF	Hypothalamus				pBluescript sk(−)
  L3658	cdA	pheochromocytoma				pTriplEx2
  L3659	CB	cord blood				pBluescript
  L3660	NP1	pituitary				pBluescript sk(−)
  L3661	NPA	pituitary				pBluescript sk(−)
  L3663	NIH_MGC_60	adenocarcinoma	prostate			pDNR-LIB
  						(Clontech)
  L3665	NIH_MGC_75		kidney			pDNR-LIB
  						(Clontech)
  L3709	CT0515		colon			puc18
  L3726	GN0038		placenta_normal			puc18
  L3729	GN0079		placenta_normal			puc18
  L3750	HT0945		head_neck			puc18
  L3783	TN0136		testis_normal			puc18
  L3796	UT0042		uterus_tumor			puc18
  L3807	UT0077		uterus_tumor			puc18
  L3811	NPC	pituitary				pBluescript sk(−)
  L3812	NPD	pituitary				pBluescript sk(−)
  L3813	TP	pituitary tumor				pTriplEx2
  L3814	BM	Bone marrow				pTriplEx2
  L3815	MDS	Bone marrow				pTriplEx2
  L3816	HEMBA1	whole embryo, mainly head				pME18SFL3
  L3817	HEMBB1	whole embryo, mainly body				pME18SFL3
  L3818	MAMMA1	mammary gland				pME18SFL3
  L3820	NIH_MGC_46	leiomyosarcoma cell line	uterus			pOTB7
  L3821	NIH_MGC_48	primary B-cells from tonsils	B-cells			pOTB7
  		(cell line)
  L3822	NIH_MGC_59	mucoepidermoid carcinoma	lung			pDNR-LIB
  						(Clontech)
  L3823	NT2RM1			NT2		pUC19FL3
  L3824	NT2RM2			NT2		pME18SFL3
  L3825	NT2RM4			NT2		pME18SFL3
  L3826	NT2RP1			NT2		pUC19FL3
  L3827	NT2RP2			NT2		pME18SFL3
  L3828	NT2RP3			NT2		pME18SFL3
  L3829	NT2RP4			NT2		pME18SFL3
  L3831	OVARC1	ovary, tumor tissue				pME18SFL3
  L3832	PLACE1	placenta				pME18SFL3
  L3833	PLACE2	placenta				pME18SFL3
  L3834	PLACE3	placenta				pME18SFL3
  L3837	THYRO1	thyroid gland				pME18SFL3
  L3872	NCI_CGAP_Skn1		skin, normal, 4			pCMV-SPORT6
  			pooled sa
  L3904	NCI_CGAP_Brn64	glioblastoma with EGFR	brain			pCMV-SPORT6
  		amplification
  L3905	NCI_CGAP_Brn67	anaplastic	brain			pCMV-SPORT6
  		oligodendroglioma with
  		1p/19q loss
  L4497	NCI_CGAP_Br22	invasive ductal carcinoma, 3	breast			pCMV-SPORT6
  		pooled samples
  L4500	NCI_CGAP_HN16	moderate to poorly	mouth			pAMP10
  		differentiated invasive
  		carcino
  L4501	NCI_CGAP_Sub8					pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L4507	NCI_CGAP_Thy6	normal epithelium	thyroid			pAMP10
  L4508	NCI_CGAP_Thy8	normal epithelium	thyroid			pAMP10
  L4537	NCI_CGAP_Thy7	follicular adenoma (benign	thyroid			pAMP10
  		lesion)
  L4556	NCI_CGAP_HN13	squamous cell carcinoma	tongue			pCMV-SPORT6
  L4557	NCI_CGAP_Adr1	neuroblastoma	adrenal gland			pCMV-SPORT6
  L4558	NCI_CGAP_Pan3		pancreas			pCMV-SPORT6
  L4559	NCI_CGAP_Thy3	follicular carcinoma	thyroid			pCMV-SPORT6
  L4560	NCI_CGAP_Ut7	tumor	uterus			pCMV-SPORT6
  L4669	NCI_CGAP_Ov41	serous papillary tumor	ovary			pCMV-SPORT6
  L4747	NCI_CGAP_Brn41	oligodendroglioma	brain			pT7T3D-Pac
  						(Pharmacia) with a
  						modified polylinker
  L4753	NCI_CGAP_HN15	leukoplakia of the buccal	mouth			pAMP10
  		mucosa
  L4775	NCI_CGAP_Thy12	papillary carcinoma	thyroid			pAMP10
  L5286	NCI_CGAP_Thy10	medullary carcinoma	thyroid			pAMP10
  L5564	NCI_CGAP_HN20		normal			pAMP1
  			head/neck tissue
  L5565	NCI_CGAP_Brn66	glioblastoma with probably	brain			pCMV-SPORT6
  		TP53 mutation and witho
  L5566	NCI_CGAP_Brn70	anaplastic	brain			pCMV-
  		oligodendroglioma				SPORT6.ccdb
  L5568	NCI_CGAP_HN21	nasopharyngeal carcinoma	head/neck			pAMP1
  L5569	NCI_CGAP_HN17	normal epithetlium	nasopharynx			pAMP10
  L5572	NCI_CGAP_Co27	adenocarcinoma (mucinous	colon			pAMP1
  		component)
  L5574	NCI_CGAP_HN19	normal epithetlium	nasopharynx			pAMP10
  L5575	NCI_CGAP_Brn65	glioblastoma without EGFR	brain			pCMV-SPORT6
  		amplification
  L5622	NCI_CGAP_Skn3		skin			pCMV-SPORT6
  L5623	NCI_CGAP_Skn4	squamous cell carcinoma	skin			pCMV-SPORT6

  
  Description of Table 5
• Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B. 1, column 9. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8, as determined using the Morbid Map database.

