JP2001512005A - 5'EST of secreted protein expressed in endoderm - Google Patents

Abstract

  (57) [Summary] Disclosed are 5'EST sequences derived from mRNA encoding secreted proteins. 5'EST is for obtaining cDNA and genomic DNA corresponding to 5'EST. 5'EST can also be used for diagnostics, forensic, gene therapy, and chromosome mapping. Upstream regulatory sequences can be obtained using 5'EST. Also, it is possible to design expression and secretion vectors using 5'EST.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION An estimated 50,000-100,000 genes scattered on human chromosomes hold considerable promise for understanding, diagnosing and treating human diseases. Probes that can specifically hybridize to loci distributed in the human genome include:
There are applications in the construction of high resolution chromosome maps and in the identification of individuals.

In the past, characterizing even a single human gene has been a painstaking process, requiring several years of effort. Recent developments in the fields of cloning vectors, DNA sequencing and computer technology have greatly accelerated the speed at which human genes can be isolated, sequenced, mapped and characterized. Cloning vectors such as yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) have a chain length of 300-1000 kilobases (kb) or 100-400k, respectively.
b) can accept DNA inserts, thereby facilitating manipulation and ordering of DNA sequences distributed over long distances of the human chromosome. Automatic DNA
The sequencer allows rapid sequencing of human genes. Bioinformatics software can compare nucleic acid and protein sequences, thereby helping to characterize human gene products.

[0002] Currently, two different methods are being explored for identifying and characterizing genes distributed in the human genome. In one method, large fragments of genomic DNA are isolated, cloned, and sequenced. Bioinformatics (Bio
(informatics) software is used to identify putative open reading frames of these genomic sequences. However, this method involves sequencing a large amount of human DNA that does not encode proteins to find protein coding sequences that are distributed throughout the genome. In addition to the need for extensive sequencing, Bioinformatics software can incorrectly characterize the resulting genomic sequence. Thus, the software may produce false positives that incorrectly characterize non-coding DNA as coding DNA or false negatives that incorrectly label coding DNA as non-coding DNA.

Another approach takes a more direct route to identifying and characterizing human genes.
In this method, an isolated messenger RNA encoding a human protein (
The complementary DNA (cDNA) is synthesized from the mRNA. Using this method, sequencing is performed only on DNA derived from the protein-coding portion of the genome. Often, only short chain length cDNAs are sequenced to obtain a sequence called an expressed sequence tag (EST). ESTs can then be used to isolate or purify extended cDNAs containing sequences flanking the EST sequences. Extended cD
NAs may include all of the sequences of the EST used to obtain them, or may include only a portion of the sequences of the ESTs used to obtain them. The extended cDNA may also include the full-length coding sequence of the gene from which the EST is derived, or may include a portion of the coding sequence of the gene from which the EST is derived.
It will be appreciated that there may be several extended cDNAs containing EST sequences as a result of alternative splicing or activity of the second promoter.

[0004] In the past, these short EST sequences have been identified as oligo-dT primed cDN.
Often obtained from the A library. Thus, they mainly corresponded to the 3 'untranslated region of the mRNA. In part, due to the prevalence of EST sequences derived from the 3 'end of mRNA, a typical technique for obtaining cDNA is to isolate a cDNA sequence derived from the 5' end of mRNA. It is a consequence of the fact that it is not very suitable (Adams et al., Nature 377: 3-174, 1).
996; Hillier et al., Genome Res. 6: 807-
828, 1996).

[0005] Also, in instances where longer cDNA sequences have been reported to have been obtained, the reported sequence generally corresponds to the coding sequence and represents the full-length 5 'untranslated region of the mRNA from which the cDNA is derived. Not included. Such an incomplete sequence may not include the first exon of the mRNA, especially if the first exon is short. Furthermore, they may not include some exons, often short ones, located upstream of the splicing site. Accordingly, there is a need to obtain sequences derived from the 5 'end of mRNA.

[0006] While many sequences derived from the human chromosome have practical uses, methods based on the identification and characterization of these chromosomal sequences encoding protein products are particularly useful for diagnostic and therapeutic applications. Is relevant. Of the genes encoding 50,000 to 100,000 proteins, a gene encoding a protein secreted from a cell in which they are synthesized, and a drug whose secreted protein itself may have a therapeutic effect It is particularly useful as Such proteins are often involved in communication between cells and are involved in producing a clinically relevant response in target cells.

[0007] Indeed, several strains, including tissue plasminogen activator, G-CSF, GM-CSF, erythropoietin, human growth hormone, insulin, interferon-α, interferon-β, interferon-γ and interleukin-2 Such secreted proteins are currently in clinical use. These proteins are
It is used to treat a wide range of conditions including acute myocardial infarction, acute ischemic stroke, anemia, diabetes, growth hormone deficiency, hepatitis, kidney cancer, neutropenia due to chemotherapy and multiple sclerosis. For these reasons, extended cDNAs encoding secreted proteins, or portions thereof, are particularly useful sources of therapeutic agents. Thus, there is a need to identify and characterize secreted proteins and the nucleic acids that encode them.

[0008] In addition to being themselves therapeutically useful, secreted proteins include a short peptide at the amino terminus called a signal peptide that directs its secretion. These signal peptides are encoded by a signal sequence located at the 5 'end of the coding sequence of the gene encoding the secreted protein. Since these signal peptides direct extracellular secretion of any protein to which they are operatively linked, the signal sequence is used to operably link the signal sequence to the gene encoding the protein for which secretion is desired. By doing so, efficient secretion of any protein can be directed. Also, portions of the signal sequence can be used to direct the transport of the peptide or protein of interest into cells. This appears to be useful in gene therapy methods where it is desired to deliver a particular gene product to cells other than the cell that produces it. Signal sequences encoding signal peptides have also found use in simplifying protein purification methods. In such applications, the extracellular secretion of the desired protein can reduce the number of unwanted proteins (from which the desired protein must be selected), making purification much easier. Accordingly, there is a need to identify and characterize the 5 'portion of the gene for secreted proteins that encodes a signal peptide.

[0009] There is very little published information on the number of human genes for which promoters and upstream regulatory regions have been identified and characterized. In part, this may be due to the difficulty in isolating such regulatory sequences. Upstream regulatory sequences, such as transcription factor binding sites, are generally too short to be used as probes to isolate promoters from human genomic libraries. Recently, several methods for isolating human promoters have been developed. One of them is to create a CpG island library (Cross et al., Natur.
e Genetics 6: 236-244, 1994). The second method is Sp
The use of an eI binding protein is to isolate a human genomic DNA sequence having a SpeI binding site. (Mortlock et al., Genome Res. 6: 327-335, 1996). All of these methods are limited by lack of specificity or comprehensiveness.

Using the 5 ′ ESTs of the present invention, it is possible to efficiently identify and isolate upstream regulatory regions that control the location, developmental stage, rate and amount of protein synthesis, and mRNA stability. it can. (Tail, BioFactors 4: 8
7-93, 1993). Once these regulatory regions have been identified and characterized, they can be used in gene therapy or protein purification methods to obtain the desired amount and location of protein synthesis, or to block the synthesis of unwanted gene products, It can be reduced or suppressed.

An EST having a 5 ′ end of a secreted protein gene may contain a sequence useful as a probe for chromosome mapping and individual identification. Thus, there is a need to identify and characterize sequences upstream of the 5 'coding sequence of a gene encoding a secreted protein. SUMMARY OF THE INVENTION The present invention relates to purified and isolated or recombinant ESTs containing sequences derived from the authentic 5 'end of the corresponding mRNA. The term "corresponding mRNA" refers to the mRNA that was the template for cDNA synthesis producing 5'EST. These sequences are hereafter referred to as "5'EST". The term "purified" as used herein does not require absolute purity; rather, it is intended to be a relative definition. Individual 5 isolated from the cDNA library
'EST clones are usually purified until electrophoretic homogeneity is obtained.
Sequences obtained from these clones could not be obtained directly from libraries or total human DNA. A cDNA clone is not naturally occurring as such, but can be obtained by manipulating partially purified natural material (messenger RNA). Conversion of mRNA to a cDNA library is performed using a synthetic substance (cDN
Including A), pure individual cDNA clones can be isolated from synthetic libraries by clonal selection. Therefore, the messenger RN
By creating a cDNA library from A and then isolating individual clones from the library, an approximately 10 ⁴ to 10 ⁶ fold purification of the native message is obtained. It is specifically intended to purify the starting or natural material by at least one order of magnitude, preferably two or three orders, more preferably up to four or five orders.

The term “isolated,” as used herein, requires that a substance be separated from its original environment (eg, the natural environment if it is natural). And For example, a native polynucleotide present in a living animal has not been isolated, but a polynucleotide isolated from some or all of the coexisting substances in a natural system has been isolated.

As used herein, the term “recombinant” means that the 5 ′ EST is adjacent to a “backbone” nucleic acid that is not adjacent in its natural environment. Also,"
To be "enriched", the 5 'EST represents more than 5% of the number of nucleic acid inserts in the population of nucleic acid backbone molecules. Scaffold molecules according to the present invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrated nucleic acids and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. Preferably, the enriched 5 'EST represents at least 15% of the number of nucleic acid inserts in the population of recombinant backbone molecules. More preferably, the enriched 5 'EST represents more than 50% of the number of nucleic acid inserts in the population of recombinant backbone molecules. In a highly preferred embodiment, the enriched 5 'EST represents more than 90% of the number of nucleic acid inserts in the population of recombinant backbone molecules.

“Stringent”, “moderate” and “low” hybridization conditions are as defined in Example 29.

[0015] Unless otherwise specified, "complementary" sequences are perfectly complementary.

Thus, 5 ′ ESTs in a cDNA library in which one or more 5 ′ ESTs account for more than 5% of the number of nucleic acid inserts in the backbone molecule are “enriched” as defined herein. Recombinant 5'EST ". Similarly, 5 ′ ESTs in a plasmid population into which one or more 5 ′ ESTs of the invention have been inserted, such that the 5 ′ ESTs of the invention are at least 5% of the number of inserts in the plasmid backbone, are described herein. "Enriched recombinant 5'EST" as defined in However, cDNA libraries in which 5 ′ ESTs make up less than 5% of the number of nucleic acid inserts in the skeletal molecule population, eg, 5 ′ ESTs in libraries where skeletal molecules with 5 ′ EST inserts are extremely rare, are “enriched” It is not "recombinant 5'EST".

In particular, the present invention relates to a 5 ′ ES derived from a gene encoding a secreted protein.
About T. As used herein, a “secreted” protein is transported across or across membranes, including as a result of transport of a signal peptide in an amino acid sequence, when expressed in a suitable host cell. It is a protein. “Secreted” proteins include, but are not limited to, proteins that are totally (eg, soluble proteins) or partially (eg, receptors) secreted from the cells in which they are expressed. "Secreted" proteins also include, but are not limited to, proteins that are transported across the endoplasmic reticulum membrane.

Such 5 ′ ESTs include a nucleic acid sequence called a signal sequence that encodes a signal peptide that directs extracellular secretion of the protein encoded by the gene from which the 5 ′ EST is derived. Generally, the signal peptide is located at the amino terminus of the secreted protein.

[0019] Secreted proteins are translated by ribosomes attached to the "rough" ER. Generally, secreted proteins are co-translatedly transported to the endoplasmic reticulum membrane. Binding of the ribosome to the endoplasmic reticulum during translation of secreted proteins is mediated by a signal peptide. Signal peptides are generally cleaved by co-translationally entering the endoplasmic reticulum. After delivery to the endoplasmic reticulum, the secreted protein may advance through the Golgi apparatus. In the Golgi apparatus, proteins can undergo post-translational modifications and then enter secretory vesicles that transport them from the cell membrane.