  TABLE 5
  OMIM
  Reference	Description
  100678	ACAT2 deficiency
  100690	Myasthenic syndrome, slow-channel congenital, 601462
  100710	Myasthenic syndrome, slow-channel congenital, 601462
  100730	Myasthenia gravis, neonatal transient
  101000	Meningioma, NF2-related, sporadic Schwannoma, sporadic
  101000	Neurofibromatosis, type 2
  101000	Neurolemmomatosis
  101000	Malignant mesothelioma, sporadic
  102200	Somatotrophinoma
  102540	Cardiomyopathy, idiopathic dilated
  102578	Leukemia, acute promyelocytic, PML/RARA type
  102700	Severe combined immunodeficiency due to ADA deficiency
  102700	Hemolytic anemia due to ADA excess
  102770	Myoadenylate deaminase deficiency
  102772	[AMP deaminase deficiency, erythrocytic]
  103000	Hemolytic anemia due to adenylate kinase deficiency
  103050	Autism, succinylpurinemic
  103050	Adenylosuccinase deficiency
  103581	Albright hereditary osteodystrophy-2
  103600	[Dysalbuminemic hyperthyroxinemia]
  103600	[Dysalbuminemic hyperzincemia], 194470
  103600	Analbuminemia
  103720	Alcoholism, susceptibility to
  103850	Aldolase A deficiency
  103950	Emphysema due to alpha-2-macroglobulin deficiency
  104150	[AFP deficiency, congenital]
  104150	[Hereditary persistence of alpha-fetoprotein]
  104311	Alzheimer disease-3
  104500	Amelogenesis imperfecta-2, hypoplastic local type
  104770	Amyloidosis, secondary, susceptibility to
  105580	Anal canal carcinoma
  105600	Dyserythropoietic anemia, congenital, type III
  106100	Angioedema, hereditary
  106150	Hypertension, essential, susceptibility to
  106150	Preeclampsia, susceptibility to
  106165	Hypertension, essential, 145500
  106180	Myocardial infarction, susceptibility to
  106210	Peters anomaly
  106210	Cataract, congenital, with late-onset corneal dystrophy
  106210	Foveal hypoplasia, isolated, 136520
  106210	Aniridia
  106300	Ankylosing spondylitis
  107250	Anterior segment mesenchymal dysgenesis
  107271	CD59 deficiency
  107300	Antithrombin III deficiency
  107470	Atypical mycobacterial infection, familial disseminated, 209950
  107470	BCG infection, generalized familial
  107470	Tuberculosis, susceptibility to
  107670	Apolipoprotein A-II deficiency
  107680	ApoA-I and apoC-III deficiency, combined
  107680	Corneal clouding, autosomal recessive
  107680	Amyloidosis, 3 or more types
  107680	Hypertriglyceridemia, one form
  107680	Hypoalphalipoproteinemia
  107720	Hypertriglyceridemia
  107741	Hyperlipoproteinemia, type III
  107777	Diabetes insipidus, nephrogenic, autosomal recessive, 222000
  107970	Arrhythmogenic right ventricular dysplasia-1
  108120	Distal arthrogryposis-1
  108725	Atherosclerosis, susceptibility to
  108730	Brody myopathy, 601003
  108800	Atrial septal defect, secundum type
  108962	Hypertension, salt-resistant
  108985	Atrophia areata
  109150	Machado-Joseph disease
  109270	Renal tubular acidosis, distal, 179800
  109270	Spherocytosis, hereditary
  109270	[Acanthocytosis, one form]
  109270	[Elliptocytosis, Malaysian-Melanesian type]
  109270	Hemolytic anemia due to band 3 defect
  109565	Lymphoma, B-cell
  109565	Lymphoma, diffuse large cell
  109690	Asthma, nocturnal, susceptibility to
  109690	Obesity, susceptibility to
  109700	Hemodialysis-related amyloidosis
  110100	Blepharophimosis, epicanthus inversus, and ptosis, type 1
  110700	Vivax malaria, susceptibility to
  112250	Bone dysplasia with medullary fibrosarcoma
  112262	Fibrodysplasia ossificans progressiva, 135100
  112410	Hypertension with brachydactyly
  113721	Breast cancer
  113811	Epidermolysis bullosa, generalized atrophic benign, 226650
  113900	Heart block, progressive familial, type I
  114130	Osteoporosis
  114208	Malignant hyperthermia susceptibility 5, 601887
  114208	Hypokalemic periodic paralysis, 170400
  114240	Muscular dystrophy, limb-girdle, type 2A, 253600
  114290	Campomelic dysplasia with autosomal sex reversal
  114350	Leukemia, acute myeloid
  114400	Lynch cancer family syndrome II
  114550	Hepatocellular carcinoma
  114835	Monocyte carboxyesterase deficiency
  115200	Cardiomyopathy, dilated, 1A
  115500	Acatalasemia
  115650	Cataract, anterior polar-1
  115660	Cataract, cerulean, type 1
  115665	Cataract, congenital, Volkmann type
  116800	Cataract, Marner type
  116806	Colorectal cancer
  116860	Cavernous angiomatous malformations
  117700	[Hypoceruloplasminemia, hereditary]
  117700	Hemosiderosis, systemic, due to aceruloplasminemia
  118210	Charcot-Marie-Tooth neuropathy-2A
  118425	Myotonia congenita, dominant, 160800
  118425	Myotonia congenita, recessive, 255700
  118425	Myotonia levior, recessive
  118485	Polycystic ovary syndrome with hyperandrogenemia
  118504	Epilepsy, benign neonatal, type 1, 121200
  118504	Epilepsy, nocturnal frontal lobe, 600513
  118511	Schizophrenia, neurophysiologic defect in
  118800	Choreoathetosis, familial paroxysmal
  119300	van der Woude syndrome
  120070	Alport syndrome, autosomal recessive, 203780
  120110	Metaphyseal chondrodysplasia, Schmid type
  120120	Epidermolysis bullosa dystrophica, dominant, 131750
  120120	Epidermolysis bullosa dystrophica, recessive, 226600
  120120	Epidermolysis bullosa, pretibial, 131850
  120131	Alport syndrome, autosomal recessive, 203780
  120131	Hematuria, familial benign
  120140	Osteoarthrosis, precocious
  120140	SED congenita
  120140	SMED Strudwick type
  120140	Stickler syndrome, type I
  120140	Wagner syndrome, type II
  120140	Achondrogenesis-hypochondrogenesis, type II
  120140	Kniest dysplasia
  120150	Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420,
  	166220
  120150	Osteoporosis, idiopathic, 166710
  120150	Ehlers-Danlos syndrome, type VIIA1, 130060
  120160	Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420,
  	166220
  120160	Osteoporosis, idiopathic, 166710
  120160	Ehlers-Danlos syndrome, type VIIA2, 130060
  120160	Marfan syndrome, atypical
  120180	Ehlers-Danlos syndrome, type III
  120180	Ehlers-Danlos syndrome, type IV, 130050
  120180	Fibromuscular dysplasia of arteries, 135580
  120180	Aneurysm, familial, 100070
  120190	Ehlers-Danlos syndrome, type I, 130000
  120215	Ehlers-Danlos syndrome, type I, 130000
  120215	Ehlers-Danlos syndrome, type II, 130010
  120220	Bethlem myopathy, 158810
  120240	Bethlem myopathy, 158810
  120260	Epiphyseal dysplasia, multiple, type 2, 600204
  120280	Stickler syndrome, type III
  120280	Marshall syndrome, 154780
  120290	OSMED syndrome, 215150
  120290	Stickler syndrome, type II, 184840
  120435	Muir-Torre syndrome, 158320
  120435	Colorectal cancer, hereditary, nonpolyposis, type 1 Ovarian cancer
  120436	Muir-Torre family cancer syndrome, 158320
  120436	Turcot syndrome with glioblastoma, 276300
  120436	Colorectal cancer, hereditary nonpolyposis, type 2
  120550	C1q deficiency, type A
  120570	C1q deficiency, type B
  120575	C1q deficiency, type C
  120580	C1r/C1s is deficiency, combined
  120620	SLE susceptibility
  120620	CR1 deficiency
  120700	C3 deficiency
  120810	C4 deficiency
  120820	C4 deficiency
  120900	C5 deficiency
  120920	Measles, susceptibility to
  120940	C9 deficiency
  120950	C8 deficiency, type I
  120960	C8 deficiency, type II
  121014	Heterotaxia, visceroatrial, autosomal recessive
  121050	Contractural arachnodactyly, congenital
  121360	Myeloid leukemia, acute, M4Eo subtype
  121800	Corneal dystrophy, crystalline, Schnyder
  122720	Nicotine addiction, protection from
  122720	Coumarin resistance, 122700
  123000	Craniometaphyseal dysplasia
  123100	Craniosynostosis, type 1
  123101	Craniosynostosis, type 2
  123270	[Creatine kinase, brain type, ectopic expression of]
  123580	Cataract, congenital, autosomal dominant
  123620	Cataract, cerulean, type 2, 601547
  123660	Cataract, Coppock-like
  123829	Melanoma
  123940	White sponge nevus, 193900
  124020	Mephenytoin poor metabolizer
  124030	Parkinsonism, susceptibility to
  124030	Debrisoquine sensitivity
  124200	Darier disease (keratosis follicularis)
  125264	Leukemia, acute nonlymphocytic
  125270	Porphyria, acute hepatic
  125270	Lead poisoning, susceptibility to
  125370	Dentatorubro-pallidoluysian atrophy
  125490	Dentinogenesis imperfecta-1
  125660	Myopathy, desminopathic
  125660	Cardiomyopathy
  125852	Insulin-dependent diabetes mellitus-2
  126060	Anemia, megaloblastic, due to DHFR deficiency
  126337	Myxoid liposarcoma
  126340	Xeroderma pigmentosum, group D, 278730
  126391	DNA ligase I deficiency
  126451	Schizophrenia, susceptibility to
  126452	Autonomic nervous system dysfunction
  126452	[Novelty seeking personality]
  126600	Drusen, radial, autosomal dominant
  126650	Chloride diarrhea, congenital, Finnish type, 214700
  126650	Colon cancer
  128100	Dystonia-1, torsion
  129010	Neuropathy, congenital hypomyelinating, 1
  129490	Ectodermal dysplasia-3, anhidrotic
  129900	EEC syndrome-1
  130410	Glutaricaciduria, type IIB
  130500	Elliptocytosis-1
  130650	Beckwith-Wiedemann syndrome
  131100	Multiple endocrine neoplasia I
  131100	Prolactinoma, hyperparathyroidism, carcinoid syndrome
  131100	Carcinoid tumor of lung
  131195	Hereditary hemorrhagic telangiectasia-1, 187300
  131210	Atherosclerosis, susceptibility to
  131242	Shah-Waardenburg syndrome, 277580
  131400	Eosinophilia, familial
  131440	Eosinophilic myeloproliferative disorder
  132700	Cylindromatosis
  132810	Diphenylhydantoin toxicity
  132810	Fetal hydantoin syndrome
  133170	Erythremia
  133171	[Erythrocytosis, familial], 133100
  133200	Erythrokeratodermia variabilis
  133430	Breast cancer
  133430	Estrogen resistance
  133450	Neuroepithelioma
  133450	Ewing sarcoma
  133510	Trichothiodystrophy
  133510	Xeroderma pigmentosum, group B
  133550	Dicarboxylicaminoaciduria, 222730
  133700	Chondrosarcoma, 215300
  133700	Exostoses, multiple, type 1
  133701	Exostoses, multiple, type 2
  133780	Vitreoretinopathy, exudative, familial
  134370	Membroproliferative glomerulonephritis
  134370	Factor H deficiency
  134370	Hemolytic-uremic syndrome, 235400
  134570	Factor XIIIA deficiency
  134580	Factor XIIIB deficiency
  134637	Autoimmune lymphoproliferative syndrome
  134790	Hyperferritinemia-cataract syndrome, 600886
  134820	Dysfibrinogenemia, alpha type, causing bleeding diathesis
  134820	Dysfibrinogenemia, alpha type, causing recurrent thrombosis
  134820	Amyloidosis, hereditary renal, 105200
  134830	Dysfibrinogenemia, beta type
  134850	Dysfibrinogenemia, gamma type
  134850	Hypofibrinogenemia, gamma type
  134934	Thanatophoric dysplasia, types I and II, 187600
  134934	Achondroplasia, 100800
  134934	Craniosynostosis, nonsyndromic
  134934	Crouzon syndrome with acanthosis nigricans
  134934	Hypochondroplasia, 146000
  135300	Fibromatosis, gingival
  135600	Ehlers-Danlos syndrome, type X
  135700	Fibrosis of extraocular muscles, congenital, 1
  135940	Ichthyosis vulgaris, 146700
  136132	[Fish-odor syndrome], 602079
  136350	Pfeiffer syndrome, 101600
  136435	Ovarian dysgenesis, hypergonadotropic, with normal karyotype, 233300
  136530	Male infertility, familial
  136836	Fucosyltransferase-6 deficiency
  137350	Amyloidosis, Finnish type, 105120
  138030	[Hyperproglucagonemia]
  138033	Diabetes mellitus, type II
  138040	Cortisol resistance
  138079	Hyperinsulinism, familial, 602485
  138079	MODY, type 2, 125851
  138130	Hyperinsulinism-hyperammonemia syndrome
  138140	Glucose transport defect, blood-brain barrier
  138160	Diabetes mellitus, noninsulin-dependent
  138160	Fanconi-Bickel syndrome, 227810
  138190	Diabetes mellitus, noninsulin-dependent
  138250	P5CS deficiency
  138300	Hemolytic anemia due to glutathione reductase deficiency
  138320	Hemolytic anemia due to glutathione peroxidase deficiency
  138491	Startle disease, autosomal recessive
  138491	Startle disease/hyperekplexia, autosomal dominant, 149400
  138491	Hyperekplexia and spastic paraparesis
  138570	Non-insulin dependent diabetes mellitus, susceptibility to
  138571	Glycogen synthase, liver, deficiency of, 240600
  138700	[Apolipoprotein H deficiency]
  138720	Bernard-Soulier syndrome, type B
  138971	Kostmann neutropenia, 202700
  138981	Pulmonary alveolar proteinosis, 265120
  139130	Hypertension, essential, susceptibility to, 145500
  139150	Basal cell carcinoma
  139190	Gigantism due to GHRF hypersecretion
  139190	Isolated growth hormone deficiency due to defect in GHRF
  139191	Growth hormone deficient dwarfism
  139250	Isolated growth hormone deficiency, Illig type with absent GH and
  	Kowarski type with bioinactive GH
  139320	Pituitary ACTH secreting adenoma
  139320	Pseudohypoparathyroidism, type Ia, 103580
  139320	Somatotrophinoma
  139320	McCune-Albright polyostotic fibrous dysplasia, 174800
  139330	Night blindness, congenital stationary
  139350	Epidermolytic hyperkeratosis, 113800
  139350	Keratoderma, palmoplantar, nonepidermolytic
  139360	Pituitary ACTH-secreting adenoma
  140100	[Anhaptoglobinemia]
  140100	[Hypohaptogloginemia]
  141750	Alpha-thalassemia/mental retardation syndrome, type 1
  141800	Methemoglobinemias, alpha-
  141800	Thalassemias, alpha-
  141800	Erythremias, alpha-
  141800	Heinz body anemias, alpha-
  141850	Thalassemia, alpha-
  141850	Erythrocytosis
  141850	Heinz body anemia
  141850	Hemoglobin H disease
  141850	Hypochromic microcytic anemia
  141900	Methemoglobinemias, beta-
  141900	Sickle cell anemia
  141900	Thalassemias, beta-
  141900	Erythremias, beta-
  141900	HPFH, deletion type
  141900	Heinz body anemias, beta-
  142000	Thalassemia due to Hb Lepore
  142000	Thalassemia, delta-
  142200	HPFH, nondeletion type A
  142250	HPFH, nondeletion type G
  142270	Hereditary persistence of fetal hemoglobin
  142335	Hereditary persistence of fetal hemoglobin, heterocellular, Indian type
  142470	[Hereditary persistence of fetal hemoglobin, heterocellular]
  142640	Thrombophilia due to elevated HRG
  142680	Periodic fever, familial
  142857	Pemphigoid, susceptibility to
  142858	Beryllium disease, chronic, susceptibility to
  142946	Holoprosencephaly-4
  142959	Hand-foot-uterus syndrome, 140000
  142989	Synpolydactyly, type II, 186000
  143100	Huntington disease
  143200	Wagner syndrome
  143200	Erosive vitreoretinopathy
  143890	Hypercholesterolemia, familial
  144200	Epidermolytic palmoplantar keratoderma
  145001	Hyperparathyroidism-jaw tumor syndrome
  145260	Pseudohypoaldosteronism, type II
  145410	Opitz G syndrome, type II
  145505	Hypertension, essential
  145981	Hypocalciuric hypercalcemia, type II
  146150	Hypomelanosis of Ito
  146150	Hypomelanosis of Ito
  146760	[IgG receptor I, phagocytic, familial deficiency of]
  146790	Lupus nephritis, susceptibility to
  147050	Atopy
  147141	Leukemia, acute lymphoblastic
  147200	[Kappa light chain deficiency]
  147280	Hepatocellular carcinoma
  147440	Growth retardation with deafness and mental retardation
  147450	Amytrophic lateral sclerosis, due to SOD1 deficiency, 105400
  147545	Diabetes mellitus, noninsulin-dependent
  147570	Interferon, immune, deficiency
  147660	Interferon, alpha, deficiency
  147670	Rabson-Mendenhall syndrome
  147670	Diabetes mellitus, insulin-resistant, with acanthosis nigricans
  147670	Leprechaunism
  147730	Interleukin-2 receptor, alpha chain, deficiency of
  147781	Atopy, susceptibility to
  147790	Leukemia, acute lymphocytic, with 4/11 translocation
  147791	Jacobsen syndrome
  148040	Epidermolysis bullosa simplex, Koebner, Dowling-Meara, and Weber-
  	Cockayne types, 131900, 131760, 131800
  148041	Pachyonychia congenita, Jadassohn-Lewandowsky type, 167200
  148043	Meesmann corneal dystrophy, 122100
  148065	White sponge nevus, 193900
  148066	Epidermolysis bullosa simplex, Koebner, Dowling-Meara, and Weber-
  	Cockayne types, 131900, 131760, 131800
  148066	Epidermolysis bullosa simplex, recessive, 601001
  148067	Nonepidermolytic palmoplantar keratoderma, 600962
  148067	Pachyonychia congenita, Jadassohn-Lewandowsky type, 167200
  148069	Pachyonychia congenita, Jackson-Lawler type, 167210
  148070	Liver disease, susceptibility to, from hepatotoxins or viruses
  148080	Epidermolytic hyperkeratosis, 113800
  148370	Keratolytic winter erythema
  148500	Tylosis with esophageal cancer
  150000	Exertional myoglobinuria due to deficiency of LDH-A
  150100	Lactate dehydrogenase-B deficiency
  150200	[Placental lactogen deficiency]
  150210	Lactoferrin-deficient neutrophils, 245480
  150230	Langer-Giedion syndrome
  150240	Cutis laxa, marfanoid neonatal type
  150250	Larsen syndrome, autosomal dominant
  150270	Laryngeal adductor paralysis
  150292	Epidermolysis bullosa, Herlitz junctional type, 226700
  150310	Epidermolysis bullosa, Herlitz junctional type, 226700
  150310	Epidermolysis bullosa, generalized atrophic benign, 226650
  151385	Leukemia, acute myeloid
  151390	Leukemia, acute T-cell
  151400	Leukemia/lymphoma, B-cell, 1
  151410	Leukemia, chronic myeloid
  151440	Leukemia, T-cell acute lymphoblastoid
  151670	Hepatic lipase deficiency
  152200	Coronary artery disease, susceptibility to
  152427	Long QT syndrome-2
  152445	Vohwinkel syndrome, 124500
  152445	Erythrokeratoderma, progressive symmetric, 602036
  152760	Hypogonadotropic hypogonadism due to GNRH deficiency, 227200
  152790	Precocious puberty, male, 176410
  152790	Leydig cell hypoplasia
  153454	Ehlers-Danlos syndrome, type VI, 225400
  153455	Cutis laxa, recessive, type I, 219100
  153700	Macular dystrophy, vitelliform type
  153880	Macular dystrophy, dominant cystoid
  153900	Stargardt disease-2
  154275	Malignant hyperthermia susceptibility 2
  154276	Malignant hyperthermia susceptibility 3
  154400	Acrofacial dysostosis, Nager type
  154500	Treacher Collins mandibulofacial dysostosis
  154550	Carbohydrate-deficient glycoprotein syndrome, type Ib, 602579
  154705	Marfan syndrome, type II
  155555	[Red hair/fair skin]
  155555	UV-induced skin damage, vulnerability to
  155600	Malignant melanoma, cutaneous
  156225	Muscular dystrophy, congenital merosin-deficient
  156232	Mesomelic dysplasia, Kantaputra type
  156570	Methylcobalamin deficiency, cbl G type
  156600	Microcoria, congenital
  156845	Tietz syndrome, 103500
  156845	Waardenburg syndrome, type IIA, 193510
  156845	Waardenburg syndrome/ocular albinism, digenic, 103470
  156850	Cataract, congenital, with microphthalmia
  157147	Abetalipoproteinemia, 200100
  157170	Holoprosencephaly-2
  157640	PEO with mitochondrial DNA deletions, type 1
  157655	Lactic acidosis due to defect in iron-sulfur cluster of complex I
  157900	Moebius syndrome
  159000	Muscular dystrophy, limb-girdle, type 1A
  159001	Muscular dystrophy, limb-girdle, type 1B
  159440	Charcot-Marie-Tooth neuropathy-1B, 118200
  159440	Dejerine-Sottas disease, myelin P-related, 145900
  159440	Hypomyelination, congenital
  159555	Leukemia, myeloid/lymphoid or mixed-lineage
  160777	Griscelli disease, 214450
  160781	Cardiomyopathy, hypertrophic, mid-left ventricular chamber type
  160900	Myotonic dystrophy
  161015	Mitochondrial complex I deficiency, 252010
  162100	Neuralgic amyotrophy with predilection for brachial plexus
  162200	Neurofibromatosis, type 1
  162200	Watson syndrome, 193520
  162400	Neuropathy, hereditary sensory and autonomic, type 1
  163729	Hypertension, pregnancy-induced
  163890	Parkinson disease, type 1, 601508
  164009	Leukemia, acute promyelocytic, NUMA/RARA type
  164040	Leukemia, acute promyelocytic, NPM/RARA type
  164160	Obesity, severe, due to leptin deficiency
  164200	Oculodentodigital dysplasia
  164200	Syndactyly, type III, 186100
  164500	Spinocerebellar ataxia-7
  164731	Ovarian carcinoma, 167000
  164759	Ovarian carcinoma
  164790	Colorectal cancer
  164860	Renal cell carcinoma, papillary, familial and sporadic
  164920	Piebaldism
  164920	Mast cell leukemia
  164920	Mastocytosis with associated hematologic disorder
  164953	Liposarcoma
  165240	Pallister-Hall syndrome, 146510
  165240	Postaxial polydactyly type A1, 174200
  165240	Greig cephalopolysyndactyly syndrome, 175700
  165320	Hepatocellular carcinoma
  165500	Optic atrophy 1
  166600	Osteopetrosis, AD, type II
  167000	Ovarian cancer, serous
  167250	Paget disease of bone
  167409	Optic nerve coloboma with renal disease, 120330
  167410	Rhabdomyosarcoma, alveolar, 268220
  167415	Hypothyroidism, congenital, due to thyroid dysgenesis or hypoplasia
  168000	Paraganglioma, familial nonchromaffin, 1
  168360	Paraneoplastic sensory neuropathy
  168450	Hypoparathyroidism, autosomal dominant
  168450	Hypoparathyroidism, autosomal recessive
  168461	Multiple myeloma, 254250
  168461	Parathyroid adenomatosis 1
  168461	Centrocytic lymphoma
  168468	Metaphyseal chondrodysplasia, Murk Jansen type, 156400
  168470	Humoral hypercalcemia of malignancy
  168500	Parietal foramina
  169600	Hailey-Hailey disease
  170261	Bare lymphocyte syndrome, type I, due to TAP2 deficiency
  170500	Myotonia congenita, atypical acetazolamide-responsive
  170500	Paramyotonia congenita, 168300
  170500	Hyperkalemic periodic paralysis
  170650	Periodontitis, juvenile
  170995	Zellweger syndrome-2
  171050	Colchicine resistance
  171060	Cholestasis, progressive familial intrahepatic, type III, 602347
  171190	Hypertension, essential, 145500
  171650	Lysosomal acid phosphatase deficiency
  171760	Hypophosphatasia, adult, 146300
  171760	Hypophosphatasia, infantile, 241500
  171860	Hemolytic anemia due to phosphofructokinase deficiency
  172400	Hemolytic anemia due to glucosephosphate isomerase deficiency
  172400	Hydrops fetalis, one form
  172411	Colorectal cancer, resistance to
  172430	Enolase deficiency
  172471	Glycogenosis, hepatic, autosomal
  172490	Phosphorylase kinase deficiency of liver and muscle, 261750
  173350	Plasminogen Tochigi disease
  173350	Plasminogen deficiency, types I and II
  173350	Thrombophilia, dysplasminogenemic
  173360	Thrombophilia due to excessive plasminogen activator inhibitor
  173360	Hemorrhagic diathesis due to PAI1 deficiency
  173370	Plasminogen activator deficiency
  173470	Glanzmann thrombasthenia, type B
  173610	Platelet alpha/delta storage pool deficiency
  173850	Polio, susceptibility to
  173870	Xeroderma pigmentosum
  173870	Fanconi anemia
  173910	Polycystic kidney disease, adult, type II
  174000	Medullary cystic kidney disease, AD
  174810	Osteolysis, familial expansile
  174900	Polyposis, juvenile intestinal
  175100	Turcot syndrome, 276300
  175100	Adenomatous polyposis coli
  175100	Adenomatous polyposis coli, attenuated
  175100	Colorectal cancer
  175100	Desmoid disease, hereditary, 135290
  175100	Gardner syndrome
  176000	Porphyria, acute intermittent
  176100	Porphyria cutanea tarda
  176100	Porphyria, hepatoerythropoietic
  176260	Episodic ataxia/myokymia syndrome, 160120
  176261	Jervell and Lange-Nielsen syndrome, 220400
  176270	Prader-Willi syndrome
  176300	[Dystransthyretinemic hyperthyroxinemia]
  176300	Carpal tunnel syndrome, familial
  176300	Amyloid neuropathy, familial, several allelic types
  176300	Amyloidosis, senile systemic
  176450	Sacral agenesis-1
  176730	Diabetes mellitus, rare form
  176730	Hyperproinsulinemia, familial
  176730	MODY, one form
  176801	Metachromatic leukodystrophy due to deficiency of SAP-1
  176801	Gaucher disease, variant form
  176830	Obesity, adrenal insufficiency, and red hair
  176830	ACTH deficiency
  176860	Purpura fulminans, neonatal
  176860	Thrombophilia due to protein C deficiency
  176880	Protein S deficiency
  176930	Dysprothrombinemia
  176930	Hypoprothrombinemia
  176943	Apert syndrome, 101200
  176943	Pfeiffer syndrome, 101600
  176943	Beare-Stevenson cutis gyrata syndrome, 123790
  176943	Crouzon craniofacial dysostosis, 123500
  176943	Jackson-Weiss syndrome, 123150
  176947	Selective T-cell defect
  176960	Pituitary tumor, invasive
  177070	Spherocytosis, hereditary, Japanese type
  177070	Hermansky-Pudlak syndrome, 203300
  177400	Apnea, postanesthetic
  177900	Psoriasis susceptibility-1
  178300	Ptosis, hereditary congenital, 1
  178600	Pulmonary hypertension, familial primary
  178640	Pulmonary alveolar proteinosis, congenital, 265120
  179095	Male infertility
  179450	Ragweed sensitivity
  179605	Retinitis pigmentosa, digenic
  179605	Retinitis pigmentosa-7, peripherin-related
  179605	Retinitis punctata albescens
  179605	Butterfly dystrophy, retinal
  179605	Macular dystrophy
  179615	Reticulosis, familial histiocytic, 267700
  179615	Severe combined immunodeficiency, B cell-negative, 601457
  179616	Severe combined immunodeficiency, B cell-negative, 601457
  179755	Renal cell carcinoma, papillary, 1
  179820	[Hyperproreninemia]
  180020	Retinal cone dystrophy-1
  180069	Retinal dystrophy, autosomal recessive, childhood-onset
  180069	Retinitis pigmentosa-20
  180069	Leber congenital amaurosis-2, 204100
  180071	Retinitis pigmentosa, autosomal recessive
  180072	Night blindness, congenital stationary, type 3, 163500
  180072	Retinitis pigmentosa, autosomal recessive
  180100	Retinitis pigmentosa-1
  180104	Retinitis pigmentosa-9
  180105	Retinitis pigmentosa-10
  180200	Osteosarcoma, 259500
  180200	Pinealoma with bilateral retinoblastoma
  180200	Retinoblastoma
  180200	Bladder cancer, 109800
  180240	Leukemia, acute promyelocytic
  180250	Retinol binding protein, deficiency of
  180297	Anemia, hemolytic, Rh-null, suppressor type, 268150
  180380	Night blindness, congenital stationery, rhodopsin-related
  180380	Retinitis pigmentosa, autosomal recessive
  180380	Retinitis pigmentosa-4, autosomal dominant
  180381	Oguchi disease-2, 258100
  180385	Leukemia, acute T-cell
  180721	Retinitis pigmentosa, digenic
  180840	Susceptibility to IDDM
  180860	Russell-Silver syndrome
  180901	Malignant hyperthermia susceptibility 1, 145600
  180901	Central core disease, 117000
  181030	Salivary gland pleomorphic adenoma
  181031	Oguchi disease-1, 258100
  181405	Scapuloperoneal spinal muscular atrophy, New England type
  181430	Scapuloperoneal syndrome, myopathic type
  181460	Schistosoma mansoni, susceptibility/resistance to
  181510	Schizophrenia
  181600	Sclerotylosis
  182138	Anxiety-related personality traits
  182279	Prader-Willi syndrome
  182280	Small-cell cancer of lung
  182380	Glucose/galactose malabsorption
  182381	Renal glucosuria, 253100
  182452	Lung cancer, small cell
  182500	Cataract, congenital
  182600	Spastic paraplegia-3A
  182601	Spastic paraplegia-4
  182860	Pyropoikilocytosis
  182860	Spherocytosis, recessive
  182860	Elliptocytosis-2
  182870	Spherocytosis-1
  182870	Elliptocytosis-3
  182870	Anemia, neonatal hemolytic, fatal and near-fatal
  182900	Spherocytosis-2
  183600	Split hand/foot malformation, type 1
  185000	Stomatocytosis I
  185430	Atherosclerosis, susceptibility to
  185470	Myopathy due to succinate dehydrogenase deficiency
  185800	Symphalangism, proximal
  186580	Arthrocutaneouveal granulomatosis
  186740	Immunodeficiency due to defect in CD3-gamma
  186770	Leukemia, T-cell acute lymphocytic
  186780	CD3, zeta chain, deficiency
  186830	Immunodeficiency, T-cell receptor/CD3 complex
  186860	Leukemia/lymphoma, T-cell
  186880	Leukemia/lymphoma, T-cell
  186921	Leukemia, T-cell acute lymphoblastic
  187040	Leukemia-1, T-cell acute lymphoblastic
  187680	6-mercaptopurine sensitivity
  188025	Thrombocytopenia, Paris-Trousseau type
  188070	Bleeding disorder due to defective thromboxane A2 receptor
  188450	Goiter, adolescent multinodular
  188450	Goiter, nonendemic, simple
  188450	Hypothyroidism, hereditary congenital
  188540	Hypothyroidism, nongoitrous
  188826	Sorsby fundus dystrophy, 136900
  189800	Preeclampsia/eclampsia
  189980	Leukemia, chronic myeloid
  190000	Atransferrinemia
  190020	Bladder cancer, 109800
  190040	Meningioma, SIS-related
  190040	Dermatofibrosarcoma protuberans
  190040	Giant-cell fibroblastoma
  190100	Geniospasm
  190160	Thyroid hormone resistance, 274300, 188570
  190182	Colon cancer
  190182	Colorectal cancer, familial nonpolyposis, type 6
  190195	Ichthyosiform erythroderma, congenital, 242100
  190195	Ichthyosis, lamellar, autosomal recessive, 242300
  190198	Leukemia, T-cell acute lymphoblastic
  190450	Hemolytic anemia due to triosephosphate isomerase deficiency
  190605	Triphalangeal thumb-polysyndactyly syndrome
  190685	Down syndrome
  190900	Colorblindness, tritan
  191010	Cardiomyopathy, familial hypertrophic, 3, 115196
  191030	Nemaline myopathy-1, 161800
  191044	Cardiomyopathy, familial hypertrophic
  191045	Cardiomyopathy, familial hypertrophic, 2, 115195
  191092	Tuberous sclerosis-2
  191100	Tuberous sclerosis-1
  191170	Colorectal cancer, 114500
  191170	Li-Fraumeni syndrome
  191181	Cervical carcinoma
  191290	Segawa syndrome, recessive
  191315	Insensitivity to pain, congenital, with anhidrosis, 256800
  191540	[Urate oxidase deficiency]
  192090	Ovarian carcinoma
  192090	Breast cancer, lobular
  192090	Endometrial carcinoma
  192090	Gastric cancer, familial, 137215
  192340	Diabetes insipidus, neurohypophyseal, 125700
  192500	Jervell and Lange-Nielsen syndrome, 220400
  192500	Long QT syndrome-1
  192974	Neonatal alloimmune thrombocytopenia
  192974	Glycoprotein Ia deficiency
  193100	Hypophosphatemic rickets, autosomal dominant
  193235	Vitreoretinopathy, neovascular inflammatory
  193300	Renal cell carcinoma
  193300	von Hippel-Lindau syndrome
  193400	von Willebrand disease
  193500	Rhabdomyosarcoma, alveolar, 268220
  193500	Waardenburg syndrome, type I
  193500	Waardenburg syndrome, type III, 148820
  193500	Craniofacial-deafness-hand syndrome, 122880
  194070	Wilms tumor, type 1
  194070	Denys-Drash syndrome
  194070	Frasier syndrome, 136680
  194071	Wilms tumor, type 2
  194071	Adrenocortical carcinoma, hereditary, 202300
  194190	Wolf-Hirschhorn syndrome
  200150	Choreoacanthocytosis
  200990	Acrocallosal syndrome
  201450	Acyl-CoA dehydrogenase, medium chain, deficiency of
  201460	Acyl-CoA dehydrogenase, long chain, deficiency of
  201470	Acyl-CoA dehydrogenase, short-chain, deficiency of
  201910	Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency
  202110	Adrenal hyperplasia, congenital, due to 17-alpha-hydroxylase
  	deficiency
  203100	Waardenburg syndrome/ocular albinism, digenic, 103470
  203100	Albinism, oculocutaneous, type IA
  203200	Albinism, ocular, autosomal recessive
  203200	Albinism, oculocutaneous, type II
  203300	Hermansky-Pudlak syndrome
  203310	Ocular albinism, autosomal recessive
  203500	Alkaptonuria
  203740	Alpha-ketoglutarate dehydrogenase deficiency
  203750	3-ketothiolase deficiency
  203800	Alstrom syndrome
  204500	Ceroid-lipofuscinosis, neuronal 2, classic late infantile
  205100	Amyotrophic lateral sclerosis, juvenile
  207750	Hyperlipoproteinemia, type Ib
  207800	Argininemia
  208100	Arthrogryposis multiplex congenita, neurogenic
  208250	Jacobs syndrome
  208400	Aspartylglucosaminuria
  209901	Bardet-Biedl syndrome 1
  211420	Breast cancer, ductal
  212138	Carnitine-acylcarnitine translocase deficiency
  214300	Klippel-Feil syndrome
  214400	Charcot-Marie-Tooth neuropathy-4A
  214500	Chediak-Higashi syndrome
  215700	Citrullinemia
  216550	Cohen syndrome
  216900	Achromatopsia
  216950	C1r/C1s deficiency, combined
  217000	C2 deficiency
  217050	C6 deficiency
  217050	Combined C6/C7 deficiency
  217070	C7 deficiency
  217800	Macular corneal dystrophy
  218000	Andermann syndrome
  218030	Apparent mineralocorticoid excess, hypertension due to
  219800	Cystinosis, nephropathic
  221770	Polycystic lipomembranous osteodysplasia with sclerosing
  	leukencephalopathy
  221820	Gliosis, familial progressive subcortical
  222100	Diabetes mellitus, insulin-dependent-1
  222600	Atelosteogenesis II, 256050
  222600	Achondrogenesis Ib, 600972
  222600	Diastrophic dysplasia
  222700	Lysinuric protein intolerance
  222800	Hemolytic anemia due to bisphosphoglycerate mutase deficiency
  222900	Sucrose intolerance
  223000	Lactase deficiency, adult, 223100
  223000	Lactase deficiency, congenital
  223360	Dopamine-beta-hydroxylase deficiency
  223900	Dysautonomia, familial
  224100	Congenital dyserythropoietic anemia II
  224120	Dyserythropoietic anemia, contenital, type I
  225500	Ellis-van Creveld syndrome
  227220	[Eye color, brown]
  227500	Factor VII deficiency
  227600	Factor X deficiency
  227646	Fanconi anemia, type D
  227650	Fanconi anemia, type A
  228960	[Kininogen deficiency]
  229300	Friedreich ataxia
  229300	Friedreich ataxia with retained reflexes
  229600	Fructose intolerance
  229700	Fructose-bisphosphatase deficiency
  229800	[Fructosuria]
  230000	Fucosidosis
  230200	Galactokinase deficiency with cataracts
  230350	Galactose epimerase deficiency
  230400	Galactosemia
  230450	Hemolytic anemia due to gamma-glutamylcysteine synthetase
  	deficiency
  230800	Gaucher disease
  230800	Gaucher disease with cardiovascular calcification
  231550	Achalasia-addisonianism-alacrimia syndrome
  231670	Glutaricaciduria, type I
  231680	Glutaricaciduria, type IIA
  231950	Glutathioninuria
  232000	Propionicacidemia, type I or pccA type
  232050	Propionicacidemia, type II or pccB type
  232300	Glycogen storage disease II
  232400	Glycogen storage disease IIIa
  232400	Glycogen storage disease IIIb
  232500	Glycogen storage disease IV
  232600	McArdle disease
  232700	Glycogen storage disease VI
  232800	Glycogen storage disease VII
  233100	[Renal glucosuria]
  233700	Chronic granulomatous disease due to deficiency of NCF-1
  233710	Chronic granulomatous disease due to deficiency of NCF-2
  234000	Factor XII deficiency
  234200	Neurodegeneration with brain iron accumulation
  235200	Hemochromatosis
  235800	[Histidinemia]
  236100	Holoprosencephaly-1
  236200	Homocystinuria, B6-responsive and nonresponsive types
  236730	Urofacial syndrome
  237300	Carbamoylphosphate synthetase I deficiency
  238300	Hyperglycinemia, nonketotic, type I
  238310	Hyperglycinemia, nonketotic, type II
  238600	Chylomicronemia syndrome, familial
  238600	Combined hyperlipemia, familial
  238600	Hyperlipoproteinemia I
  238600	Lipoprotein lipase deficiency
  238970	HHH syndrome
  239100	Van Buchem disease
  239500	Hyperprolinemia, type I
  240300	Autoimmune polyglandular disease, type I
  240400	Scurvy
  243500	Isovalericacidemia
  245000	Papillon-Lefevre syndrome
  245050	Ketoacidosis due to SCOT deficiency
  245200	Krabbe disease
  245349	Lacticacidemia due to PDX1 deficiency
  245900	Norum disease
  245900	Fish-eye disease
  246450	HMG-CoA lyase deficiency
  246530	Leukotriene C4 synthase deficiency
  246600	Pancreatic lipase deficiency
  246900	Lipoamide dehydrogenase deficiency
  247200	Miller-Dieker lissencephaly syndrome
  247640	Leukemia, acute lymphoblastic
  248510	Mannosidosis, beta-
  248600	Maple syrup urine disease, type Ia
  248610	Maple syrup urine disease, type II
  248611	Maple syrup urine disease, type Ib
  249000	Meckel syndrome
  249270	Thiamine-responsive megaloblastic anemia
  250100	Metachromatic leukodystrophy
  250250	Cartilage-hair hypoplasia
  250790	Methemoglobinemia due to cytochrome b5 deficiency
  250800	Methemoglobinemia, type I
  250800	Methemoglobinemia, type II
  251000	Methylmalonicaciduria, mutase deficiency type
  251600	Microphthalmia, autosomal recessive
  252500	Mucolipidosis II
  252500	Mucolipidosis III
  252800	Mucopolysaccharidosis Ih
  252800	Mucopolysaccharidosis Ih/s
  252800	Mucopolysaccharidosis Is
  252900	Sanfilippo syndrome, type A
  252940	Sanfilippo syndrome, type D
  253000	Mucopolysaccharidosis IVA
  253200	Maroteaux-Lamy syndrome, several forms
  253250	Mulibrey nanism
  253270	Multiple carboxylase deficiency, biotin-responsive
  253601	Miyoshi myopathy, 254130
  253601	Muscular dystrophy, limb-girdle, type 2B
  253800	Walker-Warburg syndrome, 236670
  253800	Fukuyama type congenital muscular dystrophy
  254210	Myasthenia gravis, familial infantile
  254770	Epilepsy, juvenile myoclonic
  254780	Myoclonus epilepsy, Lafora type
  255800	Schwartz-Jampel syndrome
  256030	Nemaline myopathy-2
  256100	Nephronophthisis, juvenile
  256540	Galactosialidosis
  256550	Sialidosis, type I
  256550	Sialidosis, type II
  256700	Neuroblastoma
  256731	Ceroid-lipofuscinosis, neuronal-5, variant late infantile
  256850	Giant axonal neuropathy-1
  257200	Niemann-Pick disease, type A
  257200	Niemann-Pick disease, type B
  257220	Niemann-Pick disease, type C
  257220	Niemann-Pick disease, type D, 257250
  258501	3-methylglutaconicaciduria, type III
  258870	Gyrate atrophy of choroid and retina with ornithinemia, B6 responsive
  	or unresponsive
  259700	Osteopetrosis, recessive
  259730	Renal tubular acidosis-osteopetrosis syndrome
  259770	Osteoporosis-pseudoglioma syndrome
  259900	Hyperoxaluria, primary, type 1
  261510	Pseudo-Zellweger syndrome
  261515	Peroxisomal bifunctional enzyme deficiency
  261600	Phenylketonuria
  261600	[Hyperphenylalaninemia, mild]
  261640	Phenylketonuria due to PTS deficiency
  261670	Myopathy due to phosphoglycerate mutase deficiency
  262000	Bjornstad syndrome
  263200	Polycystic kidney disease, autosomal recessive
  263700	Porphyria, congenital erythropoietic
  264300	Pseudohermaphroditism, male, with gynecomastia
  264470	Adrenoleukodystrophy, pseudoneonatal
  264700	Pseudo-vitamin D dependency rickets 1
  266100	Pyridoxine dependency with seizures
  266150	Pyruvate carboxylase deficiency
  266200	Anemia, hemolytic, due to PK deficiency
  266300	[Hair color, red]
  266600	Inflammatory bowel disease-1
  267750	Knobloch syndrome
  268900	[Sarcosinemia]
  270100	Situs inversus viscerum
  270800	Spastic paraplegia-5A
  271245	Spinocerebellar ataxia-8, infantile, with sensory neuropathy
  271900	Canavan disease
  272750	GM2-gangliosidosis, AB variant
  272800	Tay-Sachs disease
  272800	[Hex A pseudodeficiency]
  272800	GM2-gangliosidosis, juvenile, adult
  273300	Male germ cell tumor
  273800	Thrombocytopenia, neonatal alloimmune
  273800	Glanzmann thrombasthenia, type A
  274180	Thromboxane synthase deficiency
  274270	Thymine-uraciluria
  274270	Fluorouracil toxicity, sensitivity to
  274600	Pendred syndrome
  274600	Deafness, autosomal recessive 4
  275350	Transcobalamin II deficiency
  276000	Pancreatitis, hereditary, 167800
  276000	Trypsinogen deficiency
  276600	Tyrosinemia, type II
  276700	Tyrosinemia, type I
  276900	Usher syndrome, type 1A
  276901	Usher syndrome, type 2
  276902	Usher syndrome, type 3
  276903	Usher syndrome, type 1B
  276903	Deafness, autosomal dominant 11, neurosensory, 601317
  276903	Deafness, autosomal recessive 2, neurosensory, 600060
  276904	Usher syndrome, type 1C
  277700	Werner syndrome
  277730	Wernicke-Korsakoff syndrome, susceptibility to
  278000	Wolman disease
  278000	Cholesteryl ester storage disease
  278250	Wrinkly skin syndrome
  278300	Xanthinuria, type I
  278700	Xeroderma pigmentosum, group A
  278760	Xeroderma pigmentosum, group F
  300008	Nephrolithiasis, type I, 310468
  300008	Proteinuria, low molecular weight, with hypercalciuric
  	nephrocalcinosis
  300008	Dent disease, 300009
  300008	Hypophosphatemia, type III
  300011	Menkes disease, 309400
  300011	Occipital horn syndrome, 304150
  300011	Cutis laxa, neonatal
  300031	Mental retardation, X-linked, FRAXF type
  300032	Alpha-thalassemia/mental retardation syndrome, type 2, 301040
  300032	Juberg-Marsidi syndrome, 309590
  300037	Simpson dysmorphia syndrome, 312870
  300039	Deafness, X-linked 3, conductive, with stapes fixation, 304400
  300044	Wernicke-Korsakoff syndrome, susceptibility to
  300046	Mental retardation, X-linked 23, nonspecific
  300047	Mental retardation, X-linked 20
  300048	Intestinal pseudoobstruction, neuronal, X-linked
  300049	Nodular heterotopia, bilateral periventricular
  300049	BPNH/MR syndrome
  300055	Mental retardation with psychosis, pyramidal signs, and
  	macroorchidism
  300062	Mental retardation, X-linked 14
  300071	Night blindness, congenital stationary, type 2
  300076	Wood neuroimmunologic syndrome
  300077	Mental retardation, X-linked 29
  300088	Epilepsy, female restricted, with mental retardation
  300100	Adrenoleukodystrophy
  300100	Adrenomyeloneuropathy
  300104	Mental retardation, X-linked nonspecific, 309541
  300110	Night blindness, congenital stationary, X-linked incomplete, 300071
  300123	Mental retardation with isolated growth hormone deficiency
  300126	Dyskeratosis congenita-1, 305000
  300136	Diabetes mellitus, insulin-dependent, X-linked, susceptibility to
  300300	XLA and isolated growth hormone deficiency, 307200
  300300	Agammaglobulinemia, type 1, X-linked
  300600	Ocular albinism, Forsius-Eriksson type
  301000	Thrombocytopenia, X-linked, 313900
  301000	Wiskott-Aldrich syndrome
  301200	Amelogenesis imperfecta
  301201	Amelogenesis imperfecta-3, hypoplastic type
  301300	Anemia, sideroblastic/hypochromic
  301310	Anemia, sideroblastic, with spinocerebellar ataxia
  301500	Fabry disease
  301590	Anophthalmos-1
  301830	Arthrogryposis, X-linked (spinal muscular atrophy, infantile, X-linked)
  301835	Arts syndrome
  301845	Bazex syndrome
  301900	Borjeson-Forssman-Lehmann syndrome
  302060	Noncompaction of left ventricular myocardium, isolated
  302060	Barth syndrome
  302060	Cardiomyopathy, X-linked dilated, 300069
  302060	Endocardial fibroelastosis-2
  302350	Nance-Horan syndrome
  302960	Chondrodysplasia punctata, X-linked dominant
  303400	Cleft palate, X-linked
  303630	Alport syndrome, 301050
  303630	Leiomyomatosis-nephropathy syndrome, 308940
  303631	Leiomyomatosis, diffuse, with Alport syndrome
  303700	Colorblindness, blue monochromatic
  303800	Colorblindness, deutan
  303900	Colorblindness, protan
  304020	Cone dystrophy, progressive X-linked, 1
  304040	Charcot-Marie-Tooth neuropathy, X-linked-1, dominant, 302800
  304340	Mental retardation, X-linked, syndromic-5, with Dandy-Walker
  	malformation, basal ganglia disease, and seizures
  304500	Deafness, X-linked 2, perceptive congenital
  304700	Mohr-Tranebjaerg syndrome
  304700	Deafness, X-linked 1, progressive
  304700	Jensen syndrome, 311150
  304800	Diabetes insipidus, nephrogenic
  305100	Anhidrotic ectodermal dysplasia
  305400	Aarskog-Scott syndrome
  305450	FG syndrome
  305900	Favism
  305900	G6PD deficiency
  305900	Hemolytic anemia due to G6PD deficiency
  306700	Hemophilia A
  306900	Hemophilia B
  306995	[Homosexuality, male]
  307150	Hypertrichosis, congenital generalized
  307700	Hypoparathyroidism, X-linked
  308000	HPRT-related gout
  308000	Lesch-Nyhan syndrome
  308230	Immunodeficiency, X-linked, with hyper-IgM
  308240	Lymphoproliferative syndrome, X-linked
  308300	Incontinentia pigmenti, sporadic type
  308310	Incontinentia pigmenti, familial
  308380	Severe combined immunodeficiency, X-linked, 300400
  308380	Combined immunodeficiency, X-linked, moderate, 312863
  308840	Spastic paraplegia, 312900
  308840	Hydrocephalus due to aqueductal stenosis, 307000
  308840	MASA syndrome, 303350
  309000	Lowe syndrome
  309200	Manic-depressive illness, X-linked
  309300	Megalocornea, X-linked
  309470	Mental retardation, X-linked, syndromic-3, with spastic diplegia
  309500	Renpenning syndrome-1
  309545	Mental retardation, X-linked nonspecific, with aphasia
  309548	Mental retardation, X-linked, FRAXE type
  309555	Gustavson syndrome
  309605	Mental retardation, X-linked, syndromic-4, with congenital
  	contractures and low fingertip arches
  309610	Mental retardation, X-linked, syndromic-2, with dysmorphism and
  	cerebral atrophy
  309620	Mental retardation-skeletal dysplasia
  309850	Brunner syndrome
  309900	Mucopolysaccharidosis II
  310300	Emery-Dreifuss muscular dystrophy
  310400	Myotubular myopathy, X-linked
  310460	Myopia-1
  310460	Bornholm eye disease
  310490	Cowchock syndrome
  310500	Night blindness, congenital stationary, type 1
  311050	Optic atrophy, X-linked
  311200	Oral-facial-digital syndrome 1
  311300	Otopalatodigital syndrome, type I
  311510	Waisman parkinsonism-mental retardation syndrome
  311800	Myoglobinuria/hemolysis due to PGK deficiency
  311800	Hemolytic anemia due to PGK deficiency
  311850	Phosphoribosyl pyrophosphate synthetase-related gout
  311870	Muscle glycogenosis
  312000	Panhypopituitarism, X-linked
  312040	N syndrome, 310465
  312060	Properdin deficiency, X-linked
  312080	Pelizaeus-Merzbacher disease
  312080	Spastic paraplegia-2, 312920
  312600	Retinitis pigmentosa-2
  312760	Turner syndrome
  313350	Split hand/foot malformation, type 2
  313850	Thoracoabdominal syndrome
  314200	[Euthyroidal hyper- and hypothyroxinemia]
  314250	Dystonia-3, torsion, with parkinsonism, Filipino type
  314300	Goeminne TKCR syndrome
  314400	Cardiac valvular dysplasia-1
  314580	Wieacker-Wolff syndrome
  600020	Prostate cancer, 176807
  600035	Schizencephaly
  600040	Colorectal cancer
  600044	Thrombocythemia, essential, 187950
  600045	Xeroderma pigmentosum, group E, subtype 2
  600048	Breast cancer-3
  600059	Retinitis pigmentosa-13
  600065	Leukocyte adhesion deficiency, 116920
  600079	Colon cancer
  600095	Split hand/foot malformation, type 3
  600101	Deafness, autosomal dominant 2
  600105	Retinitis pigmentosa-12, autosomal recessive
  600119	Muscular dystrophy, Duchenne-like, type 2
  600119	Adhalinopathy, primary
  600138	Retinitis pigmentosa-11
  600140	Rubenstein-Taybi syndrome, 180849
  600143	Epilepsy, progressive, with mental retardation
  600151	Bardet-Biedl syndrome 3
  600160	Melanoma, 155601
  600163	Long QT syndrome-3
  600173	SCID, autosomal recessive, T-negative/B-positive type
  600175	Spinal muscular atrophy, congenital nonprogressive, of lower limbs
  600179	Leber congenital amaurosis, type I, 204000
  600184	Carnitine acetyltransferase deficiency
  600185	Pancreatic cancer
  600185	Breast cancer 2, early onset
  600194	Ichthyosis bullosa of Siemens, 146800
  600202	Dyslexia, specific, 2
  600211	Cleidocranial dysplasia, 119600
  600221	Venous malformations, multiple cutaneous and mucosal, 600195
  600223	Spinocerebellar ataxia-4
  600225	Phenylketonuria, atypical, due to GCH1 deficiency, 233910
  600225	Dystonia, DOPA-responsive, 128230
  600228	Pseudohypoaldosteronism, type I, 264350
  600231	Palmoplantar keratoderma, Bothnia type
  600234	HMG-CoA synthease-2 deficiency
  600243	Temperature-sensitive apoptosis
  600258	Colorectal cancer, hereditary nonpolyposis, type 3
  600259	Turcot syndrome with glioblastoma, 276300
  600259	Colorectal cancer, hereditary nonpolyposis, type 4
  600261	Ehlers-Danlos-like syndrome
  600266	Resistance/susceptibility to TB, etc.
  600273	Polycystic kidney disease, infantile severe, with tuberous sclerosis
  600276	Cerebral arteriopathy with subcortical infarcts and
  	leukoencephalopathy, 125310
  600281	Non-insulin-dependent diabetes mellitus, 125853
  600281	MODY, type 1, 125850
  600309	Atrioventricular canal defect-1
  600310	Pseudoachondroplasia, 177170
  600310	Epiphyseal dysplasia, multiple 1, 132400
  600319	Diabetes mellitus, insulin-dependent, 4
  600320	Insulin-dependent diabetes mellitus-5
  600321	Diabetes mellitus, insulin-dependent, 7
  600332	Rippling muscle disease-1
  600354	Spinal muscular atrophy-1, 253300
  600354	Spinal muscular atrophy-2, 253550
  600354	Spinal muscular atrophy-3, 253400
  600364	Cone dystrophy-3, 602093
  600374	Bardet-Biedl syndrome 4
  600414	Adrenoleukodystrophy, neonatal, 202370
  600415	Ataxia with isolated vitamin E deficiency, 277460
  600429	[Ii blood group, 110800]
  600509	Persistent hyperinsulinemic hypoglycemia of infancy, 256450
  600510	Pigment dispersion syndrome
  600511	Schizophrenia-3
  600512	Epilepsy, partial
  600525	Trichodontoosseous syndrome, 190320
  600528	CPT deficiency, hepatic, type I, 255120
  600536	Myopathy, congenital
  600542	Chondrosarcoma, extraskeletal myxoid
  600584	Atrial septal defect with atrioventricular conduction defects, 108900
  600593	Craniosynostosis, Adelaide type
  600617	Lipoid adrenal hyperplasia, 201710
  600618	Leukemia, acute lymphoblastic
  600623	Prostate cancer, 176807
  600624	Cone-rod retinal dystrophy-1
  600631	Enuresis, nocturnal, 1
  600650	Myopathy due to CPT II deficiency, 255110
  600650	CPT deficiency, hepatic, type II, 600649
  600698	Salivary adenoma
  600698	Uterine leiomyoma
  600698	Lipoma
  600698	Lipomatosis, mutiple, 151900
  600700	Lipoma
  600701	Lipoma
  600722	Ceroid lipofuscinosis, neuronal, variant juvenile type, with granular osmiophilic
  	deposits
  600722	Ceroid lipofuscinosis, neuronal-1, infantile, 256730
  600725	Holoprosencephaly-3, 142945
  600759	Alzheimer disease-4
  600760	Pseudohypoaldosteronism, type I, 264350
  600760	Liddle syndrome, 177200
  600761	Pseudohypoaldosteronism, type I, 264350
  600761	Liddle syndrome, 177200
  600795	Dementia, familial, nonspecific
  600807	Bronchial asthma
  600808	Enuresis, nocturnal, 2
  600811	Xeroderma pigmentosum, group E, DDB-negative subtype, 278740
  600837	Hirschsprung disease, 142623
  600839	Bartter syndrome, 241200
  600850	Schizophrenia disorder-4
  600852	Retinitis pigmentosa-17
  600856	Beckwith-Wiedemann syndrome, 130650
  600881	Cataract, congenital, zonular, with sutural opacities
  600882	Charcot-Marie-Tooth neuropathy-2B
  600883	Diabetes mellitus, insulin-dependent, 8
  600887	Endometrial carcinoma
  600897	Cataract, zonular pulverulent-1, 116200
  600900	Muscular dystrophy, limb-girdle, type 2E
  600918	Cystinuria, type III
  600923	Porphyria variegata, 176200
  600937	Persistent hyperinsulinemic hypoglycemia of infancy, 256450
  600946	Short stature, autosomal dominant, with normal serum growth hormone
  	binding protein
  600946	Short stature, idiopathic
  600946	Laron dwarfism, 262500
  600956	Persistent Mullerian duct syndrome, type II, 261550
  600957	Persistent Mullerian duct syndrome, type I, 261550
  600958	Cardiomyopathy, familial hypertrophic, 4, 115197
  600965	Deafness, autosomal dominant 6
  600968	Gitelman syndrome, 263800
  600971	Deafness, autosomal recessive 6
  600974	Deafness, autosomal recessive 7
  600975	Glaucoma 3, primary infantile, B
  600977	Cone dystrophy, progressive
  600983	Pseudohypoaldosteronism type I, autosomal dominant, 177735
  600993	Pancreatic cancer
  600995	Nephrotic syndrome, idiopathic, steroid-resistant
  600996	Arrhythmogenic right ventricular dysplasia-2
  600998	Bleeding diathesis due to GNAQ deficiency
  601002	5-oxoprolinuria, 266130
  601002	Hemolytic anemia due to glutathione synthetase deficiency, 231900
  601011	Spinocerebellar ataxia-6, 183086
  601011	Cerebellar ataxia, pure
  601011	Episodic ataxia, type 2, 108500
  601011	Hemiplegic migraine, familial, 141500
  601071	Deafness, autosomal recessive 9
  601072	Deafness, autosomal recessive 8
  601090	Iridogoniodysgenesis, 601631
  601105	Pycnodysostosis, 265800
  601107	Dubin-Johnson syndrome, 237500
  601130	Tolbutamide poor metabolizer
  601145	Epilepsy, progressive myoclonic 1, 254800
  601146	Brachydactyly, type C, 113100
  601146	Acromesomelic dysplasia, Hunter-Thompson type, 201250
  601146	Chondrodysplasia, Grebe type, 200700
  601154	Cardiomyopathy, dilated, 1E
  601199	Neonatal hyperparathyroidism, 239200
  601199	Hypocalcemia, autosomal dominant, 601198
  601199	Hypocalciuric hypercalcemia, type I, 145980
  601202	Cataract, anterior polar-2
  601208	Insulin-dependent diabetes mellitus-11
  601226	Progressive external ophthalmoplegia, type 2
  601238	Cerebellar ataxia, Cayman type
  601267	HIV infection, susceptibility/resistence to
  601277	Ichthyosis, lamellar, type 2
  601284	Hereditary hemorrhagic telangiectasia-2, 600376
  601313	Polycystic kidney disease, adult type I, 173900
  601316	Deafness, autosomal dominant 10
  601318	Diabetes mellitus, insulin-dependent, 13
  601362	DiGeorge syndrome/velocardiofacial syndrome complex-2
  601363	Wilms tumor, type 4
  601373	HIV infection, susceptibility/resistance to
  601382	Charcot-Marie-Tooth neuropathy-4B
  601385	Prostate cancer
  601387	Breast cancer
  601399	Platelet disorder, familial, with associated myeloid malignancy
  601402	Leukemia, myeloid, acute
  601406	B-cell non-Hodgkin lymphoma, high-grade
  601410	Diabetes mellitus, transient neonatal
  601411	Muscular dystrophy, limb-girdle, type 2F, 601287
  601412	Deafness, autosomal dominant 7
  601414	Retinitis pigmentosa-18
  601458	Inflammatory bowel disease-2
  601471	Moebius syndrome-2
  601493	Cardiomyopathy, dilated 1C
  601494	Cardiomyopathy, familial, dilated-2
  601498	Peroxisomal biogenesis disorder, complementation group 4
  601518	Prostate cancer, hereditary, 1, 176807
  601545	Lissencephaly-1
  601556	Spinocerebellar ataxia-1, 164400
  601567	Combined factor V and VIII deficiency, 227300
  601596	Charcot-Marie-Tooth neuropathy, demyelinating
  601604	Mycobacterial and salmonella infections, susceptibility to
  601606	Trichoepithelioma, multiple familial
  601620	Holt-Oram syndrome, 142900
  601621	Ulnar-mammary syndrome, 181450
  601622	Saethre-Chotzen syndrome, 101400
  601623	Angelman syndrome
  601649	Blepharophimosis, epicanthus inversus, and ptosis, type 2
  601650	Paraganglioma, familial nonchromaffin, 2
  601652	Glaucoma 1A, primary open angle, juvenile-onset, 137750
  601653	Branchiootic syndrome
  601653	Branchiootorenal syndrome, 113650
  601666	Insulin-dependent diabetes mellitus-15
  601669	Hirschsprung disease, one form
  601676	Acute insulin response
  601680	Distal arthrogryposis, type 2B
  601682	Glaucoma 1C, primary open angle
  601687	Meesmann corneal dystrophy, 122100
  601690	Platelet-activating factor acetylhydrolase deficiency
  601691	Retinitis pigmentosa-19, 601718
  601691	Stargardt disease-1, 248200
  601691	Cone-rod dystrophy 3
  601691	Fundus flavimaculatus with macular dystrophy, 248200
  601692	Reis-Bucklers corneal dystrophy
  601692	Corneal dystrophy, Avellino type
  601692	Corneal dystrophy, Groenouw type I, 121900
  601692	Corneal dystrophy, lattice type I, 122200
  601718	Retinitis pigmentosa-19
  601728	Bannayan-Zonana syndrome, 153480
  601728	Cowden disease, 158350
  601728	Endometrial carcinoma
  601728	Lhermitte-Duclos syndrome
  601744	Systemic lupus erythematosus, susceptibility to, 1
  601757	Rhizomelic chondrodysplasia punctata, type 1, 215100
  601768	Leukemia, acute myeloid
  601769	Osteoporosis, involutional
  601769	Rickets, vitamin D-resistant, 277440
  601771	Glaucoma 3A, primary infantile, 231300
  601777	Cone dystrophy, progressive
  601780	Ceroid-lipofuscinosis, neuronal-6, variant late infantile
  601785	Carbohydrate-deficient glycoprotein syndrome, type I, 212065
  601800	[Hair color, brown]
  601843	Hypothyroidism, congenital, 274400
  601844	Pseudohypoaldosteronism type II
  601846	Muscular dystrophy with rimmed vacuoles
  601847	Progressive intrahepatic cholestasis-2
  601850	Retinitis pigmentosa-deafness syndrome
  601863	Bare lymphocyte syndrome, complementation group C
  601868	Deafness, autosomal dominant 13
  601884	[High bone mass]
  601889	Lymphoma, diffuse large cell
  601916	Pancreatic cancer
  601928	Monilethrix, 158000
  601941	Insulin-dependent diabetes mellitus-6
  601954	Muscular dystrophy, limb-girdle, type 2G
  601969	Medulloblastoma, 155255
  601969	Glioblastoma multiforme, 137800
  601975	Ectodermal dysplasia/skin fragility syndrome
  601990	Neuroblastoma
  602014	Hypomagnesemia with secondary hypocalcemia
  602023	Bartter syndrome, type 3
  602025	Obesity/hyperinsulinism, susceptibility to
  602028	Multiple myeloma
  602066	Convulsions, infantile and paroxysmal choreoathetosis
  602067	Cardiomyopathy, dilated, 1F
  602078	Fibrosis of extraocular muscles, congenital, 2
  602080	Paget disease of bone-2
  602081	Speech-language disorder-1
  602082	Corneal dystrophy, Thiel-Behnke type
  602084	Endometrial carcinoma
  602085	Postaxial polydactyly, type A2
  602086	Arrhythmogenic right ventricular dysplasia-3
  602087	Arrhythmogenic right ventricular dysplasia-4
  602088	Nephronophthisis, infantile
  602089	Hemangioma, capillary, hereditary
  602091	Marfan syndrome, atypical
  602092	Deafness, autosomal recessive 18
  602094	Lipodystrophy, familial partial
  602096	Alzheimer disease-5
  602099	Amytrophic lateral sclerosis-5
  602116	Glioma
  602117	Prader-Willi syndrome
  602121	Deafness, autosomal dominant nonsyndromic sensorineural, 1, 124900
  602134	Tremor, familial essential, 2
  602136	Refsum disease, infantile, 266510
  602136	Zellweger syndrome-1, 214100
  602136	Adrenoleukodystrophy, neonatal, 202370
  602153	Monilethrix, 158000
  602216	Peutz-Jeghers syndrome, 175200
  602225	Cone-rod retinal dystrophy-2, 120970
  602225	Leber congenital amaurosis, type III
  602235	Epilepsy, benign, neonatal, type 1, 121200
  602279	Oculopharyngeal muscular dystorphy, 164300
  602279	Oculopharyngeal muscular dystrophy, autosomal recessive, 257950
  602280	Retinitis pigmentosa-14, 600132
  602363	Ellis-van Creveld-like syndrome
  602397	Cholestasis, benign recurrent intrahepatic, 243300
  602397	Cholestasis, progressive familial intrahepatic-1, 211600
  602403	Alzheimer disease, susceptibility to
  602404	Parkinson disease, type 3
  602447	Coronary artery disease, susceptibility to
  602460	Deafness, autosomal dominant 15, 602459
  602475	Ossification of posterior longitudinal ligament of spine
  602476	Febrile convulsions, familial, 1
  602477	Febrile convulsions, familial, 2
  602491	Hyperlipidemia, familial combined, 1
  602522	Bartter syndrome, infantile, with sensorineural deafness
  602544	Parkinson disease, juvenile, type 2, 600116
  602568	Homocystinuria-megaloblastic anemia, cbl E type, 236270
  602574	Deafness, autosomal dominant 12, 601842
  602574	Deafness, autosomal dominant 8, 601543
  602575	Nail-patella syndrome with open-angle glaucoma, 137750
  602575	Nail-patella syndrome, 161200
  602616	Carbohydrate-deficient glycoprotein syndrome, type II, 212066
  602629	Dystonia-6, torsion
  602631	Rhabdomyosarcoma, 268210
  602631	Breast Cancer
  602669	Anterior segment mesenchymal dysgenesis and cataract, 107250
  602669	Cataract, congenital
  602685	Mental retardation, severe, with spasticity and tapetoretinal
  	degeneration
  602716	Nephrosis-1, congenital, Finnish type, 256300
  602759	Prostate cancer, hereditary, 2, 176807
  602771	Muscular dystrophy, congenital, with early spine rigidity
  602772	Retinitis pitmentosa-24
  602782	Faisalabad histiocytosis
  602783	Spastic paraplegia-7

  
  Mature Polypeptides
• The present invention also encompasses mature forms of a polypeptide having the amino acid sequence of SEQ ID NO:Y and/or the amino acid sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional activities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity in detecting, preventing, treating and/or indicated disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.
• According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
• Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.
• In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1A.
• In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the predicted mature form of the polypeptide as delineated in columns 14 and 15 of Table 1A. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional activities of the full-length or mature form of the polypeptide (e.g., biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the invention antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.
• Polynucleotides encoding proteins comprising, or consisting of, the predicted mature form of polypeptides of the invention (e.g., polynucleotides having the sequence of SEQ ID NO: X (Table 1A, column 4), the sequence delineated in columns 7 and 8 of Table 1A, and a sequence encoding the mature polypeptide delineated in columns 14 and 15 of Table 1A (e.g., the sequence of SEQ ID NO:X encoding the mature polypeptide delineated in columns 14 and 15 of Table 1)) are also encompassed by the invention, as are fragments or variants of these polynucleotides (such as, fragments as described herein, polynucleotides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polynucleotides, and nucleic acids which hybridizes under stringent conditions to the complementary strand of the polynucleotide).
• As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 15 residues of the predicted cleavage point (i.e., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 more or less contiguous residues of SEQ ID NO:Y at the N-terminus when compared to the predicted mature form of the polypeptide (e.g., the mature polypeptide delineated in columns 14 and 15 of Table 1). Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
• Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.
• Polynucleotide and Polypeptide Variants
• The present invention is also directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID NO:X that encodes the polypeptide sequence as defined in columns 13 and 14 of Table 1A, nucleotide sequences encoding the polypeptide sequence as defined in columns 13 and 14 of Table 1A, the nucleotide sequence of SEQ ID NO:X encoding the polypeptide sequence as defined in column 5 of Table 1B.1, nucleotide sequences encoding the polypeptide as defined in column 6 and column 7 of Table 1B.1, the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as defined in column 6 of Table 1C, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1C, the cDNA sequence contained in ATCC Deposit NO: Z, nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in ATCC Deposit NO: Z, and/or nucleotide sequences encoding a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCC Deposit NO: Z.
• The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide as defined in columns 13 and 14 of Table 1A, the polypeptide sequence as defined in columns 6 and 7 of Table 1B.1, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table 1C, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, the polypeptide sequence encoded by the cDNA sequence contained in ATCC Deposit NO: Z and/or a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCC Deposit NO: Z.
• “Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.
• Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X or contained in the cDNA sequence of ATCC Deposit No: Z; (b) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCC Deposit No: Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCC Deposit No: Z which encodes a mature polypeptide (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)); (d) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCC Deposit No: Z, which encodes a biologically active fragment of a polypeptide; (e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCC Deposit No: Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID NO:Y (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)) or a mature polypeptide of the amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.
• The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA contained in ATCC Deposit No: Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in columns 6 and 7 of Table 1B.1 or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in column 6 and 7 of Table 1B.1 or the complementary strand thereto, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.
• In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides. In another preferred embodiment, polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; (b) the amino acid sequence of a mature (secreted) form of a polypeptide having the amino acid sequence of SEQ ID NO:Y (e.g., as delineated in columns 14 and 15 of Table 1A) or a mature form of the amino acid sequence encoded by the cDNA in ATCC Deposit No: Z mature; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC Deposit No: Z.
• The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in ATCC Deposit No: Z, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C, the amino acid sequence as defined in column 6 and 7 of Table 1B. 1, an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.
• By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to in Table 1B or 2 as the ORF (open reading frame), or any fragment specified as described herein.
• As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.
• If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
• For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
• By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence delineated in columns 14 and 15) or a fragment thereof, Table 1B.1 (e.g., the amino acid sequence identified in column 6) or a fragment thereof, Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in ATCC Deposit No: Z, or a fragment thereof, the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCC Deposit No: Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.
• If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
• For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequnce are manually corrected for. No other manual corrections are to made for the purposes of the present invention.
• The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
• Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
• Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
• Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
• Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
• Thus, the invention further includes polypeptide variants which show a functional activity (e.g., biological activity) of the polypeptides of the invention. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
• The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).
• Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity. By a polypeptide having “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein and/or a mature (secreted) protein of the invention. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an anti-polypeptide of the invention antibody], immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.
• The functional activity of the polypeptides, and fragments, variants and derivatives of the invention, can be assayed by various methods.
• For example, in one embodiment where one is assaying for the ability to bind or compete with a full-length polypeptide of the present invention for binding to an anti-polypeptide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
• In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a polypeptide of the present invention to bind to a substrate(s) of the polypeptide of the invention can be routinely assayed using techniques known in the art.
• In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of the polypeptides of the present invention and fragments, variants and derivatives thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.
• Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in ATCC Deposit No: Z, the nucleic acid sequence referred to in Table 1B (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 1A (e.g., the nucleic acid sequence delineated in columns 7 and 8), the nucleic acid sequence disclosed in Table 2 (e.g., the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides “having functional activity.” In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
• For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
• The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
• The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.
• As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
• Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
• For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).
• A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course it is highly preferable for a polypeptide to have an amino acid sequence which, for example, comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, the amino acid sequence of the mature (e.g., secreted) polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X, an amino acid sequence encoded by cDNA contained in ATCC Deposit No: Z, and/or the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCC Deposit No: Z, or a fragment thereof, which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
• In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature formand/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in ATCC Deposit No: Z or fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Polynucleotide and Polypeptide Fragments
• The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a “polynucleotide fragment” refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in ATCC Deposit No: Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCC Deposit No: Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the mature (secreted) polypeptide encoded by the cDNA contained in ATCC Deposit No: Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the mature amino acid sequence as defined in columns 14 and 15 of Table 1A or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto.
• The polynucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in ATCC Deposit No: Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length) are also encompassed by the invention.
• Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in ATCC Deposit No: Z, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1C column 6. Additional, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of a sequence delineated in column 6 of Table 1C. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C which correspond to the same ATCC Deposit No: Z (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.
• In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of the amino acid sequence contained in SEQ ID NO:Y, is a portion of the mature form of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, is a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:X, is a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, is a portion of the amino acid sequence of a mature (secreted) polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, and/or is a portion of an amino acid sequence encoded by the cDNA contained in ATCC Deposit No: Z. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
• Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
• Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
• The present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, and/or a mature polypeptide encoded by the cDNA contained in ATCC Deposit No: Z). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• The present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, and/or a mature polypeptide encoded by the cDNA contained in ATCC Deposit No: Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n corresponds to the position of amino acid residue in a polypeptide of the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in ATCC Deposit No: Z, and/or the complement thereof, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind a ligand) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.
• The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in ATCC Deposit No: Z, or the polynucleotide sequence as defined in column 6 of Table 1C, may be analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2) or the cDNA contained in ATCC Deposit No: Z may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, Wis. 53715 USA; http://www.dnastar.com/).
• Polypeptide regions that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to, Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above.
• Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
• Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g. biological activity) of the polypeptide sequence of which the amino acid sequence is a fragment. By a polypeptide displaying a “functional activity” is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.
• Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
• In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Epitopes and Antibodies
• The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereto; the polypeptide sequence encoded by the cDNA contained in ATCC Deposit No: Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in ATCC Deposit No: Z under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lower stringency hybridization conditions defined supra.
• The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)
• In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
• Non-limiting examples of epitopes of polypeptides that can be used to generate antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y specified in column 6 of Table 1B.1. These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index which is included in the DNAStar suite of computer programs. By “comprise” it is intended that a polypeptide contains at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y shown in column 6 of Table 1B.1, but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterolgous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein. In particular embodiments, epitope portions of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in column 6 of Table 1B.1.
• Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
• Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
• Fusion Proteins
• Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as targeting molecules once fused to other proteins.
• Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
• In certain preferred embodiments, proteins of the invention are fusion proteins comprising an amino acid sequence that is an N and/or C-terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the invention. Polynucleotides encoding these proteins are also encompassed by the invention.
• Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
• As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous polypeptide sequences. For example, the polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A 0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
• Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).
• Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
• Recombinant and Synthetic Production of Polypeptides of the Invention
• The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
• The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
• As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.
• Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
• The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
• Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
• In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication Number WO 96/29411; International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
• Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulos chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid charomatography (“HPLC”) is employed for purification.
• Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
• In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O₂. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O₂. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
• In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
• Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.
• In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
• In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
• In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
• The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
• Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹¹In, ¹¹²In, ^113mIn, ^115mIn), technetium (⁹⁹Tc, ^99mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.
• In specific embodiments, a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is ¹¹¹In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.
• As mentioned, the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
• Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
• As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al, Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
• The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
• One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
• As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
• The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
• The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
• The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
• Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in ATCC Deposit No: Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in ATCC Deposit No: Z). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
• Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
• Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.
• In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.
• The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
• Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
• Antibodies
• Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgG1. In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.
• Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
• Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures. Preferred epitopes of the invention include the predicted epitopes shown in column 6 of Table 1B.1, as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
• Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.
• The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
• Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
• The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).
• Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); incorporated by reference herein in its entirety.
• As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.
• The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press; 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
• Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
• Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
• Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.
• In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human×mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.
• Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.
• For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
• As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
• Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
• Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
• Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
• Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).
• Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
• Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.
• Polynucleotides Encoding Antibodies
• The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
• Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
• In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
• In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
• Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
• Methods of Producing Antibodies
• The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.
• Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
• The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
• A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
• In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
• In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
• In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.
• The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
• Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suplliers, including, for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entirities by reference herein.
• The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
• The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
• The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).
• As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J. Biochem. 270:3958-3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
• Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.
• The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.
• Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
• The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).
• Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
• Immunophenotyping
• The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
• These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
• Assays for Antibody Binding
• The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
• Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
• Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.8.1.
• ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.
• The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
• Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.
• Therapeutic Uses
• Table 1D: In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Table 1A and Table 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Table 1D) in an amount effective to treat, prevent, or ameliorate the disease or disorder.
• As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to treat the associated disease.
• The present invention encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and seccond columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.
• In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.
• The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).
• The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1 D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).
• In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.
• The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.
• The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.
• The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.
• The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.
• The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.
• The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).
• The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.
• The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.
• The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypemephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.
• The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.
• The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).
• In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.
• Table 1E also provides information regarding biological activities and preferred therapeutic uses (i.e. see, “Preferred Indications” column) for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information regarding assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA ATCC Deposit No: Z”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention as well as indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.
• The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions., and/or as described elsewhere herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in column 7 of Table 1B.1; or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
• The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
• The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
• It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.
• Gene Therapy
• In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.
• Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
• For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
• In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
• Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
• In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.
• Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
• In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
• In a preferred embodiment, the cell used for gene therapy is autologous to the patient.
• In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
• In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.
• Demonstration of Therapeutic or Prophylactic Activity
• The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• Therapeutic/Prophylactic Administration and Composition
• The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
• Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.
• In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
• In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
• Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
• In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
• The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
• In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
• The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
• The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• Diagnosis and Imaging
• Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
• The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
• Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• One facet of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
• It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
• Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
• In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
• Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
• In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
• Kits
• The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
• In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.
• In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
• In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
• In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or calorimetric substrate (Sigma, St. Louis, Mo.).
• The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
• Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
• Uses of the Polynucleotides
• Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
• The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table 1B.1, column 8 provides the chromosome location of some of the polynucleotides of the invention.
• Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably at least 15 bp (e.g., 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to SEQ ID NO:X will yield an amplified fragment.
• Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries, and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).
• Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).
• For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
• Thus, the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1B and/or Table 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.
• The polynucleotides of the present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping. For a review of these techniques and others known in the art, see, e.g. Dear, “Genome Mapping: A Practical Approach,” IRL Press at Oxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.
• Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Column 9 of Table 1B.1 provides an OMIM reference identification number of diseases associated with the cytologic band disclosed in column 8 of Table 1B.1, as determined using techniques described herein and by reference to Table 5. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
• Thus, once coinheritance is established, differences in a polynucleotide of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
• Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and prognostic methods, kits and reagents encompassed by the present invention are briefly described below and more thoroughly elsewhere herein (see e.g., the sections labeled “Antibodies”, “Diagnostic Assays”, and “Methods for Detecting Diseases”).
• Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., Example 12).
• In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31′mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.
• Where a diagnosis of a related disorder, including, for example, diagnosis of a tumor, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
• By “measuring the expression level of polynucleotides of the invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains polypeptide of the present invention or the corresponding mRNA. As indicated, biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• The method(s) provided above may preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, such as for example, in neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supra are hereby incorporated by reference in their entirety herein.
• The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
• The compounds of the present invention have uses which include, but are not limited to, detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.
• Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)
• For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO 91/15580). However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5′ end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness is not be limited to treatment, prevention, and/or prognosis of proliferative disorders of cells and tissues of hematopoietic origin, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.
• In addition to the foregoing, a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. The oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease, and in particular, for the treatment of proliferative diseases and/or conditions. Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the section labeled “Antisense and Ribozyme (Antagonists)”).
• Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled “Gene Therapy Methods”, and Examples 16, 17 and 18).
• The polynucleotides are also useful for identifying indivisuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
• The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
• Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
• There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1B. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.
• The polynucleotides of the present invention are also useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays) or cell type(s) (e.g., immunocytochemistry assays). In addition, for a number of disorders of the above tissues or cells, significantly higher or lower levels of gene expression of the polynucleotides/polypeptides of the present invention may be detected in certain tissues (e.g., tissues expressing polypeptides and/or polynucleotides of the present invention, for example, those disclosed in column 5 of Table 1B.2, and/or cancerous and/or wounded tissues) or bodily fluids (e.g., semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.
• Thus, the invention provides a diagnostic method of a disorder, which involves: (a) assaying gene expression level in cells or body fluid of an individual; (b) comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.
• In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
• Uses of the Polypeptides
• Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
• Polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry assays).
• Antibodies can be used to assay levels of polypeptides encoded by polynucleotides of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^115mIn, ^113mIn, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^99mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• In addition to assaying levels of polypeptide of the present invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
• A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In, ^99mTc, (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^115mIn, ^113mIn, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^99mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹ Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ^99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
• In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
• In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs.
• By “toxin” is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ⁹⁰Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ¹¹¹In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ¹³¹I.
• Techniques known in the art may be applied to label polypeptides of the invention (including antibodies). Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).
• Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
• Moreover, polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
• Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
• At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.
• Diagnostic Assays
• The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described in the legends for Tables 1D and 1E and as indicated in the “Preferred Indications” columns in Table 1D and Table 1E; and, also as described herein under the section heading “Biological Activities”.
• For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
• The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.
• In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
• By “assaying the expression level of the gene encoding the polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).
• The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur using any art-known method.
• Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
• The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of inteest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.
• For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
• In a preferred embodiment, antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention (shown in column 7 of Table 1B.1) may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
• In an additional preferred embodiment, antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
• The antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the gene product, or conserved variants or peptide fragments, or polypeptide binding, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
• Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
• The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled antibody or detectable polypeptide of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
• By “solid phase support or carrier” is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
• The binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
• In addition to assaying polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of the invention, polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms. In another embodiment, polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell) are used to image diseased or neoplastic cells.
• Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where in vivo imaging is used to detect enhanced levels of polypeptides for diagnosis in humans, it may be preferable to use human antibodies or “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).
• Additionally, any polypeptides of the invention whose presence can be detected, can be administered. For example, polypeptides of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.
• A polypeptide-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In, ^99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ^99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
• With respect to antibodies, one of the ways in which an antibody of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
• Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect polypeptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
• It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
• The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
• The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
• Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
• Methods for Detecting Diseases
• In general, a disease may be detected in a patient based on the presence of one or more proteins of the invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein. In addition, such proteins may be useful for the detection of other diseases and cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer. In general, polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.
• There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, supra. In general, the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
• In a preferred embodiment, the assay involves the use of a binding agent(s) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or antibodies, to which the binding agent binds, as described above.
• The solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to immobilize an adequate amount of binding agent.
• Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
• Gene Therapy Methods
• Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.
• Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.
• As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
• In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotide of the present invention can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
• The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.
• Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotide of the present invention.
• Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
• The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
• For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.
• The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
• The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.
• The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitating agents, etc. Such methods of delivery are known in the art.
• In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.
• Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
• Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.
• Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
• For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.
• The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca²⁺-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.
• Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
• U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.
• In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
• The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
• The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention.
• In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis. 109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).
• Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.
• Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest which is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.
• In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.
• For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a polypeptide of the invention.
• Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.
• Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
• The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.
• The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.
• The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.
• The polynucleotide encoding a polypeptide of the present invention may contain a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.
• Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
• A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.
• Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.
• Therapeutic compositions useful in systemic administration, include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.
• Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
• Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.
• Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans being particularly preferred.
• Biological Activities
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention, can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to treat the associated disease.
• Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with aberrant activity of secreted polypeptides.
• In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders relating to diseases and disorders of the endocrine system, the nervous system (See, for example, “Neurological Disorders” section below), and the immune system (See, for example, “Immune Activity” section below).
• In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
• Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, detection and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.
• More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with the following systems.
• Immune Activity
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
• In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies. Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.
• In specific embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing using the polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof.
• Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
• In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.
• Other immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
• In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
• In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
• Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
• Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.
• Additional disorders that are likely to have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
• Additional disorders that may have an autoimmune component that may be treated, prevented, diagnosed and/or prognosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.
• In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
• In another specific preferred embodiment, systemic lupus erythematosus is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
• In another specific preferred embodiment IgA nephropathy is treated, prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.
• In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention
• In preferred embodiments, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a immunosuppressive agent(s).
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, prognosing, and/or diagnosing diseases, disorders, and/or conditions of hematopoietic cells. Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.
• Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated, prevented, diagnosed and/or prognosed using polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
• Additionally, polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, may be used to treat, prevent, diagnose and/or prognose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate IgE concentrations in vitro or in vivo.
• Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention have uses in the diagnosis, prognosis, prevention, and/or treatment of inflammatory conditions. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).
• Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, polynucleotides, polypeptides, and antibodies of the invention, as well as agonists or antagonists thereof, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.
• In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat organ transplant rejections and graft-versus-host disease. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.
• In other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.
• Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.
• In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance tumor-specific immune responses.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
• In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia colt, Enterohemorrhagic E. coli, and Borrelia burgdorferi.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.
• In one embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell responsiveness to pathogens.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an activator of T cells.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to induce higher affinity antibodies.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to increase serum immunoglobulin concentrations.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to accelerate recovery of immunocompromised individuals.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in the pretreatment of bone marrow samples prior to transplant.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence/immunodeficiency such as observed among SCID patients.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.
• In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in one or more of the applications decribed herein, as they may apply to veterinary medicine.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.
• The polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit complement mediated cell lysis.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed to treat adult respiratory distress syndrome (ARDS).
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.
• In a specific embodiment, polynucleotides or polypeptides, and/or agonists thereof are used to diagnose, prognose, treat, and/or prevent a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be prevented, diagnosed, prognosed, and/or treated with polynucleotides or polypeptides, and/or agonists of the present invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.
• In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease (“CVID”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.
• In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to diagnose, prognose, prevent, and/or treat cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed, or treated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled “Hyperproliferative Disorders” elsewhere herein.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.
• In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
• In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.
• Antagonists of the invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of the invention include, for example, binding or stimulatory antibodies, and soluble forms of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.
• In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741). Administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention to such animals is useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention.
• Blood-Related Disorders
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, polynucleotides or polypeptides, and/or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.
• In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to prevent, diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, include, but are not limited to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
• In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to prevent, diagnose, prognose, and/or treat diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hematopoietic activity (the formation of blood cells). For example, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of anemias and leukopenias described below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the prevention, detection, diagnosis and/or treatment of leukocytoses, such as, for example eosinophilia.
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to prevent, treat, or diagnose blood dyscrasia.
• Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias. Anemias that may be treated prevented or diagnosed by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary siderob; astic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs. Additionally, rhe polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease). Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating thalassemias, including, but not limited to major and minor forms of alpha-thalassemia and beta-thalassemia.
• In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bemard-Soulier syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
• The effect of the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.
• Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.
• In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukopenia. In other specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukocytosis.
• Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).
• The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.
• In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.
• In yet another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in diagnosing, prognosing, preventing, and/or treating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies of undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, and/or diagnosing myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as a treatment prior to surgery, to increase blood cell production.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase cytokine production.
• In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, diagnosing, and/or treating primary hematopoietic disorders.
• Hyperproliferative Disorders
• In certain embodiments, polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, Polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
• For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.
• Examples of hyperproliferative disorders that can be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
• Similarly, other hyperproliferative disorders can also be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
• In another preferred embodiment, polynucleotides or polypeptides, or agonists or antagonists of the present invention are used to diagnose, prognose, prevent, and/or treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.)
• Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.
• Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
• Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
• Additional pre-neoplastic disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
• In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognose disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
• In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to those described herein. In a further preferred embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.
• Additionally, polynucleotides, polypeptides, and/or agonists or antagonists of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis. For example, diseases associated with increased cell survival or the inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
• In preferred embodiments, polynucleotides, polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
• Additional diseases or conditions associated with increased cell survival that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
• Diseases associated with increased apoptosis that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
• Hyperproliferative diseases and/or disorders that could be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
• Similarly, other hyperproliferative disorders can also be diagnosed, prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
• Another preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
• Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.
• Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
• Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
• For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.
• The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
• By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.
• Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
• The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.
• In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.
• The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example., which serve to increase the number or activity of effector cells which interact with the antibodies.
• It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragements thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragements thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.
• Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).
• Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. April 24; 111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int J Tissue React; 20(1):3-15 (1998), which are all hereby incorporated by reference).
• Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference). Such therapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
• In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
• Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
• Renal Disorders
• Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose disorders of the renal system. Renal disorders which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.
• Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.)
• In addition, compositions of the invention can be used to diagnose, prognose, prevent, and/or treat metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).
• Compositions of the invention can also be used to diagnose, prognose, prevent, and/or treat sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypemephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
• Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
• Cardiovascular Disorders
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose cardiovascular disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.
• Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
• Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
• Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
• Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
• Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
• Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.
• Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
• Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
• Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
• Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
• Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
• Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.
• Respiratory Disorders
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention may be used to treat, prevent, diagnose, and/or prognose diseases and/or disorders of the respiratory system.
• Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus(staphylococcal pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).
• Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, ftungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.; candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.
• Anti-Angiogenesis Activity
• The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
• The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
• Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near, the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
• Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
• For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.
• Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists of the invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
• Moreover, Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
• Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue which normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
• Within particularly preferred embodiments of the invention, may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
• Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
• Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.
• Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
• Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.
• Additionally, disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
• Moreover, disorders and/or states, which can be treated, prevented, diagnosed, and/or prognosed with the the polynucleotides, polypeptides, agonists and/or agonists of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.
• In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
• Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.
• Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
• Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
• Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
• The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
• Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
• Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
• Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
• A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.
• Diseases at the Cellular Level
• Diseases associated with increased cell survival or the inhibition of apoptosis that could be treated, prevented, diagnosed, and/or prognosed using polynucleotides or polypeptides, as well as antagonists or agonists of the present invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.
• In preferred embodiments, polynucleotides, polypeptides, and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
• Additional diseases or conditions associated with increased cell survival that could be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
• Diseases associated with increased apoptosis that could be treated, prevented, diagnosed, and/or prognesed using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.
• Wound Healing and Epithelial Cell Proliferation
• In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote dermal reestablishment subsequent to dermal loss
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that polynucleotides or polypeptides, agonists or antagonists of the present invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, can be used to promote skin strength and to improve the appearance of aged skin.
• It is believed that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Polynucleotides or polypeptides, agonists or antagonists of the present invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with polynucleotides or polypeptides, agonists or antagonists of the present invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat diseases associate with the under expression.
• Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to prevent and heal damage to the lungs due to various pathological states. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of aveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
• In addition, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
• Neural Activity and Neurological Diseases
• The polynucleotides, polypeptides and agonists or antagonists of the invention may be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
• In one embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.
• In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.
• In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.
• The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci., 4:17-42 (1981); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
• In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
• Further, polypeptides or polynucleotides of the invention may play a role in neuronal survival; synapse formation; conductance; neural differentiation, etc. Thus, compositions of the invention (including polynucleotides, polypeptides, and agonists or antagonists) may be used to diagnose and/or treat or prevent diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders. The compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders. Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.
• Additionally, polypeptides, polynucleotides and/or agonists or antagonists of the invention, may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).
• In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, polynucleotides, polypeptides, agonists and/or antagonists of the invention may be used to treat and/or detect neurologic diseases. Moreover, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.
• Examples of neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include brain diseases, such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central nervous system infections such as AIDS Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube defects such as anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations (such as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wemicke Aphasia, articulation disorders, communicative disorders such as speech disorders which include dysarthria, echolalia, mutism and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff-man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressive external ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders such as hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as amyotonia congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adie's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome, Chronic Progressive External Ophthalmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as diabetic foot.
• Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
• Endocrine Disorders
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.
• Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.
• Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).
• Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma—islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.
• In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.
• Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
• In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).
• Reproductive System Disorders
• The polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the diagnosis, treatment, or prevention of diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.
• Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).
• Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.
• Additionally, the compositions of the invention may be useful in the diagnosis, treatment, and/or prevention of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.
• Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.
• Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.
• Further, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.
• Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or antagonists of the invention may be useful as a marker or detector of, as well as in the diagnosis, treatment, and/or prevention of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.
• Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).
• Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).
• Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. Additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the diagnosis, treatment, and/or prevention of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders, and obstruction of the intestine.
• Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
• Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
• Other disorders and/or diseases of the female reproductive system that may be diagnosed, treated, and/or prevented by the polynucleotides, polypeptides, and agonists or antagonists of the present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.
• Infectious Disease
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
• Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.
• Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacteria, bacterial families, and fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium dificile, Clostridium perfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to: antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections, noscomial infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, diptheria, botulism, and/or meningitis type B.
• Moreover, parasitic agents causing disease or symptoms that can be treated, prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat, prevent, and/or diagnose any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat, prevent, and/or diagnose malaria.
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
• Regeneration
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
• Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.
• Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
• Similarly, nerve and brain tissue could also be regenerated by using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotides or polypeptides, as well as agonists or antagonists of the present invention.
• Gastrointestinal Disorders
• Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to treat, prevent, diagnose, and/or prognose gastrointestinal disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
• Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess,).
• Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T. solium).
• Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).
• Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).
• Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
• Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis), immunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.
• Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).
• Chemotaxis
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.
• Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
• It is also contemplated that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could be used as an inhibitor of chemotaxis.
• Binding Activity
• A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
• Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.
• Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
• The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
• Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
• Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
• Additionally, the receptor to which the polypeptide of the present invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.
• Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.
• As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.
• Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of the polypeptide of the present invention thereby effectively generating agonists and antagonists of the polypeptide of the present invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptide of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic (dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).
• Other preferred fragments are biologically active fragments of the polypeptide of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
• Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and ³[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of ³[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of ³[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.
• In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.
• All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues.
• Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.
• Targeted Delivery
• In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.
• As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
• In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.
• By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
• Drug Screening
• Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.
• This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.
• Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.
• Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
• This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
• Antisense And Ribozyme (Antagonists)
• In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to cDNA sequences contained in cDNA ATCC Deposit No: Z identified for example, in Table 1A and/or 1B. In one embodiment, antisense sequence is generated internally, by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
• For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoRI site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRI/Hind III site of the retroviral vector PMV7 (WO 91/15580).
• For example, the 5′ coding portion of a polynucleotide that encodes the polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
• In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding the polypeptide of the present invention or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.
• The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of the present invention. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
• Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of polynucleotide sequences described herein could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA of the present invention, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
• The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
• The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
• The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
• In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
• In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
• Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
• While antisense nucleotides complementary to the coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
• Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of SEQ ID NO:X. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
• As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
• Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
• The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.
• The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.
• The antagonist/agonist may also be employed to treat the diseases described herein.
• Thus, the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.
• Binding Peptides and Other Molecules
• The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind polypeptides of the invention, and the binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.
• This method comprises the steps of:
• • a. contacting polypeptides of the invention with a plurality of molecules; and
  • b. identifying a molecule that binds the polypeptides of the invention.
• The step of contacting the polypeptides of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptides on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized polypeptides of the invention. The molecules having a selective affinity for the polypeptides can then be purified by affinity selection. The nature of the solid support, process for attachment of the polypeptides to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.
• Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by the polypeptides of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the polypeptides and the individual clone. Prior to contacting the polypeptides with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for polypeptides of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the polypeptides of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.
• In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of the polypeptides of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the polypeptides of the invention or the plurality of polypeptides are bound to a solid support.
• The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind polypeptides of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.
• Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
• In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.
• By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
• The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.
• Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.
• Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.
• Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.
• In a specific embodiment, screening to identify a molecule that binds polypeptides of the invention can be carried out by contacting the library members with polypeptides of the invention immobilized on a solid phase and harvesting those library members that bind to the polypeptides of the invention. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.
• In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to polypeptides of the invention.
• Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.
• Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.
• As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.
• The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.
• Other Activities
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. The polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
• A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
• The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.
Other Preferred Embodiments
• Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No: Z.
• Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in column 5, “ORF (From-To)”, in Table 1B.1.
• Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in columns 8 and 9, “NT From” and “NT To” respectively, in Table 2.
• Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No: Z.
• Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No: Z.
• A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in column 5, “ORF (From-To)”, in Table 1B.1.
• A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in columns 8 and 9, “NT From” and “NT To”, respectively, in Table 2.
• A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC Deposit No: Z, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
• Also preferred is a composition of matter comprising a DNA molecule which comprises the cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides of the cDNA sequence contained in ATCC Deposit No: Z.
• Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of an open reading frame sequence encoded by cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCC Deposit No: Z.
• A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCC Deposit No: Z.
• A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by cDNA contained in ATCC Deposit No: Z.
• A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B. 1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCC Deposit No: Z; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
• Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
• A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of the cDNA contained in ATCC Deposit No: Z.
• The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
• Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; or the cDNA contained in ATCC Deposit No: Z which encodes a protein, wherein the method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B. 1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of cDNA contained in ATCC Deposit No: Z.
• The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.
• Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in column 5 of Table 1B.1 or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCC Deposit No: Z. The nucleic acid molecules can comprise DNA molecules or RNA molecules.
• Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a DNA microarray or “chip” of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 500, 1000, 2000, 3000, or 4000 nucleotide sequences, wherein at least one sequence in said DNA microarray or “chip” is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1A and/or 1B; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA “Clone ID” in Table 1A and/or 1B.
• Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No: Z.
• Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No: Z.
• Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC Deposit No: Z.
• Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a polypeptide encoded by contained in ATCC Deposit No: Z
• Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a portion of said polypeptide encoded by cDNA contained in ATCC Deposit No: Z; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or the polypeptide sequence of SEQ ID NO:Y.
• Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of a polypeptide encoded by cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.
• Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
• Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.
• Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleic acid sequence identified in Table 1A, 1B or Table 2 encoding a polypeptide, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.
• Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
• Also preferred is a polypeptide molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z.
• Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
• Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a human protein comprising an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC Deposit No: Z. The isolated polypeptide produced by this method is also preferred.
• Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to increase the level of said protein activity in said individual.
• Also preferred is a method of treatment of an individual in need of a decreased level of a protein activity, which method comprised administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to decrease the level of said protein activity in said individual.
• Also preferred is a method of treatment of an individual in need of a specific delivery of toxic compositions to diseased cells (e.g., tumors, leukemias or lymphomas), which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide of the invention, including, but not limited to a binding agent, or antibody of the claimed invention that are associated with toxin or cytotoxic prodrugs.
• Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
• Description of Table 6
• Table 6 summarizes some of the ATCC Deposits, Deposit dates, and ATCC designation numbers of deposits made with the ATCC in connection with the present application. These deposits were made in addition to those described in the Table 1A.