The 5 ′ EST of the present invention has several important uses. For example, using them, cDNA clones containing the full-length protein coding sequence of the corresponding gene product, including the genuine translation initiation site derived from the 5 'end of the coding sequence of the mRNA from which the 5' EST is derived. Can be obtained and expressed. These cDNAs are hereinafter referred to as "full length cDNAs". These cDNAs may also include DNA derived from mRNA sequences upstream of the translation start site. Full length cDN
The A sequence can be used to express a protein corresponding to the 5 'EST. As noted above, secreted proteins are of therapeutic importance. Therefore, the cDNA
Can be useful in treating or preventing various human conditions. 5 ′ ESTs can also be used to obtain the corresponding genomic DNA. The term “corresponding genomic DNA” refers to the mR from which the 5 ′ EST is derived.
Refers to genomic DNA encoding NA.

Alternatively, an extended cDNA encoding a portion of a secreted protein can be obtained and expressed using 5 ′ EST. This portion may include the signal peptide of the secreted protein or the mature protein produced when the signal peptide is cleaved. This portion may also include a polypeptide having at least 10 contiguous amino acids encoded by the extended or full length cDNA. Alternatively, this portion may include at least 15 contiguous amino acids encoded by the extended or full length cDNA. In some embodiments, this portion may include at least 25 contiguous amino acids encoded by the extended or full length cDNA. In other embodiments, the portion may include at least 40 contiguous amino acids encoded by the extended or full length cDNA.

The extended cDNA, the entire secreted protein encoded by the full-length cDNA, or at least 10 contiguous amino acids, at least 15 contiguous amino acids,
Antibodies that specifically recognize at least 25 contiguous amino acids or fragments thereof having at least 40 contiguous amino acids can also be obtained as set forth below. An antibody that specifically recognizes the mature protein formed when the signal peptide is cleaved can also be obtained as follows. Similarly, the extended cDNA
Alternatively, an antibody that specifically recognizes a signal peptide encoded by full-length cDNA can be obtained.

[0023] In some embodiments, the extended cDNA obtained using 5'EST includes a signal sequence. In another embodiment, the extended cDN obtained using 5'EST
A contains the entire coding sequence of the mature protein (ie, the protein formed when the signal polypeptide is cleaved). Extended cDNA obtained using 5 'ESTs may also include regulatory regions that control the amount, location or developmental stage of gene expression, upstream of the translation start site or downstream of the stop codon.

As mentioned above, secreted proteins are of therapeutic importance. Thus, proteins expressed from extended or full-length cDNAs obtained using 5 'ESTs may be useful in treating or preventing a variety of human conditions.

5 ′ ESTs (or cDNA or genomic DNA obtained therefrom) may be used to identify individuals with forensic techniques or individuals with a genetic disease due to abnormal expression of a gene corresponding to 5 ′ ESTs. Can also be used for the diagnostic method. Further, the present invention is useful for constructing a high-resolution human chromosome map.

The present invention also relates to secretion vectors capable of directing secretion of the protein of interest. Such a vector can be used in a gene therapy method in which it is desired to produce a gene product to be delivered to another site in a living body in one cell. Secretory vectors can also facilitate the purification of the protein of interest.

[0027] The present invention also relates to an expression vector capable of directing the expression of the inserted gene at a desired location, at a desired time, or in a desired amount. Such a vector may include sequences upstream of the 5 'EST, such as a promoter or upstream regulatory sequences.

Finally, the present invention can be used for gene therapy for preventing or treating a genetic disease. Signal peptides can also be fused to heterologous proteins to direct their extracellular secretion.

The 5 ′ EST of the present invention (SEQ ID NO: currently stored at 80 ° C. in 4% (v / v) glycerol in our laboratory with the name listed next to the SEQ ID NO in II) 38-184). Bacterial clone containing a Bluescript plasmid with an insert containing Inserts can be recovered from the deposit by growing an appropriate clone on a suitable medium. The Bluescript DNA can then be isolated using plasmid isolation methods well known to those skilled in the art, such as alkaline lysis miniprep or large scale alkaline lysis plasmid isolation. If desired, the plasmid DNA may be further concentrated by centrifugation on a cesium chloride gradient, size exclusion chromatography or anion exchange chromatography. The plasmid DNA obtained using these techniques can then be manipulated using standard cloning techniques well known to those skilled in the art. Or E
PCR can be performed using primers designed at both ends of the ST insert. Then, using standard cloning techniques well known to those skilled in the art, the 5'EST
Can be manipulated.

One aspect of the present invention is a purified or isolated nucleic acid having one of SEQ ID NOs: 38-184 or a sequence complementary thereto. In one embodiment, the nucleic acids are recombinant.

Another aspect of the invention is a purified or isolated nucleic acid comprising at least 10 contiguous bases of one of the sequences of SEQ ID NOs: 38-184 or a sequence complementary thereto.

[0032] Yet another aspect of the invention is a purified or isolated nucleic acid comprising at least 15 contiguous bases of one of the sequences of SEQ ID NOs: 38-184 or one of the sequences complementary thereto. In one embodiment, the nucleic acids are recombinant.

[0033] Still another aspect of the present invention is that at least one of the sequences of SEQ ID NOs: 38-184 can hybridize under stringent conditions to one of the sequences complementary to the sequence of SEQ ID NOs: 38-184. It is a purified or isolated nucleic acid of 15 bases. In one embodiment, the nucleic acids are recombinant.

Yet another aspect of the present invention is a purified or isolated nucleic acid encoding a human gene product having a sequence partially encoded by one of the sequences of SEQ ID NOs: 38-184.

Yet another aspect of the invention is a method of making a cDNA encoding a human secreted protein partially encoded by one of SEQ ID NOs: 38-184. The method comprises contacting a collection of mRNA molecules from a human cell with a primer comprising at least 15 contiguous nucleotides of a sequence complementary to one of SEQ ID NOs: 38-184, wherein the primer is contained in the collection. MRNA encoding the protein
And a first cDNA strand is prepared from the mRNA by reverse transcription of the hybridized primer, a second cDNA strand complementary to the first cDNA strand is produced, and the first cDNA strand is produced. And isolating the resulting cDNA encoding the protein, comprising the steps of:

Another aspect of the present invention encodes a protein secreted by one of SEQ ID NOs: 38-184 or a human secreted protein comprising a fragment thereof consisting of at least 10 amino acids, obtained by the method described in the preceding paragraph. It is a purified or isolated cDNA. In one embodiment, the cDNA comprises the entire protein coding sequence of said protein, the sequence of which is partially included in one of the sequences of SEQ ID NOs: 38-184.

[0037] Another aspect of the invention is a method of making a cDNA encoding a human secreted protein partially encoded by one of SEQ ID NOs: 38-184. The method comprises obtaining a cDNA comprising one of the sequences of SEQ ID NOs: 38-184 and detecting a cDNA comprising at least 15 consecutive nucleotides of said cDNA and said sequence of SEQ ID NOs: 38-184 or a sequence complementary thereto. A probe and the probe
Contacting under conditions that hybridize to the DNA, identifying a cDNA that hybridizes to the detectable probe, and isolating the cDNA that hybridizes to the probe.

Another aspect of the present invention relates to a purified or purified human secreted protein comprising a protein encoded by one of SEQ ID NOs: 38-184 or a fragment of at least 10 amino acids, obtained by the method described in the preceding paragraph. Isolated cD
NA. In one embodiment, the cDNA comprises one of the sequences of SEQ ID NOs: 38-184.
And the entire protein coding sequence partially contained therein.

Another aspect of the present invention is a method for producing a cDNA comprising one of the sequences of SEQ ID NOs: 38-184. The method comprises assembling a collection of mRNA molecules from a human cell and
Contacting a first primer capable of hybridizing to the poly-A tail of RNA, hybridizing the first primer to the poly-A tail, reverse transcription of the mRNA to produce a first cDNA strand, Generating a second cDNA strand complementary to said first cDNA strand using at least one primer comprising at least 15 nucleotides of one of the sequences of numbers 38-184;
isolating a cDNA comprising a cDNA strand and said second cDNA strand.

Another aspect of the invention is a purification of a human secreted protein comprising a protein encoded by one of SEQ ID NOs: 38-184 or a fragment thereof of at least 10 amino acids and obtained by the method described in the preceding paragraph. Alternatively, it is an isolated cDNA. In one embodiment, the cDNA comprises the entire protein coding sequence partially contained in one of the sequences of SEQ ID NOs: 38-184.

In one embodiment of the method described in the preceding two paragraphs, the second cDNA strand is prepared by adding, to the first cDNA strand, at least 15 contiguous nucleotides of one of the sequences SEQ ID NOs: 38-184. A first primer pair including a second primer including a second primer and a third primer having a sequence included in the sequence of the first primer, and using the first pair of nested primers to contact the first primer pair.
Performing a polymerase chain reaction to produce a first PCR product, wherein the first PC
The R product comprises a fourth primer comprising at least 15 contiguous nucleotides of said sequence of one of SEQ ID NOs: 38-184 and capable of hybridizing to a sequence in said first PCR product and Fifth that can hybridize to the sequence
By contacting a second pair of primers, including the first and second primers, and performing a second polymerase chain reaction to produce a second PCR product.

One aspect of the present invention is a human secreted protein comprising a protein encoded by one of SEQ ID NOs: 38-184 or a fragment thereof of at least 10 amino acids, wherein the isolation or purification is obtained by the method of the preceding paragraph. CDNA. In one embodiment, the cDNA comprises the entire protein coding sequence partially contained in one of the sequences of SEQ ID NOs: 38-184.

In another embodiment of the present invention, the second cDNA strand comprises the first cDNA strand as SEQ ID NO: 3.
Contacting a second primer comprising at least 15 contiguous nucleotides of the sequence of 8-184, hybridizing said second primer to said first cDNA strand,
The method described in the preceding four paragraphs, wherein the method is made by extending the hybridized second primer to form the second cDNA strand.

Another aspect of the present invention encodes a protein partially encoded by one of SEQ ID NOs: 38-184 or a human secreted protein comprising a fragment of at least 10 amino acids and obtained by the method described in the preceding paragraph. Isolated or purified cDNA. In one embodiment, the cDNA comprises the entire protein coding sequence partially contained in one of the sequences of SEQ ID NOs: 38-184.

Another aspect of the invention is a method of making a protein comprising one of the sequences of SEQ ID NOs: 185-331. This method comprises obtaining a cDNA encoding the entire protein sequence partially contained in one of the sequences of SEQ ID NOs: 38-184, and expressing the cDNA such that the cDNA is operably linked to a promoter. Inserting the expression vector into a host cell, thereby causing the host cell to produce the protein encoded by the cDNA, and isolating the protein.

Another aspect of the present invention is an isolated protein obtained by the method described in the preceding paragraph.

Another embodiment of the present invention is a method for obtaining a promoter DNA. This method involves the use of a DN located upstream of the nucleic acids of SEQ ID NOs: 38-184 or a sequence complementary thereto.
A, obtaining said A, screening said upstream DNA to identify a promoter capable of directing transcription initiation, and isolating said DNA containing said identified promoter. In one embodiment, the obtaining step comprises chromosomal walking from said nucleic acid of SEQ ID NOs: 38-184 or a sequence complementary thereto. In another embodiment, the screening step comprises inserting the upstream sequence into a promoter reporter vector. In another embodiment, the screening step comprises identifying a motif in said upstream DNA that is a transcription factor binding site or a transcription initiation site.

Another aspect of the present invention is an isolated promoter obtained by the above method.

Another aspect of the invention is an isolated or purified protein comprising one of the sequences of SEQ ID NOs: 185-331.

Another aspect of the present invention provides that an individual EST or an array of fragments having a chain length of at least 15 nucleotides comprises at least one of the sequences of SEQ ID NOs: 38-184.
Or one of the sequences complementary to the sequence of SEQ ID NOs: 38-184, or a fragment thereof of at least 15 contiguous nucleotides.
In one embodiment, the array is a sequence of SEQ ID NOs: 38-184, SEQ ID NOs: 38-1.
84 or at least two of its fragments of at least 15 contiguous nucleotides. In another embodiment, the array comprises at least five of the sequences of SEQ ID NOs: 38-184, a sequence complementary to the sequence of SEQ ID NOs: 38-184, or a fragment of at least 15 contiguous nucleotides.