  TABLE 6
  ATCC Deposits	Deposit Date	ATCC Designation Number
  LP01, LP02, LP03, LP04,	May-20-97	209059, 209060, 209061,
  LP05, LP06, LP07, LP08,		209062, 209063, 209064,
  LP09, LP10, LP11,		209065, 209066, 209067,
  		209068, 209069
  LP12	Jan-12-98	209579
  LP13	Jan-12-98	209578
  LP14	Jul-16-98	203067
  LP15	Jul-16-98	203068
  LP16	Feb-1-99	203609
  LP17	Feb-1-99	203610
  LP20	Nov-17-98	203485
  LP21	Jun-18-99	PTA-252
  LP22	Jun-18-99	PTA-253
  LP23	Dec-22-99	PTA-1081

EXAMPLES Example 1 Isolation of a Selected cDNA Clone from the Deposited Sample
• Each ATCC Deposit No: Z is contained in a plasmid vector. Table 7 identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The following correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 7 as being isolated in the vector “Lambda Zap,” the corresponding deposited clone is in “pBluescript.”

  Vector Used to Construct Library	Corresponding Deposited Plasmid
  Lambda Zap	pBluescript (pBS)
  Uni-Zap XR	pBluescript (pBS)
  Zap Express	pBK
  lafmid BA	plafmid BA
  pSport1	pSport1
  pCMVSport 2.0	pCMVSport 2.0
  pCMVSport 3.0	pCMVSport 3.0
  pCR ® 2.1	pCR ® 2.1

• Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4 forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S” is for SacI and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the f1 origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the f1 ori generates sense strand DNA and in the other, antisense.
• Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from Life Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from Life Technologies. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993)). Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from Life Technologies. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991)). Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 7, as well as the corresponding plasmid vector sequences designated above.
• The deposited material in the sample assigned the ATCC Deposit Number cited by reference to Tables 1, 2, 6 and 7 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC Deposit Number contain at least a plasmid for each ATCC Deposit No: Z.

  TABLE 7
  			ATCC
  Libraries owned by Catalog	Catalog Description	Vector	Deposit
  HUKA HUKB HUKC HUKD	Human Uterine Cancer	Lambda ZAP II	LP01
  HUKE HUKF HUKG
  HCNA HCNB	Human Colon	Lambda Zap II	LP01
  HFFA	Human Fetal Brain, random	Lambda Zap II	LP01
  	primed
  HTWA	Resting T-Cell	Lambda ZAP II	LP01
  HBQA	Early Stage Human Brain,	Lambda ZAP II	LP01
  	random primed
  HLMB HLMF HLMG HLMH	breast lymph node CDNA	Lambda ZAP II	LP01
  HLMI HLMJ HLMM HLMN	library
  HCQA HCQB	human colon cancer	Lamda ZAP II	LP01
  HMEA HMEC HMED	Human Microvascular	Lambda ZAP II	LP01
  HMEE HMEF HMEG HMEI	Endothelial Cells, fract. A
  HMEJ HMEK HMEL
  HUSA HUSC	Human Umbilical Vein	Lambda ZAP II	LP01
  	Endothelial Cells, fract. A
  HLQA HLQB	Hepatocellular Tumor	Lambda ZAP II	LP01
  HHGA HHGB HHGC HHGD	Hemangiopericytoma	Lambda ZAP II	LP01
  HSDM	Human Striatum Depression,	Lambda ZAP II	LP01
  	re-rescue
  HUSH	H Umbilical Vein Endothelial	Lambda ZAP II	LP01
  	Cells, frac A, re-excision
  HSGS	Salivary gland, subtracted	Lambda ZAP II	LP01
  HFXA HFXB HFXC HFXD	Brain frontal cortex	Lambda ZAP II	LP01
  HFXE HFXF HFXG HFXH
  HPQA HPQB HPQC	PERM TF274	Lambda ZAP II	LP01
  HFXJ HFXK	Brain Frontal Cortex, re-	Lambda ZAP II	LP01
  	excision
  HCWA HCWB HCWC	CD34 positive cells (Cord	ZAP Express	LP02
  HCWD HCWE HCWF	Blood)
  HCWG HCWH HCWI HCWJ
  HCWK
  HCUA HCUB HCUC	CD34 depleted Buffy Coat	ZAP Express	LP02
  	(Cord Blood)
  HRSM	A-14 cell line	ZAP Express	LP02
  HRSA	A1-CELL LINE	ZAP Express	LP02
  HCUD HCUE HCUF HCUG	CD34 depleted Buffy Coat	ZAP Express	LP02
  HCUH HCUI	(Cord Blood), re-excision
  HBXE HBXF HBXG	H. Whole Brain #2, re-	ZAP Express	LP02
  	excision
  HRLM	L8 cell line	ZAP Express	LP02
  HBXA HBXB HBXC HBXD	Human Whole Brain #2 -	ZAP Express	LP02
  	Oligo dT >1.5 Kb
  HUDA HUDB HUDC	Testes	ZAP Express	LP02
  HHTM HHTN HHTO	H. hypothalamus, frac A; re-	ZAP Express	LP02
  	excision
  HHTL	H. hypothalamus, frac A	ZAP Express	LP02
  HASA HASD	Human Adult Spleen	Uni-ZAP XR	LP03
  HFKC HFKD HFKE HFKF	Human Fetal Kidney	Uni-ZAP XR	LP03
  HFKG
  HE8A HE8B HE8C HE8D	Human 8 Week Whole	Uni-ZAP XR	LP03
  HE8E HE8F HE8M HE8N	Embryo
  HGBA HGBD HGBE HGBF	Human Gall Bladder	Uni-ZAP XR	LP03
  HGBG HGBH HGBI
  HLHA HLHB HLHC HLHD	Human Fetal Lung III	Uni-ZAP XR	LP03
  HLHE HLHF HLHG HLHH
  HLHQ
  HPMA HPMB HPMC HPMD	Human Placenta	Uni-ZAP XR	LP03
  HPME HPMF HPMG HPMH
  HPRA HPRB HPRC HPRD	Human Prostate	Uni-ZAP XR	LP03
  HSIA HSIC HSID HSIE	Human Adult Small Intestine	Uni-ZAP XR	LP03
  HTEA HTEB HTEC HTED	Human Testes	Uni-ZAP XR	LP03
  HTEE HTEF HTEG HTEH
  HTEI HTEJ HTEK
  HTPA HTPB HTPC HTPD	Human Pancreas Tumor	Uni-ZAP XR	LP03
  HTPE
  HTTA HTTB HTTC HTTD	Human Testes Tumor	Uni-ZAP XR	LP03
  HTTE HTTF
  HAPA HAPB HAPC HAPM	Human Adult Pulmonary	Uni-ZAP XR	LP03
  HETA HETB HETC HETD	Human Endometrial Tumor	Uni-ZAP XR	LP03
  HETE HETF HETG HETH
  HETI
  HHFB HHFC HHFD HHFE	Human Fetal Heart	Uni-ZAP XR	LP03
  HHFF HHFG HHFH HHFI
  HHPB HHPC HHPD HHPE	Human Hippocampus	Uni-ZAP XR	LP03
  HHPF HHPG HHPH
  HCE1 HCE2 HCE3 HCE4	Human Cerebellum	Uni-ZAP XR	LP03
  HCE5 HCEB HCEC HCED
  HCEE HCEF HCEG
  HUVB HUVC HUVD HUVE	Human Umbilical Vein,	Uni-ZAP XR	LP03
  	Endo. remake
  HSTA HSTB HSTC HSTD	Human Skin Tumor	Uni-ZAP XR	LP03
  HTAA HTAB HTAC HTAD	Human Activated T-Cells	Uni-ZAP XR	LP03
  HTAE
  HFEA HFEB HFEC	Human Fetal Epithelium	Uni-ZAP XR	LP03
  	(Skin)
  HJPA HJPB HJPC HJPD	HUMAN JURKAT	Uni-ZAP XR	LP03
  	MEMBRANE BOUND
  	POLYSOMES
  HESA	Human epithelioid sarcoma	Uni-Zap XR	LP03
  HLTA HLTB HLTC HLTD	Human T-Cell Lymphoma	Uni-ZAP XR	LP03
  HLTE HLTF
  HFTA HFTB HFTC HFTD	Human Fetal Dura Mater	Uni-ZAP XR	LP03
  HRDA HRDB HRDC HRDD	Human Rhabdomyosarcoma	Uni-ZAP XR	LP03
  HRDE HRDF
  HCAA HCAB HCAC	Cem cells cyclohexamide	Uni-ZAP XR	LP03
  	treated
  HRGA HRGB HRGC HRGD	Raji Cells, cyclohexamide	Uni-ZAP XR	LP03
  	treated
  HSUA HSUB HSUC HSUM	Supt Cells, cyclohexamide	Uni-ZAP XR	LP03
  	treated
  HT4A HT4C HT4D	Activated T-Cells, 12 hrs.	Uni-ZAP XR	LP03
  HE9A HE9B HE9C HE9D	Nine Week Old Early Stage	Uni-ZAP XR	LP03
  HE9E HE9F HE9G HE9H	Human
  HE9M HE9N
  HATA HATB HATC HATD	Human Adrenal Gland Tumor	Uni-ZAP XR	LP03
  HATE
  HT5A	Activated T-Cells, 24 hrs.	Uni-ZAP XR	LP03
  HFGA HFGM	Human Fetal Brain	Uni-ZAP XR	LP03
  HNEA HNEB HNEC HNED	Human Neutrophil	Uni-ZAP XR	LP03
  HNEE
  HBGB HBGD	Human Primary Breast	Uni-ZAP XR	LP03
  	Cancer
  HBNA HBNB	Human Normal Breast	Uni-ZAP XR	LP03
  HCAS	Cem Cells, cyclohexamide	Uni-ZAP XR	LP03
  	treated, subtra
  HHPS	Human Hippocampus,	pBS	LP03
  	subtracted
  HKCS HKCU	Human Colon Cancer,	pBS	LP03
  	subtracted
  HRGS	Raji cells, cyclohexamide	pBS	LP03
  	treated, subtracted
  HSUT	Supt cells, cyclohexamide	pBS	LP03
  	treated, differentially
  	expressed
  HT4S	Activated T-Cells, 12 hrs,	Uni-ZAP XR	LP03
  	subtracted
  HCDA HCDB HCDC HCDD	Human Chondrosarcoma	Uni-ZAP XR	LP03
  HCDE
  HOAA HOAB HOAC	Human Osteosarcoma	Uni-ZAP XR	LP03
  HTLA HTLB HTLC HTLD	Human adult testis, large	Uni-ZAP XR	LP03
  HTLE HTLF	inserts
  HLMA HLMC HLMD	Breast Lymph node cDNA	Uni-ZAP XR	LP03
  	library
  H6EA H6EB H6EC	HL-60, PMA 4 H	Uni-ZAP XR	LP03
  HTXA HTXB HTXC HTXD	Activated T-Cell	Uni-ZAP XR	LP03
  HTXE HTXF HTXG HTXH	(12 hs)/Thiouridine
  	labelledEco
  HNFA HNFB HNFC HNFD	Human Neutrophil, Activated	Uni-ZAP XR	LP03
  HNFE HNFF HNFG HNFH
  HNFJ
  HTOB HTOC	HUMAN TONSILS,	Uni-ZAP XR	LP03
  	FRACTION 2
  HMGB	Human OB MG63 control	Uni-ZAP XR	LP03
  	fraction I
  HOPB	Human OB HOS control	Uni-ZAP XR	LP03
  	fraction I
  HORB	Human OB HOS treated (10 nM	Uni-ZAP XR	LP03
  	E2) fraction I
  HSVA HSVB HSVC	Human Chronic Synovitis	Uni-ZAP XR	LP03
  HROA	HUMAN STOMACH	Uni-ZAP XR	LP03
  HBJA HBJB HBJC HBJD	HUMAN B CELL	Uni-ZAP XR	LP03
  HBJE HBJF HBJG HBJH	LYMPHOMA
  HBJI HBJJ HBJK
  HCRA HCRB HCRC	human corpus colosum	Uni-ZAP XR	LP03
  HODA HODB HODC HODD	human ovarian cancer	Uni-ZAP XR	LP03
  HDSA	Dermatofibrosarcoma	Uni-ZAP XR	LP03
  	Protuberance
  HMWA HMWB HMWC	Bone Marrow Cell Line	Uni-ZAP XR	LP03
  HMWD HMWE HMWF	(RS4; 11)
  HMWG HMWH HMWI
  HMWJ
  HSOA	stomach cancer (human)	Uni-ZAP XR	LP03
  HERA	SKIN	Uni-ZAP XR	LP03
  HMDA	Brain-medulloblastoma	Uni-ZAP XR	LP03
  HGLA HGLB HGLD	Glioblastoma	Uni-ZAP XR	LP03
  HEAA	H. Atrophic Endometrium	Uni-ZAP XR	LP03
  HBCA HBCB	H. Lymph node breast Cancer	Uni-ZAP XR	LP03
  HPWT	Human Prostate BPH, re-	Uni-ZAP XR	LP03
  	excision
  HFVG HFVH HFVI	Fetal Liver, subtraction II	pBS	LP03
  HNFI	Human Neutrophils,	pBS	LP03
  	Activated, re-excision
  HBMB HBMC HBMD	Human Bone Marrow, re-	pBS	LP03
  	excision
  HKML HKMM HKMN	H. Kidney Medulla, re-	pBS	LP03
  	excision
  HKIX HKIY	H. Kidney Cortex, subtracted	pBS	LP03
  HADT	H. Amygdala Depression,	pBS	LP03
  	subtracted
  H6AS	Hl-60, untreated, subtracted	Uni-ZAP XR	LP03
  H6ES	HL-60, PMA 4 H, subtracted	Uni-ZAP XR	LP03
  H6BS	HL-60, RA 4 h, Subtracted	Uni-ZAP XR	LP03
  H6CS	HL-60, PMA 1 d, subtracted	Uni-ZAP XR	LP03
  HTXJ HTXK	Activated T-	Uni-ZAP XR	LP03
  	cell(12 h)/Thiouridine-re-
  	excision
  HMSA HMSB HMSC HMSD	Monocyte activated	Uni-ZAP XR	LP03
  HMSE HMSF HMSG HMSH
  HMSI HMSJ HMSK
  HAGA HAGB HAGC HAGD	Human Amygdala	Uni-ZAP XR	LP03
  HAGE HAGF
  HSRA HSRB HSRE	STROMAL -	Uni-ZAP XR	LP03
  	OSTEOCLASTOMA
  HSRD HSRF HSRG HSRH	Human Osteoclastoma	Uni-ZAP XR	LP03
  	Stromal Cells - unamplified
  HSQA HSQB HSQC HSQD	Stromal cell TF274	Uni-ZAP XR	LP03
  HSQE HSQF HSQG
  HSKA HSKB HSKC HSKD	Smooth muscle, serum treated	Uni-ZAP XR	LP03
  HSKE HSKF HSKZ
  HSLA HSLB HSLC HSLD	Smooth muscle, control	Uni-ZAP XR	LP03
  HSLE HSLF HSLG
  HSDA HSDD HSDE HSDF	Spinal cord	Uni-ZAP XR	LP03
  HSDG HSDH
  HPWS	Prostate-BPH subtracted II	pBS	LP03
  HSKW HSKX HSKY	Smooth Muscle- HASTE	pBS	LP03
  	normalized
  HFPB HFPC HFPD	H. Frontal cortex, epileptic; re-	Uni-ZAP XR	LP03
  	excision
  HSDI HSDJ HSDK	Spinal Cord, re-excision	Uni-ZAP XR	LP03
  HSKN HSKO	Smooth Muscle Serum	pBS	LP03
  	Treated, Norm
  HSKG HSKH HSKI	Smooth muscle, serum	pBS	LP03
  	induced, re-exc
  HFCA HFCB HFCC HFCD	Human Fetal Brain	Uni-ZAP XR	LP04
  HFCE HFCF
  HPTA HPTB HPTD	Human Pituitary	Uni-ZAP XR	LP04
  HTHB HTHC HTHD	Human Thymus	Uni-ZAP XR	LP04
  HE6B HE6C HE6D HE6E	Human Whole Six Week Old	Uni-ZAP XR	LP04
  HE6F HE6G HE6S	Embryo
  HSSA HSSB HSSC HSSD	Human Synovial Sarcoma	Uni-ZAP XR	LP04
  HSSE HSSF HSSG HSSH
  HSSI HSSJ HSSK
  HE7T	7 Week Old Early Stage	Uni-ZAP XR	LP04
  	Human, subtracted
  HEPA HEPB HEPC	Human Epididymus	Uni-ZAP XR	LP04
  HSNA HSNB HSNC HSNM	Human Synovium	Uni-ZAP XR	LP04
  HSNN
  HPFB HPFC HPFD HPFE	Human Prostate Cancer,	Uni-ZAP XR	LP04
  	Stage C fraction
  HE2A HE2D HE2E HE2H	12 Week Old Early Stage	Uni-ZAP XR	LP04
  HE2I HE2M HE2N HE2O	Human
  HE2B HE2C HE2F HE2G	12 Week Old Early Stage	Uni-ZAP XR	LP04
  HE2P HE2Q	Human, II
  HPTS HPTT HPTU	Human Pituitary, subtracted	Uni-ZAP XR	LP04
  HAUA HAUB HAUC	Amniotic Cells - TNF	Uni-ZAP XR	LP04
  	induced
  HAQA HAQB HAQC HAQD	Amniotic Cells - Primary	Uni-ZAP XR	LP04
  	Culture
  HWTA HWTB HWTC	wilm's tumor	Uni-ZAP XR	LP04
  HBSD	Bone Cancer, re-excision	Uni-ZAP XR	LP04
  HSGB	Salivary gland, re-excision	Uni-ZAP XR	LP04
  HSJA HSJB HSJC	Smooth muscle-ILb induced	Uni-ZAP XR	LP04
  HSXA HSXB HSXC HSXD	Human Substantia Nigra	Uni-ZAP XR	LP04
  HSHA HSHB HSHC	Smooth muscle, IL1b induced	Uni-ZAP XR	LP04
  HOUA HOUB HOUC HOUD	Adipocytes	Uni-ZAP XR	LP04
  HOUE
  HPWA HPWB HPWC	Prostate BPH	Uni-ZAP XR	LP04
  HPWD HPWE
  HELA HELB HELC HELD	Endothelial cells-control	Uni-ZAP XR	LP04
  HELE HELF HELG HELH
  HEMA HEMB HEMC	Endothelial-induced	Uni-ZAP XR	LP04
  HEMD HEME HEMF HEMG
  HEMH
  HBIA HBIB HBIC	Human Brain, Striatum	Uni-ZAP XR	LP04
  HHSA HHSB HHSC HHSD	Human	Uni-ZAP XR	LP04
  HHSE	Hypothalmus, Schizophrenia
  HNGA HNGB HNGC HNGD	neutrophils control	Uni-ZAP XR	LP04
  HNGE HNGF HNGG HNGH
  HNGI HNGJ
  HNHA HNHB HNHC HNHD	Neutrophils IL-1 and LPS	Uni-ZAP XR	LP04
  HNHE HNHF HNHG HNHH	induced
  HNHI HNHJ
  HSDB HSDC	STRIATUM DEPRESSION	Uni-ZAP XR	LP04
  HHPT	Hypothalamus	Uni-ZAP XR	LP04
  HSAT HSAU HSAV HSAW	Anergic T-cell	Uni-ZAP XR	LP04
  HSAX HSAY HSAZ
  HBMS HBMT HBMU	Bone marrow	Uni-ZAP XR	LP04
  HBMV HBMW HBMX
  HOEA HOEB HOEC HOED	Osteoblasts	Uni-ZAP XR	LP04
  HOEE HOEF HOEJ
  HAIA HAIB HAIC HAID	Epithelial-TNFa and INF	Uni-ZAP XR	LP04
  HAIE HAIF	induced
  HTGA HTGB HTGC HTGD	Apoptotic T-cell	Uni-ZAP XR	LP04
  HMCA HMCB HMCC	Macrophage-oxLDL	Uni-ZAP XR	LP04
  HMCD HMCE
  HMAA HMAB HMAC	Macrophage (GM-CSF	Uni-ZAP XR	LP04
  HMAD HMAE HMAF	treated)
  HMAG
  HPHA	Normal Prostate	Uni-ZAP XR	LP04
  HPIA HPIB HPIC	LNCAP prostate cell line	Uni-ZAP XR	LP04
  HPJA HPJB HPJC	PC3 Prostate cell line	Uni-ZAP XR	LP04
  HOSE HOSF HOSG	Human Osteoclastoma, re-	Uni-ZAP XR	LP04
  	excision
  HTGE HTGF	Apoptotic T-cell, re-excision	Uni-ZAP XR	LP04
  HMAJ HMAK	H Macrophage (GM-CSF	Uni-ZAP XR	LP04
  	treated), re-excision
  HACB HACC HACD	Human Adipose Tissue, re-	Uni-ZAP XR	LP04
  	excision
  HFPA	H. Frontal Cortex, Epileptic	Uni-ZAP XR	LP04
  HFAA HFAB HFAC HFAD	Alzheimer's, spongy change	Uni-ZAP XR	LP04
  HFAE
  HFAM	Frontal Lobe, Dementia	Uni-ZAP XR	LP04
  HMIA HMIB HMIC	Human Manic Depression	Uni-ZAP XR	LP04
  	Tissue
  HTSA HTSE HTSF HTSG	Human Thymus	pBS	LP05
  HTSH
  HPBA HPBB HPBC HPBD	Human Pineal Gland	pBS	LP05
  HPBE
  HSAA HSAB HSAC	HSA 172 Cells	pBS	LP05
  HSBA HSBB HSBC HSBM	HSC172 cells	pBS	LP05
  HJAA HJAB HJAC HJAD	Jurkat T-cell G1 phase	pBS	LP05
  HJBA HJBB HJBC HJBD	Jurkat T-Cell, S phase	pBS	LP05
  HAFA HAFB	Aorta endothelial cells + TNF-a	pBS	LP05
  HAWA HAWB HAWC	Human White Adipose	pBS	LP05
  HTNA HTNB	Human Thyroid	pBS	LP05
  HONA	Normal Ovary,	pBS	LP05
  	Premenopausal
  HARA HARB	Human Adult Retina	pBS	LP05
  HLJA HLJB	Human Lung	pCMVSport 1	LP06
  HOFM HOFN HOFO	H. Ovarian Tumor, II,	pCMVSport 2.0	LP07
  	OV5232
  HOGA HOGB HOGC	OV 10-3-95	pCMVSport 2.0	LP07
  HCGL	CD34+cells, II	pCMVSport 2.0	LP07
  HDLA	Hodgkin's Lymphoma I	pCMVSport 2.0	LP07
  HDTA HDTB HDTC HDTD	Hodgkin's Lymphoma II	pCMVSport 2.0	LP07
  HDTE
  HKAA HKAB HKAC HKAD	Keratinocyte	pCMVSport 2.0	LP07
  HKAE HKAF HKAG HKAH
  HCIM	CAPFINDER, Crohn's	pCMVSport 2.0	LP07
  	Disease, lib 2
  HKAL	Keratinocyte, lib 2	pCMVSport 2.0	LP07
  HKAT	Keratinocyte, lib 3	pCMVSport 2.0	LP07
  HNDA	Nasal polyps	pCMVSport 2.0	LP07
  HDRA	H. Primary Dendritic Cells, lib 3	pCMVSport 2.0	LP07
  HOHA HOHB HOHC	Human Osteoblasts II	pCMVSport 2.0	LP07
  HLDA HLDB HLDC	Liver, Hepatoma	pCMVSport 3.0	LP08
  HLDN HLDO HLDP	Human Liver, normal	pCMVSport 3.0	LP08
  HMTA	pBMC stimulated w/ poly I/C	pCMVSport 3.0	LP08
  HNTA	NTERA2, control	pCMVSport 3.0	LP08
  HDPA HDPB HDPC HDPD	Primary Dendritic Cells, lib 1	pCMVSport 3.0	LP08
  HDPF HDPG HDPH HDPI
  HDPJ HDPK
  HDPM HDPN HDPO HDPP	Primary Dendritic cells, frac 2	pCMVSport 3.0	LP08
  HMUA HMUB HMUC	Myoloid Progenitor Cell Line	pCMVSport 3.0	LP08
  HHEA HHEB HHEC HHED	T Cell helper I	pCMVSport 3.0	LP08
  HHEM HHEN HHEO HHEP	T cell helper II	pCMVSport 3.0	LP08
  HEQA HEQB HEQC	Human endometrial stromal	pCMVSport 3.0	LP08
  	cells
  HJMA HJMB	Human endometrial stromal	pCMVSport 3.0	LP08
  	cells-treated with
  	progesterone
  HSWA HSWB HSWC	Human endometrial stromal	pCMVSport 3.0	LP08
  	cells-treated with estradiol
  HSYA HSYB HSYC	Human Thymus Stromal Cells	pCMVSport 3.0	LP08
  HLWA HLWB HLWC	Human Placenta	pCMVSport 3.0	LP08
  HRAA HRAB HRAC	Rejected Kidney, lib 4	pCMVSport 3.0	LP08
  HMTM	PCR, pBMC I/C treated	PCRII	LP09
  HMJA	H. Meniingima, M6	pSport 1	LP10
  HMKA HMKB HMKC	H. Meningima, M1	pSport 1	LP10
  HMKD HMKE
  HUSG HUSI	Human umbilical vein	pSport 1	LP10
  	endothelial cells, IL-4
  	induced
  HUSX HUSY	Human Umbilical Vein	pSport 1	LP10
  	Endothelial Cells, uninduced
  HOFA	Ovarian Tumor I, OV5232	pSport 1	LP10
  HCFA HCFB HCFC HCFD	T-Cell PHA 16 hrs	pSport 1	LP10
  HCFL HCFM HCFN HCFO	T-Cell PHA 24 hrs	pSport 1	LP10
  HADA HADC HADD HADE	Human Adipose	pSport 1	LP10
  HADF HADG
  HOVA HOVB HOVC	Human Ovary	pSport 1	LP10
  HTWB HTWC HTWD	Resting T-Cell Library, II	pSport 1	LP10
  HTWE HTWF
  HMMA	Spleen metastic melanoma	pSport 1	LP10
  HLYA HLYB HLYC HLYD	Spleen, Chronic lymphocytic	pSport 1	LP10
  HLYE	leukemia
  HCGA	CD34+ cell, I	pSport 1	LP10
  HEOM HEON	Human Eosinophils	pSport 1	LP10
  HTDA	Human Tonsil, Lib 3	pSport 1	LP10
  HSPA	Salivary Gland, Lib 2	pSport 1	LP10
  HCHA HCHB HCHC	Breast Cancer cell line, MDA	pSport 1	LP10
  	36
  HCHM HCHN	Breast Cancer Cell line,	pSport 1	LP10
  	angiogenic
  HCIA	Crohn's Disease	pSport 1	LP10
  HDAA HDAB HDAC	HEL cell line	pSport 1	LP10
  HABA	Human Astrocyte	pSport 1	LP10
  HUFA HUFB HUFC	Ulcerative Colitis	pSport 1	LP10
  HNTM	NTERA2 + retinoic acid, 14	pSport 1	LP10
  	days
  HDQA	Primary Dendritic	pSport 1	LP10
  	cells, CapFinder2, frac 1
  HDQM	Primary Dendritic Cells,	pSport 1	LP10
  	CapFinder, frac 2
  HLDX	Human Liver,	pSport 1	LP10
  	normal, CapFinder
  HULA HULB HULC	Human Dermal Endothelial	pSport 1	LP10
  	Cells, untreated
  HUMA	Human Dermal Endothelial	pSport 1	LP10
  	cells, treated
  HCJA	Human Stromal Endometrial	pSport 1	LP10
  	fibroblasts, untreated
  HCJM	Human Stromal endometrial	pSport 1	LP10
  	fibroblasts, treated w/
  	estradiol
  HEDA	Human Stromal endometrial	pSport 1	LP10
  	fibroblasts, treated with
  	progesterone
  HFNA	Human ovary tumor cell	pSport 1	LP10
  	OV350721
  HKGA HKGB HKGC HKGD	Merkel Cells	pSport 1	LP10
  HISA HISB HISC	Pancreas Islet Cell Tumor	pSport 1	LP10
  HLSA	Skin, burned	pSport 1	LP10
  HBZA	Prostate, BPH, Lib 2	pSport 1	LP10
  HBZS	Prostate BPH, Lib 2,	pSport 1	LP10
  	subtracted
  HFIA HFIB HFIC	Synovial Fibroblasts (control)	pSport 1	LP10
  HFIH HFII HFIJ	Synovial hypoxia	pSport 1	LP10
  HFIT HFIU HFIV	Synovial IL-1/TNF stimulated	pSport 1	LP10
  HGCA	Messangial cell, frac 1	pSport 1	LP10
  HMVA HMVB HMVC	Bone Marrow Stromal Cell,	pSport 1	LP10
  	untreated
  HFIX HFIY HFIZ	Synovial Fibroblasts	pSport 1	LP10
  	(Il1/TNF), subt
  HFOX HFOY HFOZ	Synovial hypoxia-RSF	pSport 1	LP10
  	subtracted
  HMQA HMQB HMQC	Human Activated Monocytes	Uni-ZAP XR	LP11
  HMQD
  HLIA HLIB HLIC	Human Liver	pCMVSport 1	LP012
  HHBA HHBB HHBC HHBD	Human Heart	pCMVSport 1	LP012
  HHBE
  HBBA HBBB	Human Brain	pCMVSport 1	LP012
  HLJA HLJB HLJC HLJD	Human Lung	pCMVSport 1	LP012
  HLJE
  HOGA HOGB HOGC	Ovarian Tumor	pCMVSport 2.0	LP012
  HTJM	Human Tonsils, Lib 2	pCMVSport 2.0	LP012
  HAMF HAMG	KMH2	pCMVSport 3.0	LP012
  HAJA HAJB HAJC	L428	pCMVSport 3.0	LP012
  HWBA HWBB HWBC	Dendritic cells, pooled	pCMVSport 3.0	LP012
  HWBD HWBE
  HWAA HWAB HWAC	Human Bone Marrow,	pCMVSport 3.0	LP012
  HWAD HWAE	treated
  HYAA HYAB HYAC	B Cell lymphoma	pCMVSport 3.0	LP012
  HWHG HWHH HWHI	Healing groin wound, 6.5	pCMVSport 3.0	LP012
  	hours post incision
  HWHP HWHQ HWHR	Healing groin wound; 7.5	pCMVSport 3.0	LP012
  	hours post incision
  HARM	Healing groin wound - zero hr	pCMVSport 3.0	LP012
  	post-incision (control)
  HBIM	Olfactory epithelium;	pCMVSport 3.0	LP012
  	nasalcavity
  HWDA	Healing Abdomen wound;	pCMVSport 3.0	LP012
  	70&90 min post incision
  HWEA	Healing Abdomen Wound; 15	pCMVSport 3.0	LP012
  	days post incision
  HWJA	Healing Abdomen	pCMVSport 3.0	LP012
  	Wound; 21&29 days
  HNAL	Human Tongue, frac 2	pSport 1	LP012
  HMJA	H. Meniingima, M6	pSport 1	LP012
  HMKA HMKB HMKC	H. Meningima, M1	pSport 1	LP012
  HMKD HMKE
  HOFA	Ovarian Tumor I, OV5232	pSport 1	LP012
  HCFA HCFB HCFC HCFD	T-Cell PHA 16 hrs	pSport 1	LP012
  HCFL HCFM HCFN HCFO	T-Cell PHA 24 hrs	pSport 1	LP012
  HMMA HMMB HMMC	Spleen metastic melanoma	pSport 1	LP012
  HTDA	Human Tonsil, Lib 3	pSport 1	LP012
  HDBA	Human Fetal Thymus	pSport 1	LP012
  HDUA	Pericardium	pSport 1	LP012
  HBZA	Prostate, BPH, Lib 2	pSport 1	LP012
  HWCA	Larynx tumor	pSport 1	LP012
  HWKA	Normal lung	pSport 1	LP012
  HSMB	Bone marrow stroma, treated	pSport 1	LP012
  HBHM	Normal trachea	pSport 1	LP012
  HLFC	Human Larynx	pSport 1	LP012
  HLRB	Siebben Polyposis	pSport 1	LP012
  HNIA	Mammary Gland	pSport 1	LP012
  HNJB	Palate carcinoma	pSport 1	LP012
  HNKA	Palate normal	pSport 1	LP012
  HMZA	Pharynx carcinoma	pSport 1	LP012
  HABG	Cheek Carcinoma	pSport 1	LP012
  HMZM	Pharynx Carcinoma	pSport 1	LP012
  HDRM	Larynx Carcinoma	pSport 1	LP012
  HVAA	Pancreas normal PCA4 No	pSport 1	LP012
  HICA	Tongue carcinoma	pSport 1	LP012
  HUKA HUKB HUKC HUKD	Human Uterine Cancer	Lambda ZAP II	LP013
  HUKE
  HFFA	Human Fetal Brain, random	Lambda ZAP II	LP013
  	primed
  HTUA	Activated T-cell labeled with	Lambda ZAP II	LP013
  	4-thioluri
  HBQA	Early Stage Human Brain,	Lambda ZAP II	LP013
  	random primed
  HMEB	Human microvascular	Lambda ZAP II	LP013
  	Endothelial cells, fract. B
  HUSH	Human Umbilical Vein	Lambda ZAP II	LP013
  	Endothelial cells, fract. A, re-
  	excision
  HLQC HLQD	Hepatocellular tumor, re-	Lambda ZAP II	LP013
  	excision
  HTWJ HTWK HTWL	Resting T-cell, re-excision	Lambda ZAP II	LP013
  HF6S	Human Whole 6 week Old	pBluescript	LP013
  	Embryo (II), subt
  HHPS	Human Hippocampus,	pBluescript	LP013
  	subtracted
  HL1S	LNCAP, differential	pBluescript	LP013
  	expression
  HLHS HLHT	Early Stage Human Lung,	pBluescript	LP013
  	Subtracted
  HSUS	Supt cells, cyclohexamide	pBluescript	LP013
  	treated, subtracted
  HSUT	Supt cells, cyclohexamide	pBluescript	LP013
  	treated, differentially
  	expressed
  HSDS	H. Striatum Depression,	pBluescript	LP013
  	subtracted
  HPTZ	Human Pituitary, Subtracted	pBluescript	LP013
  	VII
  HSDX	H. Striatum Depression, subt	pBluescript	LP013
  	II
  HSDZ	H. Striatum Depression, subt	pBluescript	LP013
  HPBA HPBB HPBC HPBD	Human Pineal Gland	pBluescript SK−	LP013
  HPBE
  HRTA	Colorectal Tumor	pBluescript SK−	LP013
  HSBA HSBB HSBC HSBM	HSC172 cells	pBluescript SK−	LP013
  HJAA HJAB HJAC HJAD	Jurkat T-cell G1 phase	pBluescript SK−	LP013
  HJBA HJBB HJBC HJBD	Jurkat T-cell, S1 phase	pBluescript SK−	LP013
  HTNA HTNB	Human Thyroid	pBluescript SK−	LP013
  HAHA HAHB	Human Adult Heart	Uni-ZAP XR	LP013
  HE6A	Whole 6 week Old Embryo	Uni-ZAP XR	LP013
  HFCA HFCB HFCC HFCD	Human Fetal Brain	Uni-ZAP XR	LP013
  HFCE
  HFKC HFKD HFKE HFKF	Human Fetal Kidney	Uni-ZAP XR	LP013
  HFKG
  HGBA HGBD HGBE HGBF	Human Gall Bladder	Uni-ZAP XR	LP013
  HGBG
  HPRA HPRB HPRC HPRD	Human Prostate	Uni-ZAP XR	LP013
  HTEA HTEB HTEC HTED	Human Testes	Uni-ZAP XR	LP013
  HTEE
  HTTA HTTB HTTC HTTD	Human Testes Tumor	Uni-ZAP XR	LP013
  HTTE
  HYBA HYBB	Human Fetal Bone	Uni-ZAP XR	LP013
  HFLA	Human Fetal Liver	Uni-ZAP XR	LP013
  HHFB HHFC HHFD HHFE	Human Fetal Heart	Uni-ZAP XR	LP013
  HHFF
  HUVB HUVC HUVD HUVE	Human Umbilical Vein, End.	Uni-ZAP XR	LP013
  	remake
  HTHB HTHC HTHD	Human Thymus	Uni-ZAP XR	LP013
  HSTA HSTB HSTC HSTD	Human Skin Tumor	Uni-ZAP XR	LP013
  HTAA HTAB HTAC HTAD	Human Activated T-cells	Uni-ZAP XR	LP013
  HTAE
  HFEA HFEB HFEC	Human Fetal Epithelium	Uni-ZAP XR	LP013
  	(skin)
  HJPA HJPB HJPC HJPD	Human Jurkat Membrane	Uni-ZAP XR	LP013
  	Bound Polysomes
  HESA	Human Epithelioid Sarcoma	Uni-ZAP XR	LP013
  HALS	Human Adult Liver,	Uni-ZAP XR	LP013
  	Subtracted
  HFTA HFTB HFTC HFTD	Human Fetal Dura Mater	Uni-ZAP XR	LP013
  HCAA HCAB HCAC	Cem cells, cyclohexamide	Uni-ZAP XR	LP013
  	treated
  HRGA HRGB HRGC HRGD	Raji Cells, cyclohexamide	Uni-ZAP XR	LP013
  	treated
  HE9A HE9B HE9C HE9D	Nine Week Old Early Stage	Uni-ZAP XR	LP013
  HE9E	Human
  HSFA	Human Fibrosarcoma	Uni-ZAP XR	LP013
  HATA HATB HATC HATD	Human Adrenal Gland Tumor	Uni-ZAP XR	LP013
  HATE
  HTRA	Human Trachea Tumor	Uni-ZAP XR	LP013
  HE2A HE2D HE2E HE2H	12 Week Old Early Stage	Uni-ZAP XR	LP013
  HE2I	Human
  HE2B HE2C HE2F HE2G	12 Week Old Early Stage	Uni-ZAP XR	LP013
  HE2P	Human, II
  HNEA HNEB HNEC HNED	Human Neutrophil	Uni-ZAP XR	LP013
  HNEE
  HBGA	Human Primary Breast	Uni-ZAP XR	LP013
  	Cancer
  HPTS HPTT HPTU	Human Pituitary, subtracted	Uni-ZAP XR	LP013
  HMQA HMQB HMQC	Human Activated Monocytes	Uni-ZAP XR	LP013
  HMQD
  HOAA HOAB HOAC	Human Osteosarcoma	Uni-ZAP XR	LP013
  HTOA HTOD HTOE HTOF	human tonsils	Uni-ZAP XR	LP013
  HTOG
  HMGB	Human OB MG63 control	Uni-ZAP XR	LP013
  	fraction I
  HOPB	Human OB HOS control	Uni-ZAP XR	LP013
  	fraction I
  HOQB	Human OB HOS treated (1 nM	Uni-ZAP XR	LP013
  	E2) fraction I
  HAUA HAUB HAUC	Amniotic Cells - TNF	Uni-ZAP XR	LP013
  	induced
  HAQA HAQB HAQC HAQD	Amniotic Cells - Primary	Uni-ZAP XR	LP013
  	Culture
  HROA HROC	HUMAN STOMACH	Uni-ZAP XR	LP013
  HBJA HBJB HBJC HBJD	HUMAN B CELL	Uni-ZAP XR	LP013
  HBJE	LYMPHOMA
  HODA HODB HODC HODD	human ovarian cancer	Uni-ZAP XR	LP013
  HCPA	Corpus Callosum	Uni-ZAP XR	LP013
  HSOA	stomach cancer (human)	Uni-ZAP XR	LP013
  HERA	SKIN	Uni-ZAP XR	LP013
  HMDA	Brain-medulloblastoma	Uni-ZAP XR	LP013
  HGLA HGLB HGLD	Glioblastoma	Uni-ZAP XR	LP013
  HWTA HWTB HWTC	wilm's tumor	Uni-ZAP XR	LP013
  HEAA	H. Atrophic Endometrium	Uni-ZAP XR	LP013
  HAPN HAPO HAPP HAPQ	Human Adult Pulmonary; re-	Uni-ZAP XR	LP013
  HAPR	excision
  HLTG HLTH	Human T-cell lymphoma; re-	Uni-ZAP XR	LP013
  	excision
  HAHC HAHD HAHE	Human Adult Heart; re-	Uni-ZAP XR	LP013
  	excision
  HAGA HAGB HAGC HAGD	Human Amygdala	Uni-ZAP XR	LP013
  HAGE
  HSJA HSJB HSJC	Smooth muscle-ILb induced	Uni-ZAP XR	LP013
  HSHA HSHB HSHC	Smooth muscle, IL1b induced	Uni-ZAP XR	LP013
  HPWA HPWB HPWC	Prostate BPH	Uni-ZAP XR	LP013
  HPWD HPWE
  HPIA HPIB HPIC	LNCAP prostate cell line	Uni-ZAP XR	LP013
  HPJA HPJB HPJC	PC3 Prostate cell line	Uni-ZAP XR	LP013
  HBTA	Bone Marrow Stroma,	Uni-ZAP XR	LP013
  	TNF&LPS ind
  HMCF HMCG HMCH HMCI	Macrophage-oxLDL; re-	Uni-ZAP XR	LP013
  HMCJ	excision
  HAGG HAGH HAGI	Human Amygdala; re-excision	Uni-ZAP XR	LP013
  HACA	H. Adipose Tissue	Uni-ZAP XR	LP013
  HKFB	K562 + PMA (36 hrs), re-	ZAP Express	LP013
  	excision
  HCWT HCWU HCWV	CD34 positive cells (cord	ZAP Express	LP013
  	blood), re-ex
  HBWA	Whole brain	ZAP Express	LP013
  HBXA HBXB HBXC HBXD	Human Whole Brain #2 -	ZAP Express	LP013
  	Oligo dT >1.5 Kb
  HAVM	Temporal cortex-Alzheizmer	pT-Adv	LP014
  HAVT	Hippocampus, Alzheimer	pT-Adv	LP014
  	Subtracted
  HHAS	CHME Cell Line	Uni-ZAP XR	LP014
  HAJR	Larynx normal	pSport 1	LP014
  HWLE HWLF HWLG	Colon Normal	pSport 1	LP014
  HWLH
  HCRM HCRN HCRO	Colon Carcinoma	pSport 1	LP014
  HWLI HWLJ HWLK	Colon Normal	pSport 1	LP014
  HWLQ HWLR HWLS	Colon Tumor	pSport 1	LP014
  HWLT
  HBFM	Gastrocnemius Muscle	pSport 1	LP014
  HBOD HBOE	Quadriceps Muscle	pSport 1	LP014
  HBKD HBKE	Soleus Muscle	pSport 1	LP014
  HCCM	Pancreatic Langerhans	pSport 1	LP014
  HWGA	Larynx carcinoma	pSport 1	LP014
  HWGM HWGN	Larynx carcinoma	pSport 1	LP014
  HWLA HWLB HWLC	Normal colon	pSport 1	LP014
  HWLM HWLN	Colon Tumor	pSport 1	LP014
  HVAM HVAN HVAO	Pancreas Tumor	pSport 1	LP014
  HWGQ	Larynx carcinoma	pSport 1	LP014
  HAQM HAQN	Salivary Gland	pSport 1	LP014
  HASM	Stomach; normal	pSport 1	LP014
  HBCM	Uterus; normal	pSport 1	LP014
  HCDM	Testis; normal	pSport 1	LP014
  HDJM	Brain; normal	pSport 1	LP014
  HEFM	Adrenal Gland, normal	pSport 1	LP014
  HBAA	Rectum normal	pSport 1	LP014
  HFDM	Rectum tumour	pSport 1	LP014
  HGAM	Colon, normal	pSport 1	LP014
  HHMM	Colon, tumour	pSport 1	LP014
  HCLB HCLC	Human Lung Cancer	Lambda Zap II	LP015
  HRLA	L1 Cell line	ZAP Express	LP015
  HHAM	Hypothalamus, Alzheimer's	pCMVSport 3.0	LP015
  HKBA	Ku 812F Basophils Line	pSport 1	LP015
  HS2S	Saos2, Dexamethosome	pSport 1	LP016
  	Treated
  HA5A	Lung Carcinoma A549	pSport 1	LP016
  	TNFalpha activated
  HTFM	TF-1 Cell Line GM-CSF	pSport 1	LP016
  	Treated
  HYAS	Thyroid Tumour	pSport 1	LP016
  HUTS	Larynx Normal	pSport 1	LP016
  HXOA	Larynx Tumor	pSport 1	LP016
  HEAH	Ea.hy.926 cell line	pSport 1	LP016
  HINA	Adenocarcinoma Human	pSport 1	LP016
  HRMA	Lung Mesothelium	pSport 1	LP016
  HLCL	Human Pre-Differentiated	Uni-Zap XR	LP017
  	Adipocytes
  HS2A	Saos2 Cells	pSport 1	LP020
  HS2I	Saos2 Cells; Vitamin D3	pSport 1	LP020
  	Treated
  HUCM	CHME Cell Line, untreated	pSport 1	LP020
  HEPN	Aryepiglottis Normal	pSport 1	LP020
  HPSN	Sinus Piniformis Tumour	pSport 1	LP020
  HNSA	Stomach Normal	pSport 1	LP020
  HNSM	Stomach Tumour	pSport 1	LP020
  HNLA	Liver Normal Met5No	pSport 1	LP020
  HUTA	Liver Tumour Met 5 Tu	pSport 1	LP020
  HOCN	Colon Normal	pSport 1	LP020
  HOCT	Colon Tumor	pSport 1	LP020
  HTNT	Tongue Tumour	pSport 1	LP020
  HLXN	Larynx Normal	pSport 1	LP020
  HLXT	Larynx Tumour	pSport 1	LP020
  HTYN	Thymus	pSport 1	LP020
  HPLN	Placenta	pSport 1	LP020
  HTNG	Tongue Normal	pSport 1	LP020
  HZAA	Thyroid Normal (SDCA2 No)	pSport 1	LP020
  HWES	Thyroid Thyroiditis	pSport 1	LP020
  HFHD	Ficolled Human Stromal	pTrip1Ex2	LP021
  	Cells, 5Fu treated
  HFHM, HFHN	Ficolled Human Stromal	pTrip1Ex2	LP021
  	Cells, Untreated
  HPCI	Hep G2 Cells, lambda library	lambda Zap-CMV	LP021
  		XR
  HBCA, HBCB, HBCC	H. Lymph node breast Cancer	Uni-ZAP XR	LP021
  HCOK	Chondrocytes	pSPORT1	LP022
  HDCA, HDCB, HDCC	Dendritic Cells From CD34	pSPORT1	LP022
  	Cells
  HDMA, HDMB	CD40 activated monocyte	pSPORT1	LP022
  	dendritic cells
  HDDM, HDDN, HDDO	LPS activated derived	pSPORT1	LP022
  	dendritic cells
  HPCR	Hep G2 Cells, PCR library	lambda Zap-CMV	LP022
  		XR
  HAAA, HAAB, HAAC	Lung, Cancer (4005313A3):	pSPORT1	LP022
  	Invasive Poorly Differentiated
  	Lung Adenocarcinoma
  HIPA, HIPB, HIPC	Lung, Cancer (4005163 B7):	pSPORT1	LP022
  	Invasive, Poorly Diff.
  	Adenocarcinoma, Metastatic
  HOOH, HOOI	Ovary, Cancer: (4004562 B6)	pSPORT1	LP022
  	Papillary Serous Cystic
  	Neoplasm, Low Malignant
  	Pot
  HIDA	Lung, Normal: (4005313 B1)	pSPORT1	LP022
  HUJA, HUJB, HUJC, HUJD, HUJE	B-Cells	pCMVSport 3.0	LP022
  HNOA, HNOB, HNOC, HNOD	Ovary, Normal: (9805C040R)	pSPORT1	LP022
  HNLM	Lung, Normal: (4005313 B1)	pSPORT1	LP022
  HSCL	Stromal Cells	pSPORT1	LP022
  HAAX	Lung, Cancer: (4005313 A3)	pSPORT1	LP022
  	Invasive Poorly-differentiated
  	Metastatic lung
  	adenocarcinoma
  HUUA, HUUB, HUUC, HUUD	B-cells (unstimulated)	pTrip1Ex2	LP022
  HWWA, HWWB, HWWC, HWWD,	B-cells (stimulated)	pSPORT1	LP022
  HWWE, HWWF, HWWG
  HCCC	Colon, Cancer: (9808C064R)	pCMVSport 3.0	LP023
  HPDO HPDP HPDQ HPDR	Ovary, Cancer (9809C332):	pSport 1	LP023
  HPD	Poorly differentiated
  	adenocarcinoma
  HPCO HPCP HPCQ HPCT	Ovary, Cancer (15395A1F):	pSport 1	LP023
  	Grade II Papillary Carcinoma
  HOCM HOCO HOCP HOCQ	Ovary, Cancer: (15799A1F)	pSport 1	LP023
  	Poorly differentiated
  	carcinoma
  HCBM HCBN HCBO	Breast, Cancer: (4004943 A5)	pSport 1	LP023
  HNBT HNBU HNBV	Breast, Normal: (4005522B2)	pSport 1	LP023
  HBCP HBCQ	Breast, Cancer: (4005522 A2)	pSport 1	LP023
  HBCJ	Breast, Cancer: (9806C012R)	pSport 1	LP023
  HSAM HSAN	Stromal cells 3.88	pSport 1	LP023
  HVCA HVCB HVCC HVCD	Ovary, Cancer: (4004332 A2)	pSport 1	LP023
  HSCK HSEN HSEO	Stromal cells (HBM3.18)	pSport 1	LP023
  HSCP HSCQ	stromal cell clone 2.5	pSport 1	LP023
  HUXA	Breast Cancer: (4005385 A2)	pSport 1	LP023
  HCOM HCON HCOO HCOP	Ovary, Cancer (4004650 A3):	pSport 1	LP023
  HCOQ	Well-Differentiated
  	Micropapillary Serous
  	Carcinoma
  HBNM	Breast, Cancer: (9802C020E)	pSport 1	LP023
  HVVA HVVB HVVC HVVD	Human Bone Marrow, treated	pSport 1	LP023
  HVVE