Another aspect of the present invention is a promoter having a sequence selected from the group consisting of SEQ ID NOs: 31, 34 and 37. Detailed Description of the Preferred Embodiments Table IV shows all human Swiss plots (SwissProt) for examining the frequency of false positives and false negatives using the signal peptide identification method described herein.
2) Analysis of the 43 amino acids at the N-terminus of the protein.

Table V shows the distribution of 5 ′ ESTs in each category described herein and the number of 5 ′ ESTs in each category with a given minimum Von Heijne score.

Table VI shows the distribution of 5 ′ ESTs in each of the categories described herein with respect to the tissue from which the corresponding mRNA 5 ′ ESTs were obtained.

Table VII shows the transcription factor binding sites present on each of these promoters. I. 'In order to obtain the 5'EST General Methods The present invention for obtaining the 5'EST derived from mRNA with terminal, complete 5' full 5 must obtain the mRNA with a terminal. Currently, there are two methods for obtaining such mRNAs having a complete 5 'end, either chemically (1) or enzymatic (2), as described below. 1. Chemical Methods for Obtaining mRNA with Complete 5 'End One of these methods is to derivatize the 5' end of
This is a chemical modification method including selecting NA. The 5 'end of eukaryotic mRNA has a structure called a "cap" containing guanosine methylated at position 7. The cap binds to the first transcribed base of the mRNA via a 5 ', 5'-triphosphate linkage. The 5 'guanosine may be methylated at both the 2 and 7 positions. Rarely, 5 'guanosine is trimethylated at positions 2, 7, and 7. In a chemical method for obtaining mRNA having a complete 5 'end, the 5' cap is specifically derivatized and attached to a reactive group on a support for immobilization. In this specific derivatization, only ribose that binds to the methylated guanosine at the 5 ′ end of mRNA and ribose that binds to the base at the 3 ′ end of mRNA are 2 ′.
, 3'-cis diol.

Optionally, chemically modifying the 2 ′, 3′-cis diol of the 3 ′ terminal ribose
By substitution, conversion or removal of mRNA having 2 ', 3'-cis diol
Only ribose binding to the methylated guanosine at the 5 'end can be left. 3
Utilizes various techniques to remove the 2 ', 3'-cis diol of the' terminal ribose
be able to. For example, using controlled alkaline hydrolysis,
Is 3'-phosphate, 2'-phosphate or (2 ', 3')-cyclo
An mRNA fragment that is a phosphate can be made. Since then
Of the fragment containing 3'-ribose with the oligo dT column
Can be removed from the mixture. Alternatively, 2 ', 3'-c
Using RNA ligase such as T4 RNA ligase
To the 3 'end of the mRNA. In Example 1 below, the message
Describes a method for ligating a nucleoside diphosphate to the 3 'end of a sender RNA.
Put on.Example 1 Ligation of nucleoside diphosphate pCp to the 3 'end of mRNA 5U T in buffer provided by the manufacturer (Gibco BRL) _Four Phage RNA ligase, 40 U RNase inhibitor RNasin (Promega) and 2 μl³²pCp (Amersham
) 1 μg RNA in 10 μl final reaction medium in the presence of # PB10208)
Incubated. Incubate at 37 ° C for 2 hours or at 7-8 ° C
Performed overnight.

After modification or removal of the 2 ′, 3′-cis diol of 3 ′ ribose, the 2 ′, 3′-cis diol present at the 5 ′ end of the mRNA is replaced with NaBH ₄ , NaBH ₃ CN or sodium periodate. Oxidation using the reagent of 2 ′,
3'-cis diol can be converted to dialdehyde. Example 2 describes the oxidation of 2 ′, 3′-cis diol at the 5 ′ end of mRNA using sodium periodate. Example 2 '2 of the 5' end of the mRNA with sodium periodate, 3'-cis Jio Le oxide 47 nucleotides of the capped oligoribonucleotides (including cap)
Or 0.1 OD of a 46 nucleotide uncapped oligoribonucleotide
The units were processed as follows. Transfer kit "Ampliscribe T7" (
Epicenter Technologies
Oligoribonucleotides were made by in vitro transcription using s)). As shown below, the DNA template of the RNA transcript contained one cytosine. In order to synthesize uncapped RNA, all four NTPs were added in
Added during the in vitro transcription reaction. To obtain capped RNA, GTP was exchanged for the cap analog m7G (5 ') ppp (5') G. This compound, recognized by the polymerase, was introduced at the 5 'end of the nascent transcript at the start of transcription, but not during the extension step. As a result, the resulting RNA had a cap at its 5 'end. The sequence of the oligoribonucleotides generated by the in vitro transcription reaction was as follows: + cap: 5'm7GpppGCAUCUCAUCCCCAUCCAAAUCCACCCU
AACUCCCUCCAUCUCCAC-3 '(SEQ ID NO: 1) -Cap: 5'-pppGCAUCUCAUCCCCAUCCAAUUCCACCCUAA
CUCCUCCCAUCUCCAC-3 ′ (SEQ ID NO: 2) Oligoribonucleotide was added to 9 μl of acetate buffer (0.1 M sodium acetate, p
H5.2) and 3 μl immediately after preparation were dissolved in 0.1 M sodium periodate solution. The mixture was incubated at 4 ° C or room temperature protected from light for 1 hour. Thereafter, 4 μl of 10% ethylene glycol was added to stop the reaction. The product was precipitated with ethanol, dissolved in at least 10 μl of water or a suitable buffer, and dialyzed against water.

The resulting aldehyde group can then be attached to a molecule having a reactive amine group such as a hydrazine, carbazide, thiocarbazide or semicarbazide group to facilitate enrichment of the 5 ′ end of the mRNA. M with a complete 5 'end
Suitable molecules having a reactive amine group for use in selecting RNA include avidin, proteins, antibodies, vitamins, ligands or oligonucleotides capable of specifically binding to receptor molecules. Example 3 describes the coupling of the resulting dialdehyde to biotin. Example 3 Coupling of dialdehyde at the 5 'end of the transcript with biotin The oxidation product obtained in Example 2 was treated with
And the formula:

[0058]

Embedded image Was dissolved in 50 μl of a freshly prepared 0.02 M solution added to a methoxyethanol / water mixture (1: 1). Example 4 Specificity of the Biotinylation Reaction of Capped Transcripts The specificity of the biotinylation reaction of capped mRNA was evaluated by gel electrophoresis of the following samples: 46 nucleotide uncapped in vitro transcript prepared as in Example 2 and labeled with ³² pCp as described in Example 1.

Sample 2. Prepared as in Example 2, labeled with ³² pCp as described in Example 1, treated with the oxidation reaction of Example 2, and subjected to the biotinylation conditions of Example 3 uncapped in vitro of 46 nucleotides. Transcript.

Sample 3. 47 nucleotide capped in vitro transcript prepared as in Example 2 and labeled with ³² pCp as described in Example 1.

Sample 4. Capped in vitro transcription of 47 nucleotides prepared as in Example 2, labeled with ³² pCp as described in Example 1, treated with the oxidation reaction of Example 2, and subjected to the biotinylation conditions of Example 3. object.

Samples 1 and 2 had the same moving speed. This indicates that the uncapped RNA was neither oxidized nor biotinylated. Sample 3 moved slower than Samples 1 and 2, while Sample 4 moved slowest. The difference in the mobility of the RNA of Samples 3 and 4 is
It shows that capped RNA was specifically biotinylated.

In some cases, complete binding of the molecule with reactive amine groups to the inside of a container containing the mRNA, magnetic beads, a chromatography matrix or a suitable solid support such as a nylon or nitrocellulose membrane is accomplished. The mRNA having the 5 'end may be concentrated. For example, when the molecule having a reactive amine group is biotin, avidin or streptavidin is bound to the solid support. Alternatively, when the molecule having a reactive amine group is an antibody or a receptor ligand, a cognate antigen or receptor is bound to the solid support. Finally, if the molecule having a reactive amine group comprises an oligonucleotide, the solid support can comprise a complementary oligonucleotide.

[0064] mRNA with a complete 5 'end can be released from the solid phase after the enrichment method. For example, if the dialdehyde binds to biotin hydrazide and the solid phase contains streptavidin, the mRNA can be released from the solid phase simply by heating to a temperature of up to 95 ° C. in 2% SDS. In some methods, after enrichment, the molecule with a reactive amine group may be cleaved from mRNA with a complete 5 'end. Example 5 describes capture of biotinylated mRNA by beads coated with streptavidin and release of biotinylated mRNA from the beads after concentration. Example 5 Capture and Release of Biotinylated mRNA Using Streptavidin-Coated Beads Streptavidin-coated magnetic beads were prepared according to the manufacturer's instructions (CPG In
c. , United States). Biotinylated mRNA was added to the hybridization buffer (1.5 M NaCl, pH 5-6). After a 30 minute incubation, unbound and unbiotinylated material was removed. Then add 1% beads
The plate was washed several times with water to which SDS was added. The beads obtained in this manner were subjected to 2% SD
Incubated for 15 minutes at 95 ° C. in water containing S.

Example 6 recovers biotinylated mRNA from streptavidin-coated beads
Clarify the efficiency ofExample 6 Recovery efficiency of biotinylated mRNA The efficiency of the recovery procedure was evaluated as follows. Capped RNA as above ³² Labeled with pCp, oxidized, biotinylated and bound to streptavidin-coated beads
I combined. Thereafter, the bound RNA was removed at 95 ° C. in the presence of 2% SDS for 5, 15, or
Was incubated for 30 minutes.

The reaction products were analyzed by 12% polyacrylamide gel electrophoresis under denaturing conditions (7M urea). The gel was subjected to autoradiography. During this operation, the hydrazone linkage was not reduced.

As the incubation time in 2% SDS increased, the amount of recovered nucleic acid increased. This indicates that the biotinylated mRNA was efficiently recovered.

Another method for obtaining mRNA with a complete 5 ′ end is to derive an oligonucleotide derivatized to contain a reactive amine group with a fully capped mR
Specific binding to NA. Preferably, as described above, prior to the step of attaching the aldehyde group to the derivatized oligonucleotide, the 3 'end of the mRNA is blocked so that the derivatized oligonucleotide does not attach to the 3' end of the mRNA. For example, using the T4 RNA ligase described in Example 1 to convert pCp to mRN
A can be attached to the 3 'end. However, as described above, the 3 'of the mRNA
The step of blocking the ends is an optional step. Derivatized oligonucleotides can be prepared as described in Example 7. Example 7 Derivatization of Oligonucleotides Oligonucleotides whose 3 ′ terminus has been phosphorylated are treated overnight at 8 ° C. with about 1-3 M hydrazine or an aqueous solution of dihydrazide of the formula H ₂ N (R1) NH ₂ at pH 4.5. By doing so, it was converted to 3 'hydrazide on the 3' side. This incubation was performed in the presence of a 0.3 M final concentration of an aqueous solution of a carbodiimide-type reagent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

[0069] The derivatized oligonucleotide was then separated from other reagents and products using standard techniques for isolating the oligonucleotide.

As described above, the mRNA to be enriched is replaced with the 3 ′ OH that may be present in the mRNA.
It can be treated to remove the group. This can be performed by enzymatic ligation of sequences without 3'OH, such as pCp, as described in Example 1. Alternatively, the 3'OH group may be removed by alkaline hydrolysis as described in Example 8 below. Example 8 Removal of the 3'OH group of mRNA using alkaline hydrolysis 1.5 [mu] g of mRNA was incubated at 4 [deg.] C for 40-60 minutes in a total volume of 100 [mu] l of 0.1 N sodium hydroxide. This solution was neutralized with acetic acid and precipitated with ethanol.

After optional removal of the 3 ′ OH group, the diol group at the 5 ′ end of the mRNA is oxidized as described in Example 9 below. Example 9 Oxidation of Diol of mRNA Up to 1 OD unit of RNA was added to 9 μl of buffer (0.1 M sodium acetate, pH
6-7) or 3 μl of water and 0.1 μM sodium periodate solution immediately after preparation. The reaction was incubated for 1 hour at 4 ° C. or room temperature protected from light. After incubation, the reaction was stopped by adding 4 μl of 10% ethylene glycol. Thereafter, the mixture was incubated at room temperature for 15 minutes. After precipitation with ethanol, the product was dissolved in at least 10 μl of water or a suitable buffer and dialyzed against water.

After oxidizing the diol group at the 5 ′ end of the mRNA, the derivatized oligonucleotide was conjugated to the aldehyde obtained as described in Example 10. Example 10 Binding of mRNA to Aldehyde Derivatized Oligonucleotides Oxidized mRNA was dissolved in 50 μl of an acidic medium such as sodium acetate pH 4-6. 50 μl of a solution of the derivatized oligonucleotide was added to give a 1:20 ratio of mRNA: derivatized oligonucleotide. The mixture was reduced with borohydride and incubated at 37 ° C. for 2 hours or at 10 ° C. overnight (14 hours). The mixture was then precipitated with ethanol, dissolved in 10 μl or more of water or a suitable buffer, and dialyzed against distilled water. If desired, the resulting product can be analyzed using acrylamide gel electrophoresis, HPLC analysis or other conventional techniques.

After binding the derivatized oligonucleotide to the mRNA, a reverse transcription reaction can be performed as described in Example 11 below. Example 11 Reverse transcribed oligodeoxyribonucleotides of mRNA bound to derivatized oligonucleotides were derivatized as follows. Sequence 5'ATCAAGAATTCGCACGAGACC having 5'OH end and 3'-P end
A 3 OD unit of the oligodeoxyribonucleotide of ATTA 3 '(SEQ ID NO: 3) was prepared in dimethylformamide / water (75:25) containing 2 μg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. Dissolved in 70 μl of 5M hydroxybenzotriazole solution pH 5.3. Mix 22
Incubation was carried out for 2 hours and 30 minutes at ℃, followed by precipitation twice with LiClO ₄ / acetone. Dissolve the pellet in 200 μl of 0.25 M hydrazine and
Incubated for 14 hours. After the hydrazine reaction, the mixture was precipitated twice with LiClO ₄ / acetone.

The messenger RNA to be reverse transcribed was stored at −80 ° C., 2 cm on a side.
Extracted from placental blocks. Total RNA using traditional acidic phenol techniques
Was extracted. The mRNA was purified using oligo-dT chromatography.
mRNA integrity was checked by Northern blot.

The diol group of 7 μg of mRNA from placenta was oxidized as described in Example 9. The derivatized oligonucleotide was bound to the mRNA as described in Example 10 above, except that the precipitation step was replaced by an exclusion chromatography step to remove derivatized oligodeoxyribonucleotides that did not bind to the mRNA. Exclusion chromatography was performed as follows: 10 ml of Ultragel A, a mixture of agarose and acrylamide.
cA34 (BioSepra # 230151) gel was run at 10 m
M Tris pH 8.0, 300 mM NaCl, 1 mM EDTA and 0.0
Equilibrated in 50 ml of 5% SDS solution. The mixture was allowed to settle. The supernatant was removed and the gel was suspended in 50 ml of buffer. This procedure was repeated two or three times.

The glass beads (diameter 3 mm) were placed in a 2 ml disposable pipette (length 25 cm). The pipette was filled with the gel suspension until the gel height stabilized at 1 cm from the top of the pipette. The column was then washed with 20 ml of equilibration buffer (10 mM
Tris HCl pH 7.4, 20 mM NaCl).

10 μl of mRNA reacted with the derivatized oligonucleotide, 39 μl of 10 mM urea, and 5 mg of bromophenol blue in 60% glycerol (v / v)
And the mixture was mixed in 2 μl of blue-glycerol buffer prepared by passing through a 0.45 μm diameter filter.

Next, the mRNA bound to the oligonucleotide was loaded on the column. Equilibration buffer was added at the same time as the sample penetrated. Then, 100 μl fractions were collected. m
Derivatized oligonucleotides that did not bind to RNA appeared in fraction 16 or later. Therefore, fractions 3 to 15 were combined and precipitated with ethanol.

To determine if the derivatized oligonucleotide did indeed bind to the mRNA, one-tenth of the combined fractions were spotted twice on a nylon membrane and radiolabeled using conventional techniques. And hybridized. The ³² P-labeled probe used for these hybridizations has a sequence 5 ′ TAATGGTC that is anticomplementary to the derivatized oligonucleotide.
It was an oligodeoxyribonucleotide of TCGTGCGAATTCTTGAT3 ′ (SEQ ID NO: 4). The signal observed by autoradiography indicated that the derivatized oligonucleotide was indeed bound to the mRNA.

The remaining 9/10 of the mRNA reacted with the derivatized oligonucleotide was reverse transcribed as follows. The reverse transcription reaction was performed according to the manufacturer's instructions using reverse transcriptase and 50 pmol of random sequence nonamers as primers.

To ensure that reverse transcription was performed through the cap structure, two types of experiments were performed.

In the first method, cDNA obtained from a reverse transcription reaction by alkaline hydrolysis
: After removing the RNA of the RNA heteroduplex, a part of the obtained single-stranded cDNA was spotted on a positively charged membrane, and the same sequence as that of the derivatized oligonucleotide was obtained using a conventional method. Was hybridized to a ³² P-labeled probe having 1 pmol, 100 fmol, 50 fmol, 10 fmol and 1 fmo of a control oligodeoxyribonucleotide having the same sequence as that of the derivatized oligonucleotide.
A control spot containing 1 was added. The signal observed in the spot containing the cDNA indicated that about 15 fmol of the derivatized oligonucleotide was reverse transcribed. These results indicate that reverse transcription is performed through the cap, especially that the reverse transcriptase crosses the 5'-PPPP-5 'linkage of the eukaryotic messenger RNA cap.

In the second type of experiment, the single-stranded cDNA obtained in the first strand synthesis was
Used as template for R reaction. Two types of reactions were performed. First, specific amplification of mRNA for α-globin, dehydrogenase, pp15 and elongation factor E4 was performed using the following oligodeoxyribonucleotide primer pairs. α-globin GLO-S: 5'CCGACAAGACCAACGTCAACGCCGC3 '(SEQ ID NO: 5) GLO-As: 5'TCACCAGCAGGCAGTGGCTTAGGAG3' (SEQ ID NO: 6) dehydrogenase 3DH-S: 5'AGTGATTCCTGCTACTTTGGATGGC3 '(SEQ ID NO: 7) 3DH-As: 5'GCTTGGTCTTGTTCTGGAGTTTAGA3' (SEQ ID NO: 8) pp15 PP15-S: 5 ′ TCCAGAATGGGAGACAAGCCAATTTT 3 ′ (SEQ ID NO: 9) PP15-As: 5 ′ AGGGAGGAGGAAACAGCGTGGAGTCC3
'(SEQ ID NO: 10) Elongation factor E4 EFA1-S: 5' ATGGGAAAGGAAAAGACTCATATCA3 '(SEQ ID NO: 11) EF1A-As: 5'AGCAGCAACAATCAGGACAGCACAG3
'(SEQ ID NO: 12) Second, a primer (5'ATC) derived from the sequence of the pair of antisense oligodeoxyribonucleotides and derivatized oligodeoxyribonucleotides described above.
Non-specific amplification was also performed using AAGAATTCGCACGAGACCATTA3 ′) (SEQ ID NO: 13).

One-twentieth of the following samples of RT-PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide.

Sample 1: Product of a PCR reaction using the globin primers of SEQ ID NOS: 5 and 6 in the presence of cDNA.

Sample 2: Product of a PCR reaction using the globin primers of SEQ ID NOs: 5 and 6 in the absence of added cDNA.

Sample 3: Product of a PCR reaction using the dehydrogenase primers of SEQ ID NOs: 7 and 8 in the presence of cDNA.

Sample 4: Product of a PCR reaction using the dehydrogenase primers of SEQ ID NOs: 7 and 8 in the absence of added cDNA.

Sample 5: Product of a PCR reaction using the pp15 primers of SEQ ID NOS: 9 and 10 in the presence of cDNA.

Sample 6: Product of a PCR reaction using the pp15 primers of SEQ ID NOs: 9 and 10 in the absence of added cDNA.

Sample 7: Product of a PCR reaction using the EIF4 primers of SEQ ID NOS: 11 and 12 in the presence of cDNA.

Sample 8: Product of a PCR reaction using the EIF4 primers of SEQ ID NOs: 11 and 12 in the absence of added cDNA.

[0093] Bands of the expected size of the PCR product were observed only in samples 1, 3, 5, and 7, indicating that the corresponding sequence was present in the cDNA population.

In addition, a PCR reaction was performed using antisense oligonucleotides of globin and dehydrogenase primers (SEQ ID NOs: 6 and 8) and oligonucleotides whose sequences corresponded to derivatized oligonucleotides. The presence of the expected size PCR product in samples equivalent to Samples 1 and 3 above indicated that the derivatized oligonucleotide had bound to the mRNA.

[0095] The above example shows that for the one with the full 5 'end, as shown in FIG.
It summarizes chemical techniques for enriching RNA. Such mRNA
Further details regarding the chemical methods for obtaining are disclosed in International Application No. WO 96/34981 published November 7, 1996, which is incorporated herein by reference. Using methods based on the above chemical modifications to the 5 'cap structure, cDNAs selected to include the 5' end of the mRNA (from which the cDNA is derived) can be made. In one form of such an approach, m
The 5 'end of the RNA is modified as described above. Thereafter, a reverse transcription reaction is performed, and m
Extend a primer complementary to the 5 'end of the RNA. Remove single-stranded RNA,
A population of cDNA / mRNA heteroduplexes whose mRNA contains the complete 5 'end is obtained. The resulting heteroduplex is captured on a solid phase. This solid phase is coated with a molecule that can interact with the molecule used to derivatize the 5 'end of the mRNA.
Thereafter, the heteroduplex is separated to form a single-stranded first cD containing the 5 'end of the mRNA.
Recover the NA chain. The second strand cDNA synthesis then proceeds using conventional techniques. For example, WO 96, the disclosure of which is incorporated herein by reference.
/ 34981 or Carninci et al., Genomics 3
7: 327-336, 1996, can be used to select a cDNA containing a sequence derived from the 5 'end of the mRNA coding sequence.

After attaching the oligonucleotide tag to the 5 ′ cap of the mRNA, a reverse transcription reaction is performed to extend a primer complementary to the mRNA to the 5 ′ end of the mRNA. After removing the RNA component of the resulting heteroduplex using standard techniques,
Second strand cDNA synthesis is performed using primers complementary to the oligonucleotide tag. 2. Enzymatic Method for Obtaining mRNA with Complete 5 'End Another technique for selecting cDNA that extends to the 5' end of the mRNA from which the cDNA is derived is a completely enzymatic method. Some forms of these techniques are:
Dumas Milne Edwards J.C., the disclosure of which is incorporated herein by reference. B. (Paris V
Doctoral dissertation from University of I, Le clone des ADNc commons
: Difficults et perspectives noubell
es. Apartments pour l'etude de la regul
ation de l'expression de la tryptoph
ane hydroxylase de rat, December 20, 1993),
EP 625572 and Kato et al., Gene 150: 24.
3-250, 1994.

Briefly, in such a method, the isolated mRNA is treated with alkaline phosphatase to remove the phosphate present at the 5 'end of the uncapped incomplete mRNA. After this procedure, the cap present on the full length mRNA is replaced by T
4 Enzymatic removal with a decapping enzyme such as polynucleotide kinase or tobacco acid pyrophosphatase. The oligonucleotide, which may be either a DNA oligonucleotide or a DNA-RNA hybrid oligonucleotide having RNA at the 3 'end, is then converted to the phosphate present at the 5' end of the uncapped mRNA using T4 RNA ligase. Join. Oligonucleotides may contain restriction sites to facilitate cloning of the cDNA after synthesis. Example 12 below describes one enzymatic method based on the doctoral dissertation of Dumas. Example 12 Enzymatic Method to Obtain 5'EST 20 μl using bovine intestinal phosphatase (Biolabs)
g of poly A + RNA was dephosphorylated. After extraction with phenol-chloroform, the cap structure of the mRNA was converted to tobacco acid pyrophosphatase (Shins
hi) et al., Biochemistry 15: 2185-2190, purified as described in 1976), and hydrolyzed to produce a non-phosphorylated 5'-end,
An adenosine ribophosphate extension at the 3 'end and EcoR near the 5' end
A hemi 5 'DNA / RNA-3' oligonucleotide having an I site was
A ligase (Biolabs) was used to ligate to the 5'P end of the mRNA. Oligonucleotides suitable for use in this technique are preferably 30-50 bases in length. By adding a fluorescent dye to the 5 'end,
Oligonucleotides having a non-phosphorylated 5 'end can be synthesized. The addition of an adenosine ribophosphate extension to the 3 'end of the oligonucleotide increases ligation efficiency. It will be appreciated that the oligonucleotide may have a cloning site other than EcoRI.

After ligation of the oligonucleotide to the phosphate present at the 5 ′ end of the uncapped mRNA, EP 625,572 and EP 625,572, the conventional methods of which or their disclosures are incorporated herein by reference. Kato et al., Supra, and Dumas Milne Edwards.
The synthesis of the first and second strand cDNAs is performed using the methods specified in, supra. The disclosure of which is then incorporated herein by reference.
Sambrook et al., Molecular Cloning: Laboratory Cloning: A Laboratory Ma.
Nual), 2nd edition, Cold Spring Harbor Laboratory Press (
Cold Spring Harbor Laboratory Press)
The resulting cDNA is ligated to a vector as disclosed in Kato et al., Supra, or other nucleic acid vectors well known to those skilled in the art, using techniques such as those described in J. Sci. be able to. II. Obtaining and Characterizing the 5 'ESTs of the Invention The 5' ESTs of the invention use the chemical and enzymatic methods described above to enrich mRNA for those having a complete 5 'end as described below. Was obtained. Obtaining 5 ′ ESTs Using mRNA with Complete 5 ′ EST First, mRNA was prepared as described in Example 13 below. Example 13 Preparation of mRNA with Complete 5 'EST Human total RNA or poly A + RNA from 29 different tissues were purchased from LABIMO and CLONETECH, respectively, and had 44 cDNAs as follows: Used to generate libraries. The purchased RNA was subjected to acidic guanidium thiocyanate-phenol-chloroform extraction (Chomczynski) and Satch (Sacc).
hi), Analytical Biochemistry 162: 156-
159, 1987) from cells or tissues. Poly A ⁺ RNA is used to remove ribosomal RNA from Aviv and Leder, Proc. Natl. Acad. Sci. USA69
: 1408-1412, 1972, isolated from total RNA (LABIMO) by two rounds of oligo dT chromatography.

The quality and integrity of poly A + RNA was examined. Northern blotting hybridized with a globin probe was used to confirm that the mRNA was not degraded. Contamination of polyA ⁺ mRNA by ribosomal sequences was examined using the Northern blot method and a probe derived from the sequence of 28S rRNA. Preparations of mRNA with less than 5% rRNA were used for library construction. Exogenous sequence (
PCR was used to examine the presence of bacterial 16S ribosomal sequences or the sequence of two highly expressed fungal mRNAs, so as not to build libraries with RNA contaminated by prokaryotes or fungi). .

After preparation of the mRNA, 5 ′ ESTs were obtained from various tissues using the chemical and / or enzymatic techniques described above to enrich the mRNA for those with a complete 5 ′ end. In both methods, the oligonucleotide tag is attached to the 5 'of the mRNA.
End bound. The oligonucleotide tag had an EcoRI site to facilitate the subsequent cloning procedure. The ligated oligonucleotides were designed using the same nucleotide sequence in a chemical and enzymatic manner to facilitate processing of the resulting single-stranded and double-stranded cDNA in the library construct. . However, in the chemical approach, the tag used was mR
Although the oligodeoxyribonucleotide was ligated to the cap of NA, but in enzymatic ligation, the tag was uncapped mRN as described in Example 12.
A was a chimeric hemi 5 'DNA / RNA 3' oligonucleotide linked to the 5 'end of A.

After binding the oligonucleotide tag to the mRNA, either by chemical or enzymatic methods, the oligonucleotide tag is probed with a probe complementary to the oligonucleotide tag.
The integrity of the mRNA was checked by performing a Northern blot with 00-500 ng of the mRNA before the first strand synthesis was performed as described in Example 14. Example 14 cDNA Synthesis Using mRNA Template with Complete 5 'End mR Attached to an Oligonucleotide Tag Using Chemical and Enzymatic Methods
For NA, Superscript II (Gibco BRL) or RNase H Minus M-MLV (Promega) reverse transcriptase and random nonammers (r
The synthesis of the first strand cDNA was carried out using an unomnonamer. To protect the internal EcoRI site of the cDNA from digestion in subsequent steps of this procedure, methylated dCTP was used for first strand synthesis. After removing the RNA by alkaline hydrolysis, the first strand cDNA was precipitated using isopropanol to remove the remaining primer.

In both chemical and enzymatic methods, Klenow (Klenow) was used using primers corresponding to the 5 ′ end of the ligated oligonucleotide described in
The second strand of the cDNA was synthesized with the Klenow fragment. Preferably,
The primer has a length of 20 to 25 bases. CDNA from digestion during cloning
Methylated dCTP was also used in the second strand synthesis to protect the internal EcoRI site therein.

After cDNA synthesis, the cDNA was converted to pBlueS as described in Example 15 below.
It was cloned into a script. Example 15 Cloning of cDNA Derived from mRNA with Complete 5 'End into BlueScript After synthesis of the second strand, both ends of the cDNA were blunted with T4 DNA polymerase (Biolabs) and the cDNA was digested with EcoRI. . cDN
Since methylated dCTP was used during A synthesis, the EcoRI site present in the tag was the only site for hemimethylation and was therefore the only site susceptible to EcoRI digestion. Subsequently, exclusion chromatography (AcA, Biosepra (B)
The size of the cDNA was fractionated using iosepra)) and the fractions corresponding to cDNA greater than 150 bp were collected and precipitated with ethanol. Phagemid p
The cDNA was directionally cloned into the SmaI and EcoRI ends of the BlueScript vector (Stratagene).
The ligation mixture was electroporated into the bacteria and grown under appropriate antibiotic selection.

As described in Example 16 below, clones with bound oligonucleotide tags were selected. Example 16 Selection of Clones with Oligonucleotide Tags Bound Plasmid DNA containing the 5 'EST library prepared as described above was purified (Qiagen). Positive selection of tagged clones was performed as follows. Briefly, in this selection procedure, the gene II endonuclease of phage F1 is converted to an exonuclease such as exonuclease III or T7 gene 6 exonuclease (Chang et al., Gen.
e 127: 95-8, 1993) to convert plasmid DNA to single-stranded DNA. Next, the obtained single-stranded DNA was subjected to Fry et al.
Purified using paramagnetic beads as described in iotechniques, 13: 124-131, 1992. In this procedure, single-stranded DNA was hybridized with a biotinylated oligonucleotide having a sequence corresponding to the three termini of the oligonucleotide described in Example 13. Preferably, the primers are 20-
It is 25 bases long. Clones containing sequences complementary to the biotinylated oligonucleotide were captured on streptavidin-coated magnetic beads by incubation and then magnetically selected. After capturing the positive clones, the plasmid DNA was released from the magnetic beads, and Amersham Pharmacia Biotech (
ThermoS from Amersham Pharmacia Biotech)
The DNA was converted to double-stranded DNA using a DNA polymethylase such as eqenase. Alternatively, Gibco BRL Gent Wrapper Kit (Gen
e Trapper kit) may be used. The double-stranded DNA was then introduced into the bacteria by electroporation. The percentage of positive clones with 5'-tagged oligonucleotides was estimated to generally range from 90-98% using dot blot analysis.

After electroporation, the libraries were arranged in 384-microtiter plates (MTP). A copy of the MTP was kept for future use. The library was then transferred to 96 MTP and sequenced as follows. Example 17 Sequencing of Inserts in Selected Clones Initially, standard SETA-A and SETA-B primers (Genset SA), AmpliTaqGold (Perkin-Elmer (P
erkin-Elmer), dNTPs (Boehringe)
r)), Perkin-Elmer C
Corporation) using the recommended buffer and cycling conditions.
9600 Thermocycler (Perkin-Elmer, Applied Biosys, Applied Biosystems Division)
PCR (Tems Division), Foster City, CA)
Amplified the plasmid insert.

The PCR products were then sequenced using an automated ABI Prism 377 sequencer (Perkin-Elmer). The sequencing reaction was performed using standard dye-primer chemistry and ThermoSeque.
case (Amersham Pharmacia Biotech (Amersham P
carried out using a PE9600 thermocycler with the aid of H. mariacia Biotech). Primers used were either T7 or 21M13 (Genset SA) as appropriate. Primers are JOE, F
Labeled with AM, ROX and TAMRA dyes. DNT used in the sequencing reaction
Ps and ddNTPs were purchased from Boehringer. Sequencing buffers, reagent concentrations and cycling conditions were as recommended by Amersham.

After the sequencing reaction, the samples were precipitated with ethanol, suspended in formamide loading buffer and loaded on a standard 4% acrylamide gel. Electrophoresis is ABI
Performed at 300 V for 2.5 hours on a 377 sequencer, sequence data collected,
Analyzed using BI Prism DNA sequencing analysis software, version 2.1.2. 2. Computer analysis of the resulting 5'EST: Construction of NetGene and SignalTag databases Sequence data of the 44 cDNA libraries prepared as described above were transferred to a proprietary database where quality control and validation steps were performed. Uni
A proprietary base-caller operating with the x system automatically flagged suspicious peaks, taking into account peak shape, peak-to-peak resolution and noise level. Proprietary base-caller
) Also performed automatic trimming. Any extension of no more than 25 bases with more than 4 suspect peaks was considered unreliable and discarded. The sequence corresponding to the cloning vector or linking oligonucleotide is automatically EST
Removed from the array. However, the obtained EST sequence may contain 1 to 5 bases belonging to the above sequence at the 5 'end. If necessary, they can be easily removed from case to case.

After sequencing as described above, the 5 ′ EST sequence was converted to NetGene ™, a proprietary database called for storage and manipulation as described below.
Added. Those of skill in the art would understand that data can be stored and manipulated in any medium that can be read and accessed by a computer. Computer readable media includes magnetically, and optionally, electronically readable media. For example, a computer-readable medium may be a hard disk, a floppy disk, a magnetic tape, a CD-ROM, a RAM or a ROM, and other types of media known to those skilled in the art.

The array data can be stored and manipulated in various formats by a data processor program. For example, sequence data is available from Microsoft
software) word (WORD) or word perfect (WORDPE)
RECT) as text in a word processing file or DB2, S
It can be stored as an ASCII file in various database programs known to those skilled in the art, such as YBASE or ORACLE.

[0110] The computer readable medium that stores the sequence information may be in a personal computer, network, server, or other computer system known to those of skill in the art. A computer or other system preferably includes the storage medium described above and a processor for accessing and manipulating the sequence data. Once the sequence data is stored, it can be manipulated and searched to locate the conserved sequence with the desired nucleic acid sequence or the protein encoding a protein with a particular functional domain. For example, conserved sequence information can be compared to other known sequences to identify motifs or structural motifs required for homology, biological function.

Programs that can be used to search or compare conserved sequences include MacPattern (EMBL), BLAST and BLAST2 program series (NCBI), nucleotides (BLASTN) and peptides (B
LASTX) Basic local alignment tool program for comparison (Altschul, J. Mol. Biol. 215: 403, 1).
990) and FASTA (Pearson and Lippman (Li
pman), Proc. Natl. Acad. Sci. USA 85: 2444
, 1988). The BLAST program extends the alignment based on defined match and mismatch criteria.

The motifs that can be detected using the programs described above and in Example 28 include leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, α-helices and β-sheets, It includes a signal sequence encoding a signal peptide that directs secretion of the encoded protein, a sequence involved in transcriptional regulation such as a homeobox, an acidic region, an enzyme active site, a substrate binding site, and an enzymatic cleavage site.

Before searching cDNAs in the NetGene ^™ database for sequence motifs of interest, identify cDNAs derived from uninteresting mRNAs and do not consider them further as described in Example 18 below. Was excluded. Example 18 Excluding Unwanted Sequences from Consideration Transfer RNA, ribosomal RNA, mitochondrial RNA, prokaryotic RNAs, fungal RNAs, Alu sequences, L1 sequences or 5 in the NetGene ^™ database from unwanted sequences such as repetitive sequences. 'ESTs were identified using the FASTA and BLASTN programs and the parameters listed in Table I.

To exclude the 5 ′ EST encoding tRNA from further consideration, the 5 ′ EST sequence was replaced with the 1190 well-known tRN from EMBL Release 38.
A sequence (100 of which are human). Comparisons were performed using FASTA on both strands of the 5'EST. Sequences with greater than 80% homology to more than 60 nucleotides were identified as tRNAs. Of the 144,341 sequences screened, 26 were identified as tRNAs and were excluded from further consideration.

To exclude the 5 ′ EST encoding rRNA from further consideration, the 5 ′ EST sequence was replaced with the 2497 known rRN from EMBL Release 38.
A sequence (73 of which are human). Comparisons were performed using BLASTN (parameter S = 108) for both strands of the 5 ′ EST. Sequences with greater than 80% homology to regions longer than 40 nucleotides were identified as rRNA. Of the 144,341 screened sequences, 3,312 were rR
NA was identified and excluded from further consideration.

In order to exclude the 5 ′ EST encoding mRNA from further consideration, this 5 ′ EST sequence was combined with two well-known mitochondrial genomic sequences from which the entire sequence of the genome was available, tRNA, All sequences transcribed from these mitochondrial genomes, including rRNA and mRNA, were compared for a total of 38 sequences. Comparisons were performed using BLASTN (parameter S = 108) for both strands of the 5 ′ EST. Sequences with greater than 80% homology to regions longer than 40 nucleotides were identified as mtRNAs. 144,341 screened
6,110 were identified as mtRNAs and excluded from further consideration.

Using BLASTN with the parameter S = 144, comparing the 5 ′ EST sequence with the release 46 of the EMBL of the bacterial and fungal departments, to allow sequences considered to be of foreign origin to be considered for further consideration. Excluded. All sequences with greater than 90% homology to at least 40 nucleotides were identified as heterologous impurities. Of the 42 cDNAs examined, the average percentage of prokaryotic and fungal sequences included was 0.2% and 0.5%, respectively. Of these sequences, only one could be identified as a fungal-specific sequence. Others were fungal or prokaryotic sequences that had homology to vertebrate sequences or contained repetitive sequences that were not masked during the computational comparison.

In addition, the 5 ′ EST is used to mask the 5 ′ EST having such a repetitive sequence.
The Alu sequence of 093 and the L1 sequence of 1115 were compared. THE and MER
5 'ESTs containing repeats, SSTR or satellite sequences, microsatellite sequences or telomere repeats were also excluded from further consideration. On average, 11.5% of the sequences in the library had repetitive sequences. Of this 11.5%, 7% is A
It contained lu repeats, 3.3% contained L1 repeats, and the remaining 1.2% were from other repetitive sequences screened. These proportions are consistent with those found in cDNA libraries prepared by other groups. For example,
The Adams et al. CDNA library contained 0% to 7.4% Alu repeats, depending on the source of RNA used to prepare the cDNA library (Adams et al., Nature 377: 174, 1996)
.

The accuracy of the above-described sequencing procedure was determined by comparing the sequence of the 5 ′ EST remaining after removing unnecessary sequences with the sequence of a known human mRNA. Example 19 Determination of Sequencing Accuracy by Comparison with Known Sequences To further determine the accuracy of the sequencing procedure described above, the sequence of the 5 'EST derived from the known sequence was identified and the original known sequence was determined. And compared. First, a FASTA analysis was performed on the 5 ′ EST using overhangs shorter than 5 base pairs at both ends to identify those that matched the entries in the public human mRNA database. Next, the 6655 5 'ESTs consistent with known human mRNAs were re-aligned with cognate mRNAs and replaced using dynamic programming with a list of "errors" that would be recognized, Insertions and deletions were included. Errors occurring in the last 10 bases of the 5 'EST sequence were ignored to avoid including the pseudo-cloning site in sequencing accuracy analysis.

In this analysis, the accuracy of the sequences recorded in the NetGene ^™ database was 99
. It was found to exceed 5%.

The following analysis was performed to determine the efficiency of selecting the cDNA containing the 5 ′ end of the corresponding mRNA in the above selection procedure. Example 20 Determination of 5 ′ EST Selection Efficiency In the above selection procedure, 5 ′ ESTs containing sequences near the 5 ′ end of mRNA were extended to determine the efficiency when isolated from their source mRNAs. The sequences at the ends of the 5 ′ ESTs from the factor 1 subunit α gene and the ferritin heavy chain gene were compared to the known cDNA sequences of these genes. Since the transcription start sites of both genes are well characterized, they can be used to determine the percentage of induced 5 'ESTs that include the true transcription start site.

For both genes, more than 95% of the resulting 5 ′ ESTs actually contained sequences near or upstream from the 5 ′ end of the corresponding mRNA.

To extend the reliability analysis of the procedure for isolating the 5 ′ EST from the ESTs in the NetGene ^™ database, a database containing human mRNA sequences extracted from GenBank database release 97 for comparison was also used. Analysis was performed. The 5 ′ ESTs from mRNA contained in the GeneBank database had more than 85% of their 5 ′ ends located near the 5 ′ end of the known sequence. Since some of the mRNA sequences available in the GenBank database are deduced from genomic sequences, 5 'ends that match these sequences are considered internal matches. Therefore, the method used here underestimates the yield of ESTs containing the true 5 'end of the corresponding mRNA.

The EST library generated above contained multiple 5 ′ ESTs from the same mRNA. The sequences of such 5 ′ ESTs were compared with each other to identify the longest 5 ′ EST for each mRNA. A continuous sequence (contig) was prepared by collecting the overlapping cDNAs. The contiguous sequence thus obtained was then compared to a public database to determine the similarity with the known sequence, as described in Example 21 below. Calculated for each EST libraries were sequenced out of novelty index for clustering and cDNA libraries Example 21 5 'ESTs, the sequence, 5' were clustered by end. Each sequence in the library was compared to the other sequences using BLASTN2 (direct strand, parameter S = 107). High scoring segment pairs (High Scoring Segmen) having at least 25 base pairs in length with 95% identical bases and starting within 10 base pairs from each EST 5 'end
ESTs containing tPair (HSP) were classified. The longest sequence present in the cluster was used as a group representative. A comprehensive clustering between libraries was then performed, leading to the definition of a supercontig.

To evaluate the yield of new sequences in the EST library, a new rate (nove
lty rate; NR) was defined as follows: NR = 100 × (number of new unique sequences identified in library / total number of sequences in library). In general,
New rates ranged from 10% to 41%, depending on the tissue from which the EST library was obtained. For most of the library, random sequencing of the 5 'EST library was continued until the new rate reached 20%.

Following the characterization described above, the 5 ′ ES in NetGene ^™ was identified to identify the 5 ′ EST carrying a potential signal sequence, as described in Example 22 below.
The T collection was screened. Example 22 Identification of a Potential Signal Sequence in the 5 ' EST To identify those with a continuous open reading frame (ORF) longer than 45 nucleotides starting at the ATG codon and extending to the end of the EST, N
The etGene ^™ database was screened for 5 'EST. About half of the NetGene ^™ cDNA sequence contained such an ORF. Then, to identify potential signal motifs, Von Heijne, Nuclei
c Acids Res. 14: 4683-4690, 1986 (the disclosures of which are incorporated herein by reference) using a modified version of the procedure disclosed in these 5'EST ORFs. did. A 5'EST sequence encoding a region of at least 15 amino acids in length with a score of at least 3.5 in the Von Heijne signal peptide identification matrix was considered to have a signal sequence. Matches a known human mRNA or EST sequence and
5 ′ ESTs with a 5 ′ end of more than 20 nucleotides downstream of the ′ end were excluded from further analysis. The remaining cDNA with the signal sequence was
It was recorded in a database called the ag ^TM.

To confirm the accuracy of the above method for identifying signal sequences, see Example 23.
Analysis was performed. Example 23 Confirmation of Accuracy of Identification of Potential Signal Sequence in 5 ′ EST Identification of a signal sequence encoding a signal peptide by applying this method to the 43 amino acids located at the N-terminus of all human SwissProt proteins The accuracy of the above procedure to perform was evaluated. The computerized Von Heijne score of each protein was compared to the characterization of known proteins as being secreted or non-secreted. In this way, 3.5
The number of non-secreted proteins with a higher score (false positive) and the number of secreted proteins with a score lower than 3.5 (false negative) could be calculated.

Using the results of the above analysis, the probability that the peptide encoded by the 5 ′ region of the mRNA is in fact a true signal peptide based on its Von Heijne score is 10% of the human protein secreted. Calculated based on either the hypothesis that it is secreted or that 20% of the human protein is secreted. The results of this analysis are shown in FIG. 2 and Table IV.

Using the secretory protein identification methods described above, 5'ESTs of the following polypeptides known to be secreted were obtained: human glucagon, gamma interferon-inducible monokine precursor, secretion Cyclophilin-like protein, human
Pleiotropin, and human biotinidase precursor. Thus, the method described above successfully identified a 5 'EST encoding a signal peptide.

In order to confirm that the signal peptide encoded by 5 ′ EST actually functions as a signal peptide, the signal sequence derived from 5 ′ EST was cloned into a vector designed for signal peptide identification. It is possible to Such vectors are designed to confer only the ability to grow on selective media only for those host cells which contain the vector with an operably linked signal sequence. For example, to confirm that 5 ′ EST encodes a true signal peptide, the signal sequence of 5 ′ EST can be modified by adding the signal sequence of US Pat. No. 5,536,637 (the disclosure of which is incorporated herein by reference). And a non-secretory yeast invertase gene in a signal peptide selection vector as described in “Incorporation”. Propagation of host cells containing the signal sequence selection vector containing the correctly inserted 5 'EST signal sequence confirms that 5' EST encodes a true signal peptide.

Alternatively, the presence of the signal peptide can be determined by using the extended cDNA obtained using EST.
Is cloned into an expression vector such as pXT1 (described in Example 30 below), or a promoter-signal sequence-reporter gene vector encoding a fusion protein between the signal peptide and the assayable reporter protein is constructed. It is possible to confirm. A suitable host cell (eg,
After introducing these vectors into COS cells or NIH 3T3 cells), the growth medium can be recovered and analyzed for the presence or absence of secreted proteins.
The media obtained from these cells is compared to media obtained from control cells containing vectors lacking the signal sequence or extended cDNA insert to identify vectors encoding functional signal peptides or true secreted proteins. I do.

The 5 ′ ESTs encoding the signal peptide determined by the method of Example 22 above were further divided into four categories based on homology with known sequences described in Example 24 below. Example 24 Classification of 5 'ESTs Encoding a Signal Peptide A 5' EST having a sequence that does not match any of the known vertebrate sequences or any of the officially available EST sequences is referred to as "new." Of the sequences in the SignalTag ^™ database, 947 5 'ESTs with a Von Heijne score of at least 3.5 fell into this category.

A 5 ′ EST having a sequence that does not match any of the vertebrate sequences, but has a sequence that extends the known EST sequence by at least 40 nucleotides in the 5 ′ direction and that is consistent with an officially known EST is referred to as “EST”. -Ext ". One of the sequences in the SignalTag ^™ database that has a Von Heijne score of at least 3.5
Fifty 5 'ESTs fell into this category.

A 5 ′ EST that has a sequence that does not match any of the vertebrate sequences but has a sequence that does not extend the known EST by at least 40 nucleotides in the 5 ′ direction and that is officially known as the EST ". Of the sequences in the SignalTag ^™ database, 599 5 'ESTs with a Von Heijne score of at least 3.5 fell into this category.

A 5 ′ EST that matches a human mRNA sequence but has the known sequence extended by at least 40 nucleotides in the 5 ′ direction is referred to as “VERT-ext”. Among the sequences in the SignalTag ^™ database, Von Heijne score of at least 3
. 23 5 'ESTs, which are 5, entered this category. Included in this category were 5 'ESTs, which extended the known sequence of human translocase mRNA by more than 200 bases in the 5' direction. A 5 ′ EST in which the sequence of the human tumor suppressor gene was extended in the 5 ′ direction was also identified.

Table V shows the distribution of 5 'ESTs in each category and the number of 5' ESTs with a predetermined minimum Von Heijne score in each category. As described in spatial and temporal outgoing current Example 25 Evaluation following of mRNA corresponding to 3.5'EST or extended cDNA, each of 5 'ESTs, based on the corresponding mRNA were obtained tissue Classified similarly. Example 25 The classification table VI of the expression pattern is a diagram showing the distribution of 5 ′ ESTs in each of the above categories for each tissue from which the corresponding mRNA 5 ′ EST was collected.

Table II lists the number of sequence identifications of 5 ′ EST sequences derived from endoderm, the categories in which these sequences fall, and the von H of the signal peptide encoded by them.
The eijne score is shown. The attached sequence listing shows the 5 ′ EST sequence and the amino acid sequence coded for it. Table III shows the 5 ′ EST SEQ ID NO and the sequence of the signal peptide encoded thereby. The sequence listing attached to this specification shows the 5'EST sequence and the sequence of the polypeptide encoded thereby.

Since the sequences of DNA SEQ ID NOs: 38-184 can be readily screened for errors involved, resequencing fragments containing such errors or ambiguities on both strands will result in a sequence Can be resolved. Such fragments can be obtained from plasmids stored in our laboratory, or can be isolated using the techniques described herein. Resolving such ambiguities or errors is facilitated by using primers that hybridize to sequences that are located near the ambiguous or erroneous sequence. For example, primers may have 50
It may be hybridized to a sequence of up to 75 bases. Once the error or ambiguity is resolved, a corresponding correction can be made in the protein sequence encoded by the DNA containing the error or ambiguity.

In addition to classifying the 5 ′ ESTs for the tissue from which they originated, as described in Example 26 below, the spatial and temporal expression patterns of the mRNA corresponding to the 5 ′ ESTs As well as the level of expression. As will be discussed in more detail below, characterizing the spatial, temporal and temporal expression patterns of these mRNs can be accomplished in a desired spatial or temporal manner, with a desired level of gene product Is useful for constructing an expression vector capable of producing E. coli. In addition, it is possible to identify 5 ′ ESTs whose corresponding mRNA is involved in the disease state. For example, certain diseases may be due to lack of expression, overexpression, or underexpression of mRNA corresponding to the 5'EST. By comparing the expression pattern and amount of mRNA in a sample collected from a healthy person with those of an individual suffering from a specific disease, it is possible to identify the 5 ′ EST responsible for the disease.

It will be apparent that the results of the characterization procedure for the 5 ′ EST described above also apply to extended cDNAs containing sequences adjacent to the 5 ′ EST (obtained as described below). Also, if desired, rather than characterizing the EST itself, the extended cDNA
It will also be clear that the characterization may be delayed until the following is obtained. Long described in Example 26 5 'ESTs or expression level of mRNA corresponding to extended cDNA and expression evaluation patterns International Patent Application No. WO 97/05277 (incorporated herein by reference in its entirety) In solution hybridization using a probe, 5
It is possible to analyze the expression levels and expression patterns of mRNA corresponding to 'EST or extended cDNA (obtained as described in Example 27 below).
Briefly, a 5 'EST, an extended cDNA, or a fragment thereof, corresponding to the gene encoding the mRNA to be characterized, is cloned into a cloning site immediately downstream of the bacteriophage (T3, T7 or SP6) RNA polymerase promoter. To create an antisense RNA. Preferably, the 5 'EST or extended cDNA has 100 or more nucleotides. Plasmids were linearized and modified ribonucleotides (ie, biotin-UTP and DIG-UTP)
Is transcribed in the presence of ribonucleotides containing This double-labeled excess RNA
Is hybridized in solution with mRNA isolated from the cell or tissue of interest. Under standard stringent conditions (80% formaldehyde, 0.4M
The hybridization is performed in a NaCl buffer (pH 7 to 8 at 40 to 50 ° C. for 16 hours). Ribonuclease specific for single-stranded RNA (ie, RN
Non-hybridized probe is removed by digestion with ase CL3, T1, PhyM, U2 or A). The presence of the biotin-UTP modification makes it possible to capture the hybrid on a streptavidin-coated microtiter plate. The presence of the DIG modification allows the hybrids to be detected and quantified by ELISA using an anti-DIG antibody conjugated to alkaline phosphatase.

As disclosed in UK Patent Application No. 2 305 241A, which is incorporated herein by reference in its entirety, 5 ′ ESTs, extended cDNAs, or fragments thereof, have a sequence of gene expression Nucleotide sequences for analysis (SAGE) can also be tagged. In this way, cDNA is prepared from cells, tissues, organisms, or other sources of nucleic acid whose gene expression pattern must be determined. The cDNA obtained in this way is divided into two pools. The cDNA in each pool is cut with a first restriction endonuclease (called an anchoring enzyme) having a recognition site likely to be present in at least one of the majority of cDNAs. Cut cD
The fragment rich in the 5 'or 3' region of NA is isolated by binding to a capture medium such as streptavidin-coated beads. A first oligonucleotide linker having a first sequence for hybridizing amplification primers and an internal restriction site for a so-called tagged endonuclease is ligated to the digested cDNA in the first pool. After digestion with a second endonuclease, the cDNA
Generate short tagged fragments from A second oligonucleotide having a second sequence for hybridizing the amplification primer and an internal restriction site is ligated to the digested cDNA in the second pool.
The cDNA fragment of the second pool was also digested with tagged endonuclease
Generate a short tag fragment derived from the cDNA of the pool. The tags generated by digestion of the first and second pools with an anchoring enzyme and a tagged endonuclease are ligated together to produce a so-called ditag. In some embodiments, the ditag is concatamerized to form the ditag from 2 to 200
The resulting ligation reaction product is generated. The tag sequence is then determined and compared to the sequence of the 5 'EST or extended cDNA, which 5' EST or extended cDNA is expressed in the cell, tissue, organism, or other source of nucleic acid from which the tag is derived. Decide what you want. In this way, the expression pattern of the 5 'EST or extended cDNA in a cell, tissue, organism, or other source of nucleic acid is obtained.

[0142] Quantitative analysis of gene expression can also be performed using arrays. As used herein, the term “array” refers to a full-length cDNA (ie, a coding sequence for a signal peptide, a coding sequence for a mature protein, and an extended cD containing stop codons).
NA), extended cDNA, 5'EST, or one-, two-, or multi-dimensional sequences (arra) of those fragments long enough to allow specific detection of gene expression.
n). Preferably, the fragments are at least 15 nucleotides in length. More preferably, the fragments are at least 100 nucleotides in length. More preferably, the fragments are greater than 100 nucleotides in length.
In some embodiments, fragments may be greater than 500 nucleotides in length.

For example, see Schena et al. (Science 270: 467-470, 1).
995; Proc. Natl. Acad. Sci. U. S. A. 9
3: 10614-10619, 1996) to perform quantitative analysis of gene expression using the following full-length cDNAs, extended cDNAs, 5'ESTs, or fragments thereof in a complementary DNA microarray: Is possible. Full-length cDNA, extended cDNA, 5 'EST or fragments thereof were amplified by PCR and sequenced using high-speed robotics from 96-well microtiter plates onto silylated microscope slides. The printed array was incubated in a humid chamber to re-hydrate the array elements, and the 0.2% SD
S once per minute in water, twice per minute in water and 5 times in sodium borohydride solution.
Rinse once per minute. The array is submerged in hot water at 95 ° C for 2 minutes, transferred to 0.2% SDS for 1 minute, rinsed twice with water, air-dried, and stored in the dark at 25 ° C.

Isolate or purchase cell or tissue mRNA and prepare probes by one round of reverse transcription. Probes are hybridized to a 1 cm ² microarray under a 14 x 14 mm cover glass at 60 ° C for 6-12 hours. Arrays were incubated in low stringency wash buffer (1 × SSC / 0.2% SDS) at 25 ° C. for 5 minutes, then high stringency wash buffer (0.1 × SSC / 0.2%).
% SDS) at room temperature for 10 minutes. The array is scanned in 0.1 × SSC using a fluorescent laser scanner fitted with a custom filter set. Taking the average of the ratios of the two independent hybridizations provides an accurate measure of differential expression.

Pietu et al. (Genome Research 6: 492-503, 1
As described by 996), it is also possible to carry out a quantitative analysis of the expression of the gene using the full-length cDNA, the extended cDNA, 5'EST, or their fragments in a complementary DNA array. Full-length cDNA, extended cDNA, 5'EST or fragments thereof are PCR amplified and spotted on a membrane. The mRNA from various tissues or cells is then labeled with a radionucleotide. After hybridizing and washing under control conditions, the hybridized mRNA is detected by phospho-imaging or autoradiography. The experiment is performed twice and then a quantitative analysis of the differentially expressed mRNA is performed.

Alternatively, Lockhart et al. (Nature Biotechnology 14: 1675-1680, 1996) and Sosnowsky et al.
rc. Natl. Acad. Sci. 94: 1119-1123,
1997), high density nucleotide arrays provide 5
'Expression analysis of EST or extended cDNA can be performed. Oligonucleotides of 15 to 50 nucleotides corresponding to the sequence of the 5 'EST or extended cDNA are synthesized directly on the chip (Lockhart et al., Supra) or synthesized and processed into the chip (Sosnowsky et al., Supra). The oligonucleotide has a length of about 20
Nucleotides are preferred.

C labeled with a suitable compound such as biotin, digoxigenin or a fluorescent dye
DNA probes are synthesized from the appropriate mRNA population and then randomly fragmented to an average size of 50-100 nucleotides. Next, the aforementioned probe is hybridized to the chip. After washing as described in Lockhart et al. (Supra) and applying a different electric field (Snowsky et al., Supra), the dye or labeled compound is detected and quantified. Hybridization is performed twice. Comparative analysis of the intensity of the signal from the cDNA probe on the same target oligonucleotide in different cDNA samples provided a differential analysis of the mRNA corresponding to the 5 'EST or extended cDNA from which the oligonucleotide sequence was designed. You can see the target occurrence. III. 'Using the above procedure using the EST, once, 5 of the corresponding mRNA' 5 for cloning cloning and the corresponding genomic cDNA extension cDNA 5 'E containing terminal
Once STs have been selected, they can be used to isolate extended cDNAs containing sequences flanking the 5 'EST. The extended cDNA may contain the entire coding sequence of the protein encoded by the corresponding mRNA, including the true translation initiation site, the signal sequence, and the sequence encoding the mature protein remaining after cleavage of the signal peptide. . Such an extended cDNA is referred to herein as "full length cDNA". Alternatively, the extended cDNA may include only the sequence encoding the mature protein remaining after cleavage of the signal peptide, or may include only the sequence encoding the signal peptide.

Example 27 below describes a general method for obtaining extended cDNA using 5 ′ EST. Example 28 below uses the method described in Example 27, which describes the entire coding sequence for several secreted proteins and several extended cDNAs containing the true 5 'end of the corresponding mRNA. Provides the results of the experiment.

Using the methods of Examples 27, 28, and 29, extended cDNAs can be obtained that encode less than the entire coding sequence of the secreted protein encoded by the gene corresponding to the 5 'EST. . In some embodiments, the extended cDNA isolated using these methods encodes at least 10 amino acids of one of the proteins encoded by the sequences of SEQ ID NOs: 38-184. In a further embodiment, the extended cDNA encodes at least 20 amino acids of the protein encoded by the sequences of SEQ ID NOs: 38-184. In a further embodiment, the extended cDNA comprises at least 30 of the sequence of SEQ ID NOs: 38-184.
Encodes amino acids. In a preferred embodiment, the extended cDNA comprises SEQ ID NO: 38
Encodes a full-length protein sequence, including ~ 184 protein coding sequence. Example 27 entire coding region and the corresponding mRNA common method true 5 of the corresponding mRNA for use EST '5 to cloning and sequencing of the cDNA, including the terminal' true 5 'terminus and total protein The following general method is used to rapidly and efficiently isolate an extended cDNA containing a coding sequence and containing a sequence adjacent to the sequence of the 5'EST used to obtain the extended cDNA. It has been. This method uses a 5 'encoding a polypeptide belonging to a secreted protein.
It can be used to obtain extended cDNA for any 5 ′ EST in the NetGene ^™ database, including EST. The method is summarized in FIG. 1. Obtaining Extended cDNA a) First Strand Synthesis This method utilizes the known 5 'sequence of mRNA. A reverse transcription reaction is performed on the purified mRNA using a poly 14dT primer containing a 49 nucleotide sequence at the 5 'end capable of adding a known sequence to the end of the cDNA corresponding to the 3' end of the mRNA. . For example, the primer has the following sequence: 5'-ATC GTT GA
G ACT CGT ACC AGC AGA GTC ACG AGA GAG ACT
It may have ACA CGG TAC TGG TTT TTT TTT TTT TTVN-3 ′ (SEQ ID NO: 14). One skilled in the art will appreciate that other sequences can also be added to the poly dT sequence and used to initiate first strand synthesis.
This primer and Superscript II (Gibco BRL) or RN
Reverse transcriptase, such as the ase H Minus M-MLV (Promega) enzyme, is used to generate a reverse transcript immobilized at the 3 'polyA site of the RNA.

After removing the mRNA hybridized to the first cDNA strand by alkaline hydrolysis, the product of alkaline hydrolysis and the residual poly dT primer were removed by an exclusion column such as the AcA34 (Biosepra) matrix described in Example 11. To remove. b) Second strand synthesis Based on the known 5 'sequence of the 5' EST and the known 3 'end added by the poly dT primer used for the first strand synthesis, a pair of nested (ne
(sted) Design the primer. The software used to design primers is based on GC content and oligonucleotide melting temperature (eg, OSP software; Illier and Green, PCR Meth. Ap.
pl. 1: 124-128, 1991), for example, PC-Rare (http
// bioinformatics. weizmann. ac. il / sof
tware / PC-Rare / doc / manuel. html) (Griffais et al., Nucleic A).
cids Res. 19: 3887-3891, 1991).

The 5'-end nested primers are preferably 4 to 9 bases apart from each other. The 5 'primer sequence can be selected to have a suitable melting temperature and specificity for use in PCR.

The nested primers at the 3 ′ end are preferably 4 to 9 bases apart from each other. For example, the nested 3 'primer has the following sequences: (5'-CCA GC AGAG TCA CGA GAG AGA CTA CAC GG -3' (SEQ ID NO: 15), and 5'-CAC GAG AGA GAC TAC ACG GTAC CT
GG-3 ′ (SEQ ID NO: 16). These primers were chosen because they have suitable melting temperatures and specificities for use in PCR. However, one skilled in the art will appreciate that other sequences can also be used as primers.

Using the outer primers from Advantage Tth Polymerase Mix (Clontech) and each of the nested primer pairs, the first
Was performed for 25 cycles. Then, 1/2500 of the first PCR product
, A second 20 cycles of PCR using inner primers from each of the same enzyme and nested primer pairs. Thereafter, the primers and nucleotides are removed. 2. Sequencing the Full Length Extended cDNA or Fragment There are no positional constraints on the design of 5 'nested primers suitable for PCR using OSP software, resulting in two types of amplicons. The second 5 'primer is located upstream of the translation initiation codon and, therefore, preferably produces a nested PCR product containing the entire coding sequence. Such full-length extended cDNA is subjected to the direct cloning procedure described in section a.
However, in some cases, the second 5 'primer is located downstream of the translation initiation codon, resulting in a PCR product containing only a portion of the ORF. Such an incomplete P
The CR product is subjected to the improved procedure described in section b. a) Nested PCR product containing the complete ORF When the resulting nested PCR product contains the complete coding sequence expected from the 5 'EST sequence, as described in Section 3, the PCR product is used as pED6dpc2 Clone with an appropriate vector. b) Nested PCR products containing incomplete ORFs When the amplicon does not contain the complete coding sequence, an intermediate step is necessary to obtain both the complete coding sequence and the PCR product containing the entire coding sequence. As described in the next section, a complete coding sequence can be assembled from several partial sequences determined directly from different PCR products.

[0154] Once the entire coding sequence has been completely determined, new primers are then designed for PCR to obtain an amplicon containing the entire coding region. However, in such cases, the 3 'primer that is suitable for PCR is located inside the 3' UTR of the corresponding mRNA, and therefore, as illustrated in FIG. And sometimes amplicons lacking the polyadenylation signal). The full-length extended cDNA is then cloned into a suitable vector as described in section 3. c) Sequencing of extended cDNA Extended cD using the Die Terminator method using the AmpliTaq DNA polymerase FS kit available from Perkin Elmer.
Perform NA sequencing.

To determine the sequence of the PCR fragment, an O
ASMG (Sutton et al., Genome Science T) to construct a contig of the walking sequence containing the first 5 'tag using software such as SP and a minimal overlap of 32 nucleotides.
echnol. 1: 9-19, 1995) to perform primer-walking. It is preferred to perform primer-walk until the full-length cDNA sequence is obtained.

The completion of sequencing a given extended cDNA fragment is assessed as follows. In the case of the non-cloned product, the sequence located after the polyA section is difficult to determine accurately, so that when the polyA section is identified in the extended cDNA obtained as described in case b. Stop sequencing and primer-walking for PCR products. The length of the sequence is compared to the size of the nested PCR product obtained as described above. The sequence is considered to be complete if the size of the obtained sequence is at least 70% of the size of the first nested PCR product due to the limited accuracy of PCR product size determination by gel electrophoresis. . If the sequence length determined by computer analysis is not at least 70% of the length of the nested PCR products, these PCR products are cloned and the insert is sequenced. When Northern blot data is available, the mR detected for a given PCR product
The size of the NA is used to ultimately assess that the sequence is complete. Sequences that do not meet the above criteria are discarded and subjected to a new isolation procedure.

[0157] All extended cDNA sequence data is then transferred to a proprietary database where quality control and verification steps are performed as described in Example 15. 3. Cloning of full-length extended cDNA The PCR product containing the entire coding sequence is then cloned in a suitable vector. For example, as described below, the extended cDNA is transformed into the expression vector pED6dpc2.
(DiscoverEase, Genetics Institute, C
ambridge, MA). After performing EcoRI digestion, blunt-ended pED6dpc2 vector DNA is prepared by performing a filling in reaction. Dephosphorylate the vector with blunt ends. After removal of the PCR primers and ethanol precipitation, a PC containing the entire coding sequence or extended cDNA obtained as described above
The R product is phosphorylated with a kinase and subsequently removed by phenol-Sevag extraction and precipitation. Next, the double-stranded elongated cDNA is ligated to a vector, and the obtained expression plasmid is introduced into a suitable host cell.

Since the PCR products obtained as described above are blunt-ended molecules that can be cloned in any direction, the orientation of several clones is determined for each PCR product. The 4-10 clones were then aligned on a microtiter plate and the first primer and m located on the vector near the cloning site.
The RNA is subjected to a PCR reaction using a second primer located at a part of the extended cDNA corresponding to the 3 'end of the RNA. This second primer may be an antisense primer used for anchored PCR in the case of direct cloning (case a) or 3 in the case of indirect cloning (case b).
'An antisense primer located inside the UTR may be used. Storing a clone in which the start codon of the extended cDNA is operably linked to the vector promoter so that the protein encoded by the extended cDNA can be expressed;
Sequence. In addition to the ends of the cDNA insert, about 50 base pairs of vector DNA on each side of the cDNA insert are also sequenced.

The cloned PCR product is then completely sequenced according to the procedure described above.
In this case, for the uncloned PCR product, long fragments are contigated using the walking sequence that has already been contiguous during primer-walking. Sequencing of the cloned amplicon is complete when the resulting contig contains the entire coding region as well as overlapping sequences with vector DNA at both ends. 4. Computer analysis of full-length extended cDNA The sequence of all full-length extended cDNAs is then further analyzed as described below.
Before searching the extended full-length cDNA for the sequence of interest, the 5 ′ of Example 18
Using methods essentially similar to those described for ESTs, the extended cDNAs other than the target (vector RNA, transfer RNA, ribosomal RNA, mitochondrial RNA, prokaryotic RNA and fungal RNA) are removed. a) Identification of Structural Characteristics Next, the structural characteristics of the sequence of the full-length elongated cDNA, for example, poly A tail and polyadenylation signal are determined as follows.

A poly A tail is defined as where A is at least 11 homopolymer stretches with at most one alternative base therein. The poly A tail search is restricted to the last 100 nucleotides (nt) of the sequence and has 11 consecutive A
After such a polyA extension, since often the sequencing reaction cannot be read. Using BLAST2N,
Extensions with greater than 90% homology of 8 nucleotides or more are identified as poly A tails.

To search for polyadenylation signals, the polyA tail is cut from the full length sequence. The 50 bases before the poly A tail are replaced with the defined polyadenylation AAUAA
Search first for the A signal, and if no defined signal is detected, search for an alternative AUUAAA signal (Sheets et al., Nuc. Acids Res. 18: 579).
9-5805, 1990). If neither of these consensus sporiadenylation signals are found, the defined motif is searched again, revealing that one mismatch is the cause of possible sequencing errors. The polyadenylation signals identified were greater than 85% of either type, with 10% or less of the polyA tail.
It actually ends with 30 base pairs. Alternatively, the AUUAAA signal represents about 15% of the total number of polyadenylation signals identified. b) Identification of Functional Characteristics Next, the functional characteristics of the sequence of the full-length extended cDNA, for example, the ORF and the signal sequence, were determined as described below.

Search for the three top strand frames of the extended cDNA for the ORF, which is defined as the longest fragment that starts at the translation start codon and ends at the stop codon. ORFs encoding at least 20 amino acids are preferred.

Then, as described in Example 22, von Heijne (Nuc. Aci
ds Res. 14: 4683-4690, 1986) (the disclosure of which is incorporated herein by reference) using the matrix method of the first 50 amino acids, or, where appropriate, the ORF of the ORF. Each ORF identified for the presence or absence of a signal peptide in a shorter region up to 20 amino acids
Is scanned. c) Homology with either nucleotide or protein sequence It is possible to perform a classification of the full-length sequence using essentially the same procedure as described in Example 24 for the 5 'EST.

Subsequently, an extended cDNA prepared as described above using conventional techniques such as subcloning, PCR, or in vitro oligonucleotide synthesis.
To obtain a nucleic acid containing a desired portion of the extended cDNA. For example, using techniques well known to those of skill in the art, a nucleic