• Two nonlimiting examples are provided below for isolating a particular clone from the deposited sample of plasmid cDNAs cited for that clone in Table 7. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to the nucleotide sequence of SEQ ID NO:X.
• Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with ³²P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (Stratagene)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.
• Alternatively, two primers of 17-20 nucleotides derived from both ends of the nucleotide sequence of SEQ ID NO:X are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.
• Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).
• Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.
• This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
• This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.
Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide
• A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the sequence corresponding to SEQ ID NO:X according to the method described in Example 1. (See also, Sambrook.)
Example 3 Tissue Specific Expression Analysis
• The Human Genome Sciences, Inc. (HGS) database is derived from sequencing tissue and/or disease specific cDNA libraries. Libraries generated from a particular tissue are selected and the specific tissue expression pattern of EST groups or assembled contigs within these libraries is determined by comparison of the expression patterns of those groups or contigs within the entire database. ESTs and assembled contigs which show tissue specific expression are selected.
• The original clone from which the specific EST sequence was generated, or in the case of an assembled contig, the clone from which the 5′ most EST sequence was generated, is obtained from the catalogued library of clones and the insert amplified by PCR using methods known in the art. The PCR product is denatured and then transferred in 96 or 384 well format to a nylon membrane (Schleicher and Scheull) generating an array filter of tissue specific clones. Housekeeping genes, maize genes, and known tissue specific genes are included on the filters. These targets can be used in signal normalization and to validate assay sensitivity. Additional targets are included to monitor probe length and specificity of hybridization.
• Radioactively labeled hybridization probes are generated by first strand cDNA synthesis per the manufacturer's instructions (Life Technologies) from mRNA/RNA samples prepared from the specific tissue being analyzed (e.g., prostate, prostate cancer, ovarian, ovarian cancer, etc.). The hybridization probes are purified by gel exclusion chromatography, quantitated, and hybridized with the array filters in hybridization bottles at 65° C. overnight. The filters are washed under stringent conditions and signals are captured using a Fuji phosphorimager.
• Data is extracted using AIS software and following background subtraction, signal normalization is performed. This includes a normalization of filter-wide expression levels between different experimental runs. Genes that are differentially expressed in the tissue of interest are identified.
Example 4 Chromosomal Mapping of the Polynucleotides
• An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95° C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 bp PCR fragment in the particular somatic cell hybrid.
Example 5 Bacterial Expression of a Polypeptide
• A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Amp^r), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
• The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan^r). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
• Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.
• Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4° C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
• Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
• The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4° C. or frozen at −80° C.
• In addition to the above expression vector, the present invention further includes an expression vector, called pHE4a (ATCC Accession Number 209645, deposited on Feb. 25, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgamo sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter and operator sequences are made synthetically.
• DNA can be inserted into the pHE4a by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.
• The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.
Example 6 Purification of a Polypeptide from an Inclusion Body
• The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10° C.
• Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10° C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
• The cells are then lysed by passing the solution through a microfluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
• The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4° C. overnight to allow further GuHCl extraction.
• Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4° C. without mixing for 12 hours prior to further purification steps.
• To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 μm membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
• Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀ monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
• The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 7 Cloning and Expression of a Polypeptide in a Baculovirus Expression System
• In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
• Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
• Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon, is amplified using the PCR protocol described in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
• The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
• The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
• The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
• Five μg of a plasmid containing the polynucleotide is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of the plasmid are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27° C. for four days.
• After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C.
• To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
• Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.
Example 8 Expression of a Polypeptide in Mammalian Cells
• The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
• Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CVI, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
• Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.
• The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
• Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), the expression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCC Accession No. 209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.
• Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.
• A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)
• The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
• The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
• Chinese hamster ovary cells lacking an active DHFR gene is used for transfection. Five μg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 n™, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 9 Protein Fusions
• The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.
• Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.
• For example, if pC4 (ATCC Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
• If the naturally occurring signal sequence is used to produce the polypeptide of the present invention, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)

  Human IgG Fc region:

  GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACAC	(SEQ ID NO:1)
  ATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCA
  CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACA
  CCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGT
  GGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAG
  TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
  CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
  GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG
  GACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCT
  CCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCAT
  CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
  TACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA
  ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA
  TCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG
  CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC
  TGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
  CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
  TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
  ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA
  ATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Production of an Antibody from a Polypeptide
• a) Hybridoma Technology
• The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of a polypeptide of the present invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
• Monoclonal antibodies specific for a polypeptide of the present invention are prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with a polypeptide of the present invention or, more preferably, with a secreted polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.
• The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide of the present invention.
• Alternatively, additional antibodies capable of binding to a polypeptide of the present invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the polypeptide-specific antibody can be blocked by said polypeptide. Such antibodies comprise anti-idiotypic antibodies to the polypeptide-specific antibody and are used to immunize an animal to induce formation of further polypeptide-specific antibodies.
• For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).
• B) Isolation of Antibody Fragments Directed Against a Polypeptide of the Present Invention from a Library of scFvs
• Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against a polypeptide of the present invention to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).
• Rescue of the Library. A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage displaying antibody fragments, approximately 10⁹ E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see International Publication No. WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are prepared as described in International Publication No. WO 92/01047.
• M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage (mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanarnycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 10¹³ transducing units/ml (ampicillin-resistant clones).
• Panning of the Library. Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 10¹³ TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 11.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
• Characterization of Binders. Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., International Publication No. WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
Example 11 Method of Determining Alterations in a Gene Corresponding to a Polynucleotide
• RNA isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease) is isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X; and/or the nucleotide sequence of the cDNA contained in ATCC Deposit No: Z. Suggested PCR conditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120 seconds at 70 degrees C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).
• PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase (Epicentre Technologies). The intron-exon boundaries of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of the direct sequencing.
• PCR products are cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
• Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.
• Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991)). Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.
Example 12 Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample
• A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
• For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.
• The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbound polypeptide.
• Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbound conjugate.
• Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.
Example 13 Formulation
• The invention also provides methods of treatment and/or prevention of diseases or disorders (such as, for example, any one or more of the diseases or disorders disclosed herein) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).
• The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.
• As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.
• Therapeutics can be are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).
• Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).
• In a preferred embodiment, polypeptide, polynucleotide, and antibody compositions of the invention are formulated in a biodegradable, polymeric drug delivery system, for example as described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and 5,487,897 and in International Publication Numbers WO01/35929, WO00/24374, and WO00/06117 which are hereby incorporated by reference in their entirety. In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).
• Examples of biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions, include but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials. The preferred polymers are those that have a lower degree of crystallization and are more hydrophobic. These polymers and copolymers are more soluble in the biocompatible solvents than the highly crystalline polymers such as polyglycolide and chitin which also have a high degree of hydrogen-bonding. Preferred materials with the desired solubility parameters are the polylactides, polycaprolactones, and copolymers of these with glycolide in which there are more amorphous regions to enhance solubility. In specific preferred embodiments, the biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions are poly(lactide-co-glycolides). Polymer properties such as molecular weight, hydrophobicity, and lactide/glycolide ratio may be modified to obtain the desired polypeptide, polynucleotide, or antibody release profile (See, e.g., Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000), which is hereby incorporated by reference in its entirety).
• It is also preferred that the solvent for the biodegradable polymer be non-toxic, water miscible, and otherwise biocompatible. Examples of such solvents include, but are not limited to, N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, and tetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15 alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone; C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate; alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such as dimethylformamide, dimethylacetamide and caprolactam; C3 to C20 ethers such as tetrahydrofuran, or solketal; tweens, triacetin, propylene carbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid, 1-dodecylazacycloheptan-2-one, Other preferred solvents are benzyl alchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate, glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate, and triethyl citrate. The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin, and propylene carbonate because of the solvating ability and their compatibility.
• Additionally, formulations comprising polypeptide, polynucleotide, and antibody compositions and a biodegradable polymer may also include release-rate modification agents and/or pore-forming agents. Examples of release-rate modification agents include, but are not limited to, fatty acids, triglycerides, other like hydrophobic compounds, organic solvents, plasticizing compounds and hydrophilic compounds. Suitable release rate modification agents include, for example, esters of mono-, di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and the like; polyhydroxy alcohols, such as propylene glycol, polyethylene glycol, glycerin, sorbitol, and the like; fatty acids; triesters of glycerol, such as triglycerides, epoxidized soybean oil, and other epoxidized vegetable oils; sterols, such as cholesterol; alcohols, such as C.sub.6-C.sub.12 alkanols, 2-ethoxyethanol. The release rate modification agent may be used singly or in combination with other such agents. Suitable combinations of release rate modification agents include, but are not limited to, glycerin/propylene glycol, sorbitol/glycerine, ethylene oxide/propylene oxide, butylene glycol/adipic acid, and the like. Preferred release rate modification agents include, but are not limited to, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitable pore-forming agents that may be used in the polymer composition include, but are not limited to, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, polymers such as hydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone. Solid crystals that will provide a defined pore size, such as salt or sugar, are preferred.
• In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the BEMA™ BioErodible Mucoadhesive System, MCA™ MucoCutaneous Absorption System, SMP™ Solvent MicroParticle System, or BCP™ BioCompatible Polymer System of Atrix Laboratories, Inc. (Fort Collins, Colo.).
• Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.
• In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
• Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).
• For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.
• Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.
• The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
• The Therapeutic is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.
• Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
• Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.
• The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.
• The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (e.g., THERACYS®), MPL and nonviable prepartions of Corynebacterium parvum. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
• The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
• In one embodiment, the Therapeutics of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADON™), acenocoumarol (e.g., nicoumalone, SINTHROME™), indan-1,3-dione, phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.
• In another embodiment, the Therapeutics of the invention are administered in combination with thrombolytic drugs. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KABIKINASE™), antiresplace (e.g., EMINASE™), tissue plasminogen activator (t-PA, altevase, ACTIVASE™), urokinase (e.g., ABBOKINASE™), sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICAR™). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.
• In another embodiment, the Therapeutics of the invention are administered in combination with antiplatelet drugs. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINE™), and ticlopidine (e.g., TICLID™).
• In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the prevention, diagnosis, and/or treatment of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).
• In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (larnivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VICEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.
• Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL™ (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/Abbott); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/Abbott); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN™ (abacavir/159 U89; Glaxo Wellcome Inc.); CS-87 (3′azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).
• Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE™ (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation, Agouron); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; Pharmacia & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DuPont); GW-420867× (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).
• Additional protease inhibitors include LOPINAVIR™ (ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide; Bristol-Myres Squibb); TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; Pharmacia & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; Agouron); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Welcome); CGP61755 (Ciba); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).
• Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).
• Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibit fusion.
• Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIR™ (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.
• Additional antiretroviral agents include hydroxyurea-like compounds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors sucha as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; Roche).
• Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.
• Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™ (aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).
• In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
• In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.
• In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.
• In other embodiments, the Therapeutics of the invention are administered in combination with immunestimulants. Immunostimulants that may be administered in combination with the Therapeutics of the invention include, but are not limited to, levamisole (e.g., ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).
• In other embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
• In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
• In certain embodiments, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.
• In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (Entremed, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.
• Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
• Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
• Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
• A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.
• Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (Celgene, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J. Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM11; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and 5-Fluorouracil.
• Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons of the invention include, but are not lmited to, AG-3340 (Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.), BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A (Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and Metastat (Aetema, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not limited to, EMD-121974 (Merck KcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons of the invention include, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (Genentech, S. San Francisco, Calif.), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. San Francisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.), and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (Cytran, Kirkland, Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).
• In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.
• In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.
• In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
• In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).
• In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as Remicade™ Centocor, Inc.), Trocade (Roche, RO-32-3555), Leflunomide (also known as Arava™ from Hoechst Marion Roussel), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from Amgen, Inc.)
• In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.
• In another specific embodiment, the compositions of the invention are administered in combination Zevalin™. In a further embodiment, compositions of the invention are administered with Zevalin™ and CHOP, or Zevalin™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. Zevalin™ may be associated with one or more radisotopes. Particularly preferred isotopes are ⁹⁰Y and ¹¹¹In.
• In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines. Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.
• In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.
• In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.
• In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
• In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINE™, PROKINE™), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of IL-1 through IL-12, interferon-gamma, or thrombopoietin.
• In certain embodiments, Therapeutics of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.
• In another embodiment, the Therapeutics of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecainide, lidocaine, mexiletine, moricizine, phenyloin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocainide, and verapamil).
• In another embodiment, the Therapeutics of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na⁺—K⁺-2Cl⁻ symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).
• In one embodiment, the Therapeutics of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, ¹²⁷I, radioactive isotopes of iodine such as ¹³¹I and ¹²³¹I; recombinant growth hormone, such as HUMATROPE™ (recombinant somatropin); growth hormone analogs such as PROTROPIN™ (somatrem); dopamine agonists such as PARLODEL™ (bromocriptine); somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropin preparations such as PREGNYL™, A.P.L.™ and PROFASI™ (chorionic gonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™ (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTREL™ and LUTREPULSE™ (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™ (nafarelin acetate), and ZOLADEX™ (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRH™ and THYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™ (levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroine sodium), and THYROLAR™ (liotrix); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodium ipodate).
• Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congugated estrogens such as ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol), PREMARIN™, ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™ (quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™ (estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™ (estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen), SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™ (hydroxyprogesterone caproate), MPA™ and DEPO-PROVERA™ (medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™ (megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ and AYGESTIN™ (norethindrone acetate); progesterone implants such as NORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins such as RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™ (norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device that releases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™, NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinyl estradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ and TRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™ (ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodiol diacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™ (norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinyl estradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinyl estradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), and OVRETTE™ (norgestrel).
• Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate), DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosterone cypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETON METHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™ (oxandrolone); testosterone transdermal systems such as TESTODERM™; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™ (finasteride); adrenocorticotropic hormone preparations such as CORTROSYN™ (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™ (amcinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate), CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasone benzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™ (betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasone sodium phosphate and acetate), BETA-VAL™ and VALISONE™ (betamethasone valerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolone pivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)), HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™ (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™ (cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol (hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate), DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone), DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate), DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodium phosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEF ACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™ (flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™ (fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™ (flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone), MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™ (methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™ (methylprednisolone sodium succinate), ELOCON™ (mometasone furoate), HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone), ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodium phosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™ (prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™ (triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™ (triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™ (trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ and TOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide, MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide), and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATIN™ (octreotide); and diazoxides such as PROGLYCEM™ (diazoxide).
• In one embodiment, the Therapeutics of the invention are administered in combination with treatments for uterine motility disorders. Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN® and ESTRATAB®), estradiols (e.g., CLIMARA® and ALORA®), estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate), PROMETRIUM® progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogens/medroxyprogesterone (e.g., PREMPRO™ and PREMPHASE®) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRT™).
• In an additional embodiment, the Therapeutics of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOL™), ferrous fumarate (e.g., FEOSTAT™), ferrous gluconate (e.g., FERGON™), polysaccharide-iron complex (e.g., NIFEREX™), iron dextran injection (e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B₁₂, cyancobalamin injection (e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g., FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.
• In certain embodiments, the Therapeutics of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the Therapeutics of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).
• In other embodiments, the Therapeutics of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the Therapeutics of the invention include, but are not limited to, antiepileptic agents (e.g., carbarnazepine, clonazepam, ethosuximide, phenobarbital, phenyloin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).
• In another embodiment, Therapeutics of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., isosorbide dinitrate, isosorbide mononitrate, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.
• In certain embodiments, the Therapeutics of the invention are administered in combination with treatments for gastrointestinal disorders. Treatments for gastrointestinal disorders that may be administered with the Therapeutic of the invention include, but are not limited to, H₂ histamine receptor antagonists (e.g., TAGAMET™ (cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™ (nizatidine)); inhibitors of H⁺, K⁺ ATPase (e.g., PREVACID™ (lansoprazole) and PRILOSEC™ (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOL™ (bismuth subsalicylate) and DE-NOL™ (bismuth subcitrate)); various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTEC™ (misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMOTIL™ (diphenoxylate), MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide), antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide and chlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.
• In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
Example 14 Method of Treating Decreased Levels of the Polypeptide
• The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an agonist of the invention (including polypeptides of the invention). Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a polypeptide of the present invention in an individual can be treated by administering the agonist or antagonist of the present invention. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the agonist or antagonist to increase the activity level of the polypeptide in such an individual.
• For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 ug/kg of the agonist or antagonist for six consecutive days. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 13.
Example 15 Method of Treating Increased Levels of the Polypeptide
• The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).
• In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, due to a variety of etiologies, such as cancer.
• For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The antisense polynucleotides of the present invention can be formulated using techniques and formulations described herein (e.g. see Example 13), or otherwise known in the art.
Example 16 Method of Treatment Using Gene Therapy-Ex Vivo
• One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.
• At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.
• pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.
• The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.
• The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).
• Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.
• The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
Example 17 Gene Therapy Using Endogenous Genes Corresponding To Polynucleotides of the Invention
• Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.
• Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.
• The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.
• In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.
• Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.
• Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×10⁶ cells/ml. Electroporation should be performed immediately following resuspension.
• Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′ end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′ end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′end and a HindIII site at the 3′ end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2—BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.
• Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.
• Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.
• The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.
Example 18 Method of Treatment Using Gene Therapy—In Vivo
• Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to (i.e., associated with) a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).
• The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
• The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.
• The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.
• The polynucleotide construct can be delivered to the interstitial space of tissues within an animal, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.
• For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.
• The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.
• Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.
• After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.
Example 19 Transgenic Animals
• The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
• Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.
• Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).
• The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
• Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
• Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
• Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 20 Knock-Out Animals
• Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.
• In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
• Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).
• When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
• Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
Example 21 Assays Detecting Stimulation or Inhibition of B cell Proliferation and Differentiation
• Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.
• One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.
• In Vitro Assay—Agonists or antagonists of the invention can be assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the agonists or antagonists of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).
• Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 10⁵ B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.
• In vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of agonists or antagonists of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with agonists or antagonists of the invention identify the results of the activity of the agonists or antagonists on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.
• Flow cytometric analyses of the spleens from mice treated with agonist or antagonist is used to indicate whether the agonists or antagonists specifically increases the proportion of ThB+, CD45R(B220) dull B cells over that which is observed in control mice.
• Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and agonists or antagonists-treated mice.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 22 T Cell Proliferation Assay
• A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of ³H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4 degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of agonists or antagonists of the invention (total volume 200 ul). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37 degrees C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatant is removed and stored −20 degrees C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 ul of medium containing 0.5 uCi of ³H-thymidine and cultured at 37 degrees C. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative control for the effects of agonists or antagonists of the invention.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 23 Effect of Agonists or Antagonists of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells
• Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγ RII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.
• FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of agonist or antagonist of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
• Effect on the production of cytokines. Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Thl helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (10⁶/ml) are treated with increasing concentrations of agonists or antagonists of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.
• Effect on the expression of MHC Class II, costimulatory and adhesion molecules. Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increased expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.
• FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of agonists or antagonists of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).
• Monocyte activation and/or increased survival. Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Agonists or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a Histopaque gradient (Sigma). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.
• Monocyte Survival Assay. Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated processes (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.
• Effect on cytokine release. An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×10⁵ cells/ml with increasing concentrations of agonists or antagonists of the invention and under the same conditions, but in the absence of agonists or antagonists. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in the presence of agonist or antagonist of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.
• Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵ cell/well. Increasing concentrations of agonists or antagonists of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37° C. for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H₂O₂ produced by the macrophages, a standard curve of a H₂O₂ solution of known molarity is performed for each experiment.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 24 Biological Effects of Agonists or Antagonists of the Invention
• Astrocyte and Neuronal Assays
• Agonists or antagonists of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate an agonist or antagonist of the invention's activity on these cells.
• Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of an agonist or antagonist of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.
• Fibroblast and Endothelial Cell Assays
• Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CytoFluor fluorescence reader. For the PGE₂ assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or agonists or antagonists of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without agonists or antagonists of the invention IL-1a for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).
• Human lung fibroblasts are cultured with FGF-2 or agonists or antagonists of the invention for 3 days in basal medium before the addition of Alamar Blue to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with agonists or antagonists of the invention.
• Parkinson Models.
• The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released. Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP⁺ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.
• It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).
• Based on the data with FGF-2, agonists or antagonists of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of an agonist or antagonist of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.
• Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if an agonist or antagonist of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the agonist or antagonist may be involved in Parkinson's Disease.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 25 The Effect of Agonists or Antagonists of the Invention on the Growth of Vascular Endothelial Cells
• On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. An agonist or antagonist of the invention, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.
• An increase in the number of HUVEC cells indicates that the compound of the invention may proliferate vascular endothelial cells, while a decrease in the number of HUVEC cells indicates that the compound of the invention inhibits vascular endothelial cells.
• The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.
Example 26 Rat Corneal Wound Healing Model
• This animal model shows the effect of an agonist or antagonist of the invention on neovascularization. The experimental protocol includes:
• a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer.
• b) Inserting a spatula below the lip of the incision facing the outer corner of the eye.
• c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).
• d) Positioning a pellet, containing 50 ng-5 ug of an agonist or antagonist of the invention, within the pocket.
• e) Treatment with an agonist or antagonist of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 27 Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
• Diabetic db+/db+Mouse Model.
• To demonstrate that an agonist or antagonist of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).
• The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).
• The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).
• Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
• Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
• Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
• An agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
• Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
• Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.
• Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:
  [Open area on day 8]−[Open area on day 1]/[Open area on day 1]
• Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an agonist or antagonist of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.
• Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.
• Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.
• Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
• Steroid Impaired Rat Model
• The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impaired wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
• To demonstrate that an agonist or antagonist of the invention can accelerate the healing process, the effects of multiple topical applications of the agonist or antagonist on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.
• Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
• The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.
• Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.
• The agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.
• Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.
• Three groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.
• Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:
  [Open area on day 8]−[Open area on day 1]/[Open area on day 1]
• Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an agonist or antagonist of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.
• Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 28 Lymphadema Animal Model
• The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of an agonist or antagonist of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.
• Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ˜350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.
• Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated or suture ligated.
• Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.
• Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of 0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.
• To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect of plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.
• Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people and those 2 readings are averaged. Readings are taken from both control and edematous limbs.
• Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), and both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into instrument to each marked level then measured by Buxco edema software (Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.
• Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca²⁺ comparison.
• Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.
• Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80 EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 29 Suppression of TNF Alpha-Induced Adhesion Molecule Expression by an Agonist or Antagonist of the Invention
• The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
• Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.
• The potential of an agonist or antagonist of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.
• To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO₂. HUVECs are seeded in 96-well plates at concentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.
• Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 ul of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 ul volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.
• Fixative is then removed from the wells and wells are washed 1× with PBS (+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10 μl of diluted primary antibody to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed X3 with PBS (+Ca,Mg)+0.5% BSA.
• Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed X3 with PBS (+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰)>10^−0.5>10⁻¹>10^−1.50.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
• The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).
Example 30 Production Of Polypeptide of the Invention For High-Throughput Screening Assays
• The following protocol produces a supernatant containing polypeptide of the present invention to be tested. This supernatant can then be used in the Screening Assays described in Examples 32-41.
• First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 ul of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.
• Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS (14-503F Biowhittaker)/1× Penstrep (17-602E Biowhittaker). Let the cells grow overnight.
• The next day, mix together in a sterile solution basin: 300 ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 ug of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8-10, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150 ul Optimem I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.
• Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a12-channel pipetter with tips on every other channel, adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degree C. for 6 hours.
• While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl₂ (anhyd); 0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L of FeSO₄.7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L of MgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L of NaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002 mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂0; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H₂0; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂0; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂0; and 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamine and 1× penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in IL DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15 ml polystyrene conical.
• The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each well. Incubate at 37 degree C. for 45 or 72 hours depending on the media used: 1% BSA for 45 hours or CHO-5 for 72 hours.
• On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 32-39.
• It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide of the present invention directly (e.g., as a secreted protein) or by polypeptide of the present invention inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.
Example 31 Construction of GAS Reporter Construct
• One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.
• GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.
• The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.
• The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO: 2)).
• Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.

  	JAKs

  Ligand	tyk2	Jak1	Jak2	Jak3	STATS	GAS(elements) or ISRE
  IFN family
  IFN-a/B	+	+	−	−	1, 2, 3	ISRE
  IFN-g		+	+	−	1	GAS (IRF1 > Lys6 > IFP)
  Il-10	+	?	?	−	1, 3
  gp130 family
  IL-6 (Pleiotropic)	+	+	+	?	1, 3	GAS (IRF1 > Lys6 > IFP)
  Il-11 (Pleiotropic)	?	+	?	?	1, 3
  OnM (Pleiotropic)	?	+	+	?	1, 3
  LIF (Pleiotropic)	?	+	+	?	1, 3
  CNTF (Pleiotropic)	−/+	+	+	?	1, 3
  G-CSF (Pleiotropic)	?	+	?	?	1, 3
  IL-12 (Pleiotropic)	+	−	+	+	1, 3
  g-C family
  IL-2 (lymphocytes)	−	+	−	+	1, 3, 5	GAS
  IL-4 (lymph/myeloid)	−	+	−	+	6	GAS (IRF1 = IFP >> Ly6)(IgH)
  IL-7 (lymphocytes)	−	+	−	+	5	GAS
  IL-9 (lymphocytes)	−	+	−	+	5	GAS
  IL-13 (lymphocyte)	−	+	?	?	6	GAS
  IL-15	?	+	?	+	5	GAS
  gp140 family
  IL-3 (myeloid)	−	−	+	−	5	GAS (IRF1 > IFP >> Ly6)
  IL-5 (myeloid)	−	−	+	−	5	GAS
  GM-CSF (myeloid)	−	−	+	−	5	GAS
  Growth hormone family
  GH	?	−	+	−	5
  PRL	?	+/−	+	−	1, 3, 5
  EPO	?	−	+	−	5	GAS(B-CAS > IRF1 = IFP >> Ly6)
  Receptor Tyrosine Kinases
  EGF	?	+	+	−	1, 3	GAS (IRF1)
  PDGF	?	+	+	−	1, 3
  CSF-1	?	+	+	−	1, 3	GAS (not IRF1)

• To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 32-33, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is:

  5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCC	(SEQ ID NO:3)
  CGAAATGATTTCCCCGAAATGATTTCCCCGAAATATC
  TGCCATCTCAATTAG:3′

• The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)
• PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (Stratagene.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence:

  5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAA	(SEQ ID NO:5)
  ATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCC
  ATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAAC
  TCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCC
  CATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTT
  ATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATT
  CCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGC
  TTTTGCAAAAAGCTT:3′

• With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.
• The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from Clontech using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
• Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (Clontech), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 32-33.
• Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing EGR and NF-KB promoter sequences are described in Examples 34 and 35. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, 11-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.
Example 32 High-Throughput Screening Assay for T-Cell Activity
• The following protocol is used to assess T-cell activity by identifying factors, and determining whether supernate containing a polypeptide of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 31. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582) cells can also be used.
• Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (Life Technologies)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.
• Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 ul of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C and incubate at room temperature for 15-45 mins.
• During the incubation period, count cell concentration, spin down the required number of cells (10⁷ per transfection), and resuspend in OPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of 1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degree C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.
• The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptide of the present invention or polypeptide of the present invention induced polypeptides as produced by the protocol described in Example 30.
• On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.
• Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 ul of cells into each well (therefore adding 100,000 cells per well).
• After all the plates have been seeded, 50 ul of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.
• The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 ul samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at −20 degree C. until SEAP assays are performed according to Example 36. The plates containing the remaining treated cells are placed at 4 degree C. and serve as a source of material for repeating the assay on a specific well if desired.
• As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.
• The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.
Example 33 High-Throughput Screening Assay Identifying Myeloid Activity
• The following protocol is used to assess myeloid activity of polypeptide of the present invention by determining whether polypeptide of the present invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 31. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.
• To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 31, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10⁷ U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.
• Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂. Incubate at 37 degrees C. for 45 min.
• Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degree C. for 36 hr.
• The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 ug/ml G418 for couple of passages.
• These cells are tested by harvesting 1×10⁸ cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×10⁵ cells/ml. Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).
• Add 50 ul of the supernatant prepared by the protocol described in Example 30. Incubate at 37 degree C for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 36.
Example 34 High-Throughput Screening Assay Identifying Neuronal Activity
• When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGRL (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed by polypeptide of the present invention.
• Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by polypeptide of the present invention can be assessed.
• The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers:

  5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-	(SEQ ID NO:6)
  3′
  5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′	(SEQ ID NO:7)

• Using the GAS:SEAP/Neo vector produced in Example 31, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.
• To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.
• PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 ug/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.
• Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamine protocol described in Example 30. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 ug/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 ug/ml G418 for couple of passages.
• To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.
• The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×10⁵ cells/ml.
• Add 200 ul of the cell suspension to each well of 96-well plate (equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced by Example 30, 37 degree C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 36.
Example 35 High-Throughput Screening Assay for T-Cell Activity
• NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.
• In non-stimulated conditions, NF-KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.
• Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 30. Activators or inhibitors of NF-KB would be useful in treating, preventing, and/or diagnosing diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.
• To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO: 8), 18 bp of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site:

  5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCG	(SEQ ID NO:9)
  GGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATT
  AG:3′

• The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site:

  5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′	(SEQ ID NO:4)

• PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from Clontech. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence:

  5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGA	(SEQ ID NO:10)
  CTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTC
  AGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCC
  GCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCC
  CCATGGCTGACTAATTTTTTTTATTTATGCAGAGGC
  CGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTA
  GTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA
  AAAGCTT:3′

• Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.
• In order to generate stable mammalian cell lines, the NF-KB/SV46/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.
• Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 32. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 32. As a positive control, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.
Example 36 Assay for SEAP Activity
• As a reporter molecule for the assays described in Examples 32-35, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.
• Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of 2.5× dilution buffer into Optiplates containing 35 ul of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.
• Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 ul Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set 10 minutes later.
• Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.

  Reaction Buffer Formulation:

  # of plates	Rxn buffer diluent (ml)	CSPD (ml)

  10	60	3
  11	65	3.25
  12	70	3.5
  13	75	3.75
  14	80	4
  15	85	4.25
  16	90	4.5
  17	95	4.75
  18	100	5
  19	105	5.25
  20	110	5.5
  21	115	5.75
  22	120	6
  23	125	6.25
  24	130	6.5
  25	135	6.75
  26	140	7
  27	145	7.25
  28	150	7.5
  29	155	7.75
  30	160	8
  31	165	8.25
  32	170	8.5
  33	175	8.75
  34	180	9
  35	185	9.25
  36	190	9.5
  37	195	9.75
  38	200	10
  39	205	10.25
  40	210	10.5
  41	215	10.75
  42	220	11
  43	225	11.25
  44	230	11.5
  45	235	11.75
  46	240	12
  47	245	12.25
  48	250	12.5
  49	255	12.75
  50	260	13

Example 37 High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability
• Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.
• The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.
• For adherent cells, seed the cells at 10,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO₂ incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after the final wash.
• A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 ul of buffer.
• For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100 ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 ul, followed by an aspiration step to 100 ul final volume.
• For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The supernatant is added to the well, and a change in fluorescence is detected.
• To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increased emission at 530 nm indicates an extracellular signaling event caused by the a molecule, either polypeptide of the present invention or a molecule induced by polypeptide of the present invention, which has resulted in an increase in the intracellular Ca⁺⁺ concentration.
Example 38 High-Throughput Screening Assay Identifying Tyrosine Kinase Activity
• The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.
• Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
• Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether polypeptide of the present invention or a molecule induced by polypeptide of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.
• Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well Loprodyne Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of alamarBlue as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.
• To prepare extracts, A431 cells are seeded onto the nylon membranes of Loprodyne plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example 30, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of protease inhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C. at 16,000×g.
• Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.
• Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from Boehringer Mannheim.
• The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 ul of 5 uM Biotinylated Peptide, then 10 ul ATP/IMg₂+(5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate (1 mM), and then 5 ul of water. Mix the components gently and preincubate the reaction mix at 30 degree C. for 2 min. Initial the reaction by adding 10 ul of the control enzyme or the filtered supernatant.
• The tyrosine kinase assay reaction is then terminated by adding 10 ul of 120 mm EDTA and place the reactions on ice.
• Tyrosine kinase activity is determined by transferring 50 ul aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C. for 20 min. This allows the streptavidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 ul of anti-phospotyrosine antibody conjugated to horse radish peroxidase (anti-P-Tyr-POD (0.5u/ml)) to each well and incubate at 37 degree C. for one hour. Wash the well as above.
• Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.
Example 39 High-Throughput Screening Assay Identifying Phosphorylation Activity
• As a potential alternative and/or complement to the assay of protein tyrosine kinase activity described in Example 38, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38. MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.
• Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (10 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degree C. until use.
• A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 30 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.
• After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by polypeptide of the present invention or a molecule induced by polypeptide of the present invention.
Example 40 Assay for the Stimulation of Bone Marrow CD34+Cell Proliferation
• This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of isolated polypeptides expressed in mammalian cells to stimulate proliferation of CD34+cells.
• It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of polypeptides on hematopoietic activity of a wide range of progenitor cells, the assay contains a given polypeptide in the presence or absence of other hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a “survival” factor. However, combined with any factor exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested polypeptide has a stimulatory effect on hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect of a given polypeptide, or agonists or antagonists thereof, might not be detected. Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+IL+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.
• Briefly, CD34+cells are isolated using methods known in the art. The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md. Cat# 160-204-101). After several gentle centrifugation steps at 200×g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5×10⁵ cells/ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with a given polypeptide in this assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat# 255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat# 203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μl of the supernatants prepared in Example 30 (supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 μl. The plates are then placed in a 37° C./5% CO₂ incubator for five days.
• Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H] Thymidine is added in a 10 μl volume to each well to determine the proliferation rate. The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OmniFilter assemblies consisting of one OmniFilter plate and one OmniFilter Tray. 60 μL Microscint is added to each well and the plate sealed with TopSeal-A press-on sealing film A bar code 15 sticker is affixed to the first plate for counting. The sealed plates are then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.
• The studies described in this example test the activity of a given polypeptide to stimulate bone marrow CD34+cell proliferation. One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof. As a nonlimiting example, potential antagonists tested in this assay would be expected to inhibit cell proliferation in the presence of cytokines and/or to increase the inhibition of cell proliferation in the presence of cytokines and a given polypeptide. In contrast, potential agonists tested in this assay would be expected to enhance cell proliferation and/or to decrease the inhibition of cell proliferation in the presence of cytokines and a given polypeptide.
• The ability of a gene to stimulate the proliferation of bone marrow CD34+cells indicates that polynucleotides and polypeptides corresponding to the gene are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein.
Example 41 Assay for Extracellular Matrix Enhanced Cell Response (EMECR)
• The objective of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to identify gene products (e.g., isolated polypeptides) that act on the hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.
• Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fn is mediated by the α₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM environment and are responsible for stimulating stem cell self-renewal have a not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications
• Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fn fragment at a coating concentration of 0.2 μg/cm². Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Gene products of the invention (e.g., including, but not limited to, polynucleotides and polypeptides of the present invention, and supernatants produced in Example 30), are tested with appropriate negative controls in the presence and absence of SCF (5.0 ng/ml), where test factor supernatants represent 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment (5% CO₂, 7% O₂, and 88% N₂) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thymidine incorporation into cellular DNA. Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.
• One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
• If a particular polypeptide of the present invention is found to be a stimulator of hematopoietic progenitors, polynucleotides and polypeptides corresponding to the gene encoding said polypeptide may be useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein. The gene product may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
• Additionally, the polynucleotides and/or polypeptides of the gene of interest and/or agonists and/or antagonists thereof, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.
• Moreover, polynucleotides and polypeptides corresponding to the gene of interest may also be useful for the treatment and diagnosis of hematopoietic related disorders such as, for example, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.
Example 42 Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation
• The polypeptide of interest is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the polypeptide of interest on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity.
• Briefly, on day 1,96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AOSMC) in 100 μl culture media. NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5% FBS. After incubation at 37° C. for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37° C. until day 2.
• On day 2, serial dilutions and templates of the polypeptide of interest are designed such that they always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add 1/3 vol media containing controls or polypeptides of the present invention and incubate at 37 degrees C./5% CO₂ until day 5.
• Transfer 60 μl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4 degrees C. until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add Alamar Blue in an amount equal to 10% of the culture volume (10 μl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CytoFluor. This yields the growth stimulation/inhibition data.
• On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 ul/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.
• On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.
• Plates are washed with wash buffer and blotted on paper towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover the plate and incubate 1 h at RT. Plates are again washed with wash buffer and blotted on paper towels.
• Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.
• A positive result in this assay suggests AoSMC cell proliferation and that the polypeptide of the present invention may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of polypeptides, polynucleotides, agonists and/or antagonists of the polynucleotide/polypeptide of the present invention which gives a positive result. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, polypeptides of the present invention and polynucleotides of the present invention may be used in wound healing and dermal regeneration, as well as the promotion of vasculogenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, antagonists of polypeptides and polynucleotides of the invention may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular agent (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, antagonists of polypeptides and polynucleotides of the invention may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.
• One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
Example 43 Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells
• The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.
• Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are then incubated at 37° C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min. Fixative is removed from the wells and wells are washed 1× with PBS (+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed three times with PBS (+Ca,Mg)+0.5% BSA. 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000 dilution, referred to herein as the working dilution) are added to each well and incubated at 37° C. for 30 min. Wells are washed three times with PBS (+Ca,Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰)>10^−0.5>10⁻¹>10^1.50.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.
Example 44 Alamar Blue Endothelial Cells Proliferation Assay
• This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard Alamar Blue Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of the protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.
• Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37 degreesC overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the protein of interest or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C. incubator for three days. After three days 10 ml of stock alamar blue (Biosource Cat# DAL100) is added to each well and the plate(s) is/are placed back in the 37° C. incubator for four hours. The plate(s) are then read at 530 nm excitation and 590 nm emission using the CytoFluor fluorescence reader. Direct output is recorded in relative fluorescence units.
• Alamar blue is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form (i.e., stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity). The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.
Example 45 Detection of Inhibition of a Mixed Lymphocyte Reaction
• This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides). Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by these polypeptides since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.
• Polypeptides of interest found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.
• Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor is adjusted to 2×10⁵ cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of test materials (50 μl) is added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC of [³H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.
• Samples of the protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.
• One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.
Example 46 Assays for Protease Activity
• The following assay may be used to assess protease activity of the polypeptides of the invention.
• Gelatin and casein zymography are performed essentially as described (Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are run on 10% polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in 2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3 at 37° C. 5 to 16 hours. After staining in amido black areas of proteolysis apear as clear areas agains the blue-black background. Trypsin (Sigma T8642) is used as a positive control.
• Protease activity is also determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500. Reactions are set up in (25 mMNaPO₄, 1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples are added and the change in adsorbance at 260 nm is monitored on the Beckman DU-6 spectrophotometer in the time-drive mode. Trypsin is used as a positive control.
• Additional assays based upon the release of acid-soluble peptides from casein or hemoglobin measured as adsorbance at 280 nm or colorimetrically using the Folin method are performed as described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assays involve the solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).
Example 47 Identifying Serine Protease Substrate Specificity
• Methods known in the art or described herein may be used to determine the substrate specificity of the polypeptides of the present invention having serine protease activity. A preferred method of determining substrate specificity is by the use of positional scanning synthetic combinatorial libraries as described in GB 2 324 529 (incorporated herein in its entirety).
Example 48 Ligand Binding Assays
• The following assay may be used to assess ligand binding activity of the polypeptides of the invention.
• Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for a polypeptide is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its polypeptide. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
Example 49 Functional Assay in Xenopus Oocytes
• Capped RNA transcripts from linearized plasmid templates encoding the polypeptides of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocytc) are injected in a 50 nl bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response polypeptides and polypeptide agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.
Example 50 Microphysiometric Assays
• Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of detecting the activation of polypeptide which is coupled to an energy utilizing intracellular signaling pathway.
Example 51 Extract/Cell Supernatant Screening
• A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligands banks as identified to date. Accordingly, the polypeptides of the invention can also be functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify its natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.
Example 52 Calcium and cAMP Functional Assays
• Seven transmembrane receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day >150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.
Example 53 ATP-Binding Assay
• The following assay may be used to assess ATP-binding activity of polypeptides of the invention.
• ATP-binding activity of the polypeptides of the invention may be detected using the ATP-binding assay described in U.S. Pat. No. 5,858,719, which is herein incorporated by reference in its entirety. Briefly, ATP-binding to polypeptides of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay. Reaction mixtures containing 1 mg/ml of the ABC transport protein of the present invention are incubated with varying concentrations of ATP, or the non-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at 4° C. A mixture of 8-azido-ATP (Sigma Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP (³²P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to a final concentration of 100 μM and 0.5 ml aliquots are placed in the wells of a porcelain spot plate on ice. The plate is irradiated using a short wave 254 nm UV lamp at a distance of 2.5 cm from the plate for two one-minute intervals with a one-minute cooling interval in between. The reaction is stopped by addition of dithiothreitol to a final concentration of 2 mM. The incubations are subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. Protein bands corresponding to the particular polypeptides of the invention are excised, and the radioactivity quantified. A decrease in radioactivity with increasing ATP or adenly-5′-imidodiphosphate provides a measure of ATP affinity to the polypeptides.
Example 54 Small Molecule Screening
• This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and polypeptide of the invention.
• Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the invention. These methods comprise contacting such an agent with a polypeptide of the invention or fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the invention.
• Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is herein incorporated by reference in its entirety. Briefly stated, large numbers of different small molecule test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with polypeptides of the invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.
• This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.
Example 55 Phosphorylation Assay
• In order to assay for phosphorylation activity of the polypeptides of the invention, a phosphorylation assay as described in U.S. Pat. No. 5,958,405 (which is herein incorporated by reference) is utilized. Briefly, phosphorylation activity may be measured by phosphorylation of a protein substrate using gamma-labeled ³²P-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. The polypeptides of the invention are incubated with the protein substrate, ³²P-ATP, and a kinase buffer. The ³²P incorporated into the substrate is then separated from free ³²P-ATP by electrophoresis, and the incorporated ³²P is counted and compared to a negative control. Radioactivity counts above the negative control are indicative of phosphorylation activity of the polypeptides of the invention.
Example 56 Detection of Phosphorylation Activity (Activation) of the Polypeptides of the Invention in the Presence of Polypeptide Ligands
• Methods known in the art or described herein may be used to determine the phosphorylation activity of the polypeptides of the invention. A preferred method of determining phosphorylation activity is by the use of the tyrosine phosphorylation assay as described in U.S. Pat. No. 5,817,471 (incorporated herein by reference).
Example 57 Identification of Signal Transduction Proteins that Interact with Polypeptides of the Present Invention
• The purified polypeptides of the invention are research tools for the identification, characterization and purification of additional signal transduction pathway proteins or receptor proteins. Briefly, labeled polypeptides of the invention are useful as reagents for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptides of the invention are covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as carcinoma tissues, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 58 IL-6 Bioassay
• To test the proliferative effects of the polypeptides of the invention, the IL-6 Bioassay as described by Marz et al. is utilized (Proc. Natl. Acad. Sci., U.S.A., 95:3251-56 (1998), which is herein incorporated by reference). Briefly, IL-6 dependent B9 murine cells are washed three times in IL-6 free medium and plated at a concentration of 5,000 cells per well in 50 μl, and 50 μl of the IL-6-like polypeptide is added. After 68 hrs. at 37° C., the number of viable cells is measured by adding the tetrazolium salt thiazolyl blue (MTT) and incubating for a further 4 hrs. at 37° C. B9 cells are lysed by SDS and optical density is measured at 570 nm. Controls containing IL-6 (positive) and no cytokine (negative) are utilized. Enhanced proliferation in the test sample(s) relative to the negative control is indicative of proliferative effects mediated by polypeptides of the invention.
Example 59 Support of Chicken Embryo Neuron Survival
• To test whether sympathetic neuronal cell viability is supported by polypeptides of the invention, the chicken embryo neuronal survival assay of Senaldi et al is utilized (Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998), which is herein incorporated by reference). Briefly, motor and sympathetic neurons are isolated from chicken embryos, resuspended in L15 medium (with 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; Life Technologies, Rockville, Md.) and Dulbecco's modified Eagles medium [with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2); Life Technologies, Rockville, Md.], respectively, and incubated at 37° C. in 5% CO₂ in the presence of different concentrations of the purified IL-6-like polypeptide, as well as a negative control lacking any cytokine. After 3 days, neuron survival is determined by evaluation of cellular morphology, and through the use of the calorimetric assay of Mosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)). Enhanced neuronal cell viability as compared to the controls lacking cytokine is indicative of the ability of the inventive purified IL-6-like polypeptide(s) to enhance the survival of neuronal cells.
Example 60 Assay for Phosphatase Activity
• The following assay may be used to assess serine/threonine phosphatase (PTPase) activity of the polypeptides of the invention.
• In order to assay for serine/threonine phosphatase (PTPase) activity, assays can be utilized which are widely known to those skilled in the art. For example, the serine/threonine phosphatase (PSPase) activity is measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine residues with cAMP-dependent Protein Kinase in the presence of [³²P]ATP. Protein serine/threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from ³²P-labeled MyBP.
Example 61 Interaction of Serine/Threonine Phosphatases with other Proteins
• The polypeptides of the invention with serine/threonine phosphatase activity as determined in Example 60 are research tools for the identification, characterization and purification of additional interacting proteins or receptor proteins, or other signal transduction pathway proteins. Briefly, labeled polypeptide(s) of the invention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptide of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as neural or liver cells, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The polypeptides of the invention-complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.
Example 62 Assaying for Heparanase Activity
• In order to assay for heparanase activity of the polypeptides of the invention, the heparanase assay described by Vlodavsky et al is utilized (Vlodavsky, I., et al., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditioned media or intact cells (1×10⁶ cells per 35-mm dish) are incubated for 18 hrs at 37° C., pH 6.2-6.6, with ³⁵S-labeled ECM or soluble ECM derived peak I proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a Sepharose CL-6B column (0.9×30 cm). Fractions are eluted with PBS and their radioactivity is measured. Degradation fragments of heparan sulfate side chains are eluted from Sepharose 6B at 0.5<K_av<0.8 (peak II). Each experiment is done at least three times. Degradation fragments corresponding to “peak II,” as described by Vlodavsky et al., is indicative of the activity of the polypeptides of the invention in cleaving heparan sulfate.
Example 63 Immobilization of Biomolecules
• This example provides a method for the stabilization of polypeptides of the invention in non-host cell lipid bilayer constucts (see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999), hereby incorporated by reference in its entirety herein) which can be adapted for the study of polypeptides of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine a biotin tag to the extracellular domain of the polypeptides of the invention, thus allowing uniform orientation upon immobilization. A 50 uM solution of polypeptides of the invention in washed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150 ul). Then the sample is dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford Ill.) at 4C first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl2, 10 mM sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).
Example 64 TAQMAN
• Quantitative PCR (QPCR). Total RNA from cells in culture are extracted by Trizol separation as recommended by the supplier (LifeTechnologies). (Total RNA is treated with DNase I (Life Technologies) to remove any contaminating genomic DNA before reverse transcription.) Total RNA (50 ng) is used in a one-step, 50 ul, RT-QPCR, consisting of Taqman Buffer A (Perkin-Elmer; 50 mM KCl/10 mM Tris, pH 8.3), 5.5 mM MgCl₂, 240 μM each dNTP, 0.4 units RNase inhibitor (Promega), 8% glycerol, 0.012% Tween-20, 0.05% gelatin, 0.3 uM primers, 0.1 uM probe, 0.025 units Amplitaq Gold (Perkin-Elmer) and 2.5 units Superscript II reverse transcriptase (Life Technologies). As a control for genomic contamination, parallel reactions are setup without reverse transcriptase. The relative abundance of (unknown) and 18S RNAs are assessed by using the Applied Biosystems Prism 7700 Sequence Detection System (Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W. & Deetz, K. (1995) PCR Methods Appl. 4, 357-362). Reactions are carried out at 48° C. for 30 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s, 60° C. for 1 min. Reactions are performed in triplicate.
• Primers (f & r) and FRET probes sets are designed using Primer Express Software (Perkin-Elmer). Probes are labeled at the 5′-end with the reporter dye 6-FAM and on the 3′-end with the quencher dye TAMRA (Biosource International, Camarillo, Calif. or Perkin-Elmer).
Example 65 Assays for Metalloproteinase Activity
• Metalloproteinases (EC 3.4.24.-) are peptide hydrolases which use metal ions, such as Zn²⁺, as the catalytic mechanism. Metalloproteinase activity of polypeptides of the present invention can be assayed according to the following methods.
• Proteolysis of Alpha-2-Macroglobulin
• To confirm protease activity, purified polypeptides of the invention are mixed with the substrate alpha-2-macroglobulin (0.2 unit/ml; Boehringer Mannheim, Germany) in 1× assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at 37° C. for 1-5 days. Trypsin is used as positive control. Negative controls contain only alpha-2-macroglobulin in assay buffer. The samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresis the proteins are visualized by silver staining. Proteolysis is evident by the appearance of lower molecular weight bands as compared to the negative control.
• Inhibition of Alpha-2-Macroglobulin Proteolysis by Inhibitors of Metalloproteinases
• Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, AND HgCl₂), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2), and commercial small molecule MMP inhibitors) are used to characterize the proteolytic activity of polypeptides of the invention. The three synthetic MMP inhibitors used are: MMP inhibitor I, [IC₅₀=1.0 μM against MMP-1 and MMP-8; IC₅₀=30 μM against MMP-9; IC₅₀=150 μM against MMP-3]; MMP-3 (stromelysin-1) inhibitor I [IC₅₀=5 μM against MMP-3], and MMP-3 inhibitor II [K_i=130 nM against MMP-3]; inhibitors available through Calbiochem, catalog # 444250, 444218, and 444225, respectively). Briefly, different concentrations of the small molecule MMP inhibitors are mixed with purified polypeptides of the invention (50 μg/ml) in 22.9 μl of 1× HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl of substrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at 37° C. for 20-hr. The reactions are stopped by adding 4× sample buffer and boiled immediately for 5 minutes. After SDS-PAGE, the protein bands are visualized by silver stain.
• Synthetic Fluorogenic Peptide Substrates Cleavage Assay
• The substrate specificity for polypeptides of the invention with demonstrated metalloproteinase activity can be determined using synthetic fluorogenic peptide substrates (purchased from BACHEM Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four are MMP substrates and the last one is a substrate of tumor necrosis factor-α (TNF-α) converting enzyme (TACE). All the substrates are prepared in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are 50-500 μM. Fluorescent assays are performed by using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nm. Briefly, the assay is carried out by incubating 176 μl 1× HEPES buffer (0.2 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, and then adding 20 μl of a purified polypeptide of the invention into the assay cuvett. The final concentration of substrate is 1 μM. Initial hydrolysis rates are monitored for 30-min.
Example 66 Characterization of the cDNA Contained in a Deposited Plasmid
• The size of the cDNA insert contained in a deposited plasmid may be routinely determined using techniques known in the art, such as PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the cDNA sequence. For example, two primers of 17-30 nucleotides derived from each end of the cDNA (i.e., hybridizable to the absolute 5′ nucleotide or the 3′ nucleotide end of the sequence of SEQ ID NO:X, respectively) are synthesized and used to amplify the cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 ul of reaction mixture with 0.5 ug of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
INCORPORATION BY REFERENCE
• The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. In addition, the sequence listing submitted herewith is incorporated herein by reference in its entirety. The specification and sequence listing of each of the following U.S. and PCT applications are herein incorporated by reference in their entirety (filing dates shown in format “year-month-day” (yyyy-mm-dd)): Application No. 60/278,650 filed on 2001-03-27, application Ser. No. 09/950,082 filed on 2001-09-12, application Ser. No. 09/950,083 filed on 2001-09-12, Application No. 60/306,171 filed on 19 Jul. 2001, application Ser. No. 09/833,245 filed on 2001-04-12, Application No. PCT/US01/11988 filed on 2001-04-12, Application No. 60/331,287 filed on 2001-11-13, Application No. 60/277,340 filed on 2001-03-21, Application No. PCT/US00/06043 filed on 2000-03-09, Application No. PCT/US00/06012 filed on 2000-03-09, Application No. PCT/US00/06058 filed on 2000-03-09, Application No. PCT/US00/06044 filed on 2000-03-09, Application No. PCT/US00/06059 filed on 2000-03-09, Application No. PCT/US00/06042 filed on 2000-03-09, Application No. PCT/US00/06014 filed on 2000-03-09, Application No. PCT/US00/06013 filed on 2000-03-09, Application No. PCT/US00/06049 filed on 2000-03-09, Application No. PCT/US00/06057 filed on 2000-03-09, Application No. PCT/US00/06824 filed on 2000-03-16, Application No. PCT/US00/06765 filed on 2000-03-16, Application No. PCT/US00/06792 filed on 2000-03-16, Application No. PCT/US00/06830 filed on 2000-03-16, Application No. PCT/US00/06782 filed on 2000-03-16, Application No. PCT/US00/06822 filed on 2000-03-16, Application No. PCT/US00/06791 filed on 2000-03-16, Application No. PCT/US00/06828 filed on 2000-03-16, Application No. PCT/US00/06823 filed on 2000-03-16, Application No. PCT/US00/06781 filed on 2000-03-16, Application No. PCT/US00/07505 filed on 2000-03-22, Application No. PCT/US00/07440 filed on 2000-03-22, Application No. PCT/US00/07506 filed on 2000-03-22, Application No. PCT/US00/07507 filed on 2000-03-22, Application No. PCT/US00/07535 filed on 2000-03-22, Application No. PCT/US00/07525 filed on 2000-03-22, Application No. PCT/US00/07534 filed on 2000-03-22, Application No. PCT/US00/07483 filed on 2000-03-22, Application No. PCT/IUS00/07526 filed on 2000-03-22, Application No. PCT/US00/07527 filed on 2000-03-22, Application No. PCT/US00/07661 filed on 2000-03-23, Application No. PCT/US00/07579 filed on 2000-03-23, Application No. PCT/US00/07723 filed on 2000-03-23, Application No. PCT/US00/07724 filed on 2000-03-23, Application No. PCT/US00/14929 filed on 2000-06-01, Application No. PCT/US00/07722 filed on 2000-03-23, Application No. PCT/US00/07578 filed on 2000-03-23, Application No. PCT/US00/07726 filed on 2000-03-23, Application No. PCT/US00/07677 filed on 2000-03-23, Application No. PCT/US00/07725 filed on 2000-03-23, Application No. PCT/US00/09070 filed on 2000-04-06, Application No. PCT/US00/08982 filed on 2000-04-06, Application No. PCT/US00/08983 filed on 2000-04-06, Application No. PCT/US00/09067 filed on 2000-04-06, Application No. PCT/US00/09066 filed on 2000-04-06, Application No. PCT/US00/09068 filed on 2000-04-06, Application No. PCT/US00/08981 filed on 2000-04-06, Application No. PCT/US00/08980 filed on 2000-04-06, Application No. PCT/US00/09071 filed on 2000-04-06, Application No. PCT/US00/09069 filed on 2000-04-06, Application No. PCT/US00/15136 filed on 2000-06-01, Application No. PCT/US00/14926 filed on 2000-06-01, Application No. PCT/US00/14963 filed on 2000-06-01, Application No. PCT/US00/15135 filed on 2000-06-01, Application No. PCT/US00/14934 filed on 2000-06-01, Application No. PCT/US00/14933 filed on 2000-06-01, Application No. PCT/US00/15137 filed on 2000-06-01, Application No. PCT/US00/14928 filed on 2000-06-01, Application No. PCT/US00/14973 filed on 2000-06-01, Application No. PCT/US00/14964 filed on 2000-06-01, Application No. PCT/US00/26376 filed on 2000-09-26, Application No. PCT/US00/26371 filed on 2000-09-26, Application No. PCT/US00/26324 filed on 2000-09-26, Application No. PCT/US00/26323 filed on 2000-09-26, Application No. PCT/US00/26337 filed on 2000-09-26, Application No. PCT/US01/13318 filed on 2001-04-27, Application No. U.S. 60/124,146 filed on 1999-03-12, Application No. U.S. 60/167,061 filed on 1999-11-23, Application No. U.S. 60/124,093 filed on 1999-03-12, Application No. U.S. 60/166,989 filed on 1999-11-23, Application No. U.S. 60/124,145 filed on 1999-03-12, Application No. U.S. 60/168,654 filed on 1999-12-03, Application No. U.S. 60/124,099 filed on 1999-03-12, Application No. U.S. 60/168,661 filed on 1999-12-03, Application No. U.S. 60/124,096 filed on 1999-03-12, Application No. U.S. 60/168,622 filed on 1999-12-03, Application No. U.S. 60/124,143 filed on 1999-03-12, Application No. U.S. 60/168,663 filed on 1999-12-03, Application No. U.S. 60/124,095 filed on 1999-03-12, Application No. U.S. 60/138,598 filed on 1999-06-11, Application No. U.S. 60/168,665 filed on 1999-12-03, Application No. U.S. 60/125,360 filed on 1999-03-19, Application No. U.S. 60/138,626 filed on 1999-06-11, Application No. U.S. 60/168,662 filed on 1999-12-03, Application No. U.S. 60/124,144 filed on 1999-03-12, Application No. U.S. 60/138,574 filed on 1999-06-11, Application No. U.S. 60/168,667 filed on 1999-12-03, Application No. U.S. 60/124,142 filed on 1999-03-12, Application No. U.S. 60/138,597 filed on 1999-06-11, Application No. U.S. 60/168,666 filed on 1999-12-03, Application No. U.S. 60/125,359 filed on 1999-03-19, Application No. U.S. 60/168,664 filed on 1999-12-03, Application No. U.S. 60/126,051 filed on 1999-03-23, Application No. U.S. 60/169,906 filed on 1999-12-10, Application No. U.S. 60/125,362 filed on 1999-03-19, Application No. U.S. 60/169,980 filed on 1999-12-10, Application No. U.S. 60/125,361 filed on 1999-03-19, Application No. U.S. 60/169,910 filed on 1999-12-10, Application No. U.S. 60/125,812 filed on 1999-03-23, Application No. U.S. 60/169,936 filed on 1999-12-10, Application No. U.S. 60/126,054 filed on 1999-03-23, Application No. U.S. 60/169,916 filed on 1999-12-10, Application No. U.S. 60/125,815 filed on 1999-03-23, Application No. U.S. 60/169,946 filed on 1999-12-10, Application No. U.S. 60/125,358 filed on 1999-03-19, Application No. U.S. 60/169,616 filed on 1999-12-08, Application No. U.S. 60/125,364 filed on 1999-03-19, Application No. U.S. 60/169,623 filed on 1999-12-08, Application No. U.S. 60/125,363 filed on 1999-03-19, Application No. U.S. 60/169,617 filed on 1999-12-08, Application No. U.S. 60/126,502 filed on 1999-03-26, Application No. U.S. 60/172,410 filed on 1999-12-17, Application No. U.S. 60/126,503 filed on 1999-03-26, Application No. U.S. 60/172,409 filed on 1999-12-17, Application No. U.S. 60/126,505 filed on 1999-03-26, Application No. U.S. 60/172,412 filed on 1999-12-17, Application No. U.S. 60/126,594 filed on 1999-03-26, Application No. U.S. 60/172,408 filed on 1999-12-17, Application No. U.S. 60/126,511 filed on 1999-03-26, Application No. U.S. 60/172,413 filed on 1999-12-17, Application No. U.S. 60/126,595 filed on 1999-03-26, Application No. U.S. 60/171,549 filed on 1999-12-22, Application No. U.S. 60/126,598 filed on 1999-03-26, Application No. U.S. 60/171,504 filed on 1999-12-22, Application No. U.S. 60/126,596 filed on 1999-03-26, Application No. U.S. 60/171,552 filed on 1999-12-22, Application No. U.S. 60/126,600 filed on 1999-03-26, Application No. U.S. 60/171,550 filed on 1999-12-22, Application No. U.S. 60/126,501 filed on 1999-03-26, Application No. U.S. 60/171,551 filed on 1999-12-22, Application No. U.S. 60/126,504 filed on 1999-03-26, Application No. U.S. 60/174,847 filed on 2000-01-07, Application No. U.S. 60/126,509 filed on 1999-03-26, Application No. U.S. 60/174,853 filed on 2000-01-07, Application No. U.S. 60/126,506 filed on 1999-03-26, Application No. U.S. 60/174,852 filed on 2000-01-07, Application No. U.S. 60/242,710 filed on 2000-10-25, Application No. U.S. 60/126,510 filed on 1999-03-26, Application No. U.S. 60/174,850 filed on 2000-01-07, Application No. U.S. 60/138,573 filed on 1999-06-11, Application No. U.S. 60/174,851 filed on 2000-01-07, Application No. U.S. 60/126,508 filed on 1999-03-26, Application No. U.S. 60/174,871 filed on 2000-01-07, Application No. U.S. 60/126,507 filed on 1999-03-26, Application No. U.S. 60/174,872 filed on 2000-01-07, Application No. U.S. 60/126,597 filed on 1999-03-26, Application No. U.S. 60/174,877 filed on 2000-01-07, Application No. U.S. 60/126,601 filed on 1999-03-26, Application No. U.S. 60/154,373 filed on 1999-09-17, Application No. U.S. 60/176,064 filed on 2000-01-14, Application No. U.S. 60/126,602 filed on 1999-03-26, Application No. U.S. 60/176,063 filed on 2000-01-14, Application No. U.S. 60/128,695 filed on 1999-04-09, Application No. U.S. 60/176,052 filed on 2000-01-14, Application No. U.S. 60/128,696 filed on 1999-04-09, Application No. U.S. 60/176,069 filed on 2000-01-14, Application No. U.S. 60/128,703 filed on 1999-04-09, Application No. U.S. 60/176,068 filed on 2000-01-14, Application No. U.S. 60/128,697 filed on 1999-04-09, Application No. U.S. 60/176,929 filed on 2000-01-20, Application No. U.S. 60/128,698 filed on 1999-04-09, Application No. U.S. 60/176,926 filed on 2000-01-20, Application No. U.S. 60/128,699 filed on 1999-04-09, Application No. U.S. 60/177,050 filed on 2000-01-20, Application No. U.S. 60/128,701 filed on 1999-04-09, Application No. U.S. 60/177,166 filed on 2000-01-20, Application No. U.S. 60/128,700 filed on 1999-04-09, Application No. U.S. 60/176,930 filed on 2000-01-20, Application No. U.S. 60/128,694 filed on 1999-04-09, Application No. U.S. 60/176,931 filed on 2000-01-20, Application No. U.S. 60/128,702 filed on 1999-04-09, Application No. U.S. 60/177,049 filed on 2000-01-20, Application No. U.S. 60/138,629 filed on 1999-06-11, Application No. U.S. 60/138,628 filed on 1999-06-11, Application No. U.S. 60/138,631 filed on 1999-06-11, Application No. U.S. 60/138,632 filed on 1999-06-11, Application No. U.S. 60/138,599 filed on 1999-06-11, Application No. U.S. 60/138,572 filed on 1999-06-11, Application No. U.S. 60/138,625 filed on 1999-06-11, Application No. U.S. 60/138,633 filed on 1999-06-11, Application No. U.S. 60/138,630 filed on 1999-06-11, Application No. U.S. 60/138,627 filed on 1999-06-11, Application No. U.S. 60/155,808 filed on 1999-09-27, Application No. U.S. 60/155,804 filed on 1999-09-27, Application No. U.S. 60/155,807 filed on 1999-09-27, Application No. U.S. 60/155,805 filed on 1999-09-27, Application No. U.S. 60/155,806 filed on 1999-09-27, Application No. U.S. 60/201,194 filed on 2000-05-O₂, and Application No. U.S. 60/212,142 filed on 2000-06-16.

  LENGTHY TABLE
  The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070048297A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

Claims (32)

1. Use of a polypeptide for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition, wherein said polypeptide comprises an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

2. Use of the polypeptide of claim 1, wherein said wherein said polypeptide comprises a heterologous amino acid sequence.

3. Use of a polypeptide for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

4. Use of the polypeptide of claim 3, wherein said polypeptide comprises a heterologous amino acid sequence.

5. Use of an antibody or fragment thereof for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition, wherein said antibody or fragment thereof binds a polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO: Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

6. Use of an antibody or fragment thereof for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition, wherein said antibody or fragment thereof binds a polypeptide selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

7. Use of a nucleic acid molecule for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition, wherein said nucleic acid molecule comprises a polynucleotide sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a polynucleotide fragment of SEQ ID NO:X as referenced in Table 1A;

(b) a polynucleotide encoding a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polynucleotide encoding a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(e) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(f) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(g) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(h) a polynucleotide encoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

8. Use of the nucleic acid molecule of claim 7, wherein said nucleic acid molecule comprises a heterologous polynucleotide sequence.

9. Use of a nucleic acid molecule for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition, wherein said nucleic acid molecule comprises a polynucleotide sequence selected from the group consisting of:

(a) a polynucleotide fragment of SEQ ID NO:X as referenced in Table 1A;

(b) a polynucleotide encoding a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polynucleotide encoding a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(e) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(f) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(g) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(h) a polynucleotide encoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

10. Use of the nucleic acid molecule of claim 9, wherein said nucleic acid molecule comprises a heterologous polynucleotide sequence.

11. Use of an agonist or antagonist for the preparation of a pharmaceutical composition for treating a medical condition, wherein said agonist or antagonist binds a polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

12. Use of an agonist or antagonist for the preparation of a pharmaceutical composition for treating a medical condition, wherein said agonist or antagonist binds a polypeptide selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

13. A polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

14. The polypeptide of claim 13, wherein said polypeptide comprises a heterologous amino acid sequence.

15. Use of the polypeptide of claim 13 for identifying a binding partner comprising:

(a) contacting the polypeptide of claim 13 with a binding partner; and

(b) determining whether the binding partner increases or decreases activity of the polypeptide.

16. A polypeptide comprising an amino acid sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

17. The polypeptide of claim 16, wherein said polypeptide comprises a heterologous polypeptide sequence.

18. Use of the polypeptide of claim 16 for identifying a binding partner comprising:

(a) contacting the polypeptide of claim 16 with a binding partner; and

(b) determining whether the binding partner increases or decreases activity of the polypeptide.

19. An antibody or fragment thereof that binds a polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

20. An antibody or fragment thereof that binds a polypeptide selected from the group consisting of:

(a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

21. A nucleic acid molecule comprising a polynucleotide sequence at least 95% identical to a sequence selected from the group consisting of:

(a) a polynucleotide fragment of SEQ ID NO:X as referenced in Table 1A;

(b) a polynucleotide encoding a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polynucleotide encoding a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(e) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(f) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(g) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(h) a polynucleotide encoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

22. The nucleic acid molecule of claim 21, wherein said nucleic acid molecule comprises a heterologous polynucleotide sequence.

23. A recombinant vector comprising the nucleic acid molecule of claim 21.

24. A recombinant vector comprising the nucleic acid molecule of claim 22.

25. A recombinant host cell comprising the recombinant vector of claim 23.

26. A recombinant host cell comprising the recombinant vector of claim 24.

27. A nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of:

(a) a polynucleotide fragment of SEQ ID NO:X as referenced in Table 1A;

(b) a polynucleotide encoding a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(c) a polynucleotide encoding a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(d) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A;

(e) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No: Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity;

(f) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B;

(g) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and

(h) a polynucleotide encoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.

28. The nucleic acid molecule of claim 27, wherein said nucleic acid molecule comprises a heterologous polynucleotide sequence.

29. A recombinant vector comprising the nucleic acid molecule of claim 27.

30. A recombinant vector comprising the nucleic acid molecule of claim 28.

31. A recombinant host cell comprising the recombinant vector of claim 29.

32. A recombinant host cell comprising the recombinant vector of claim 30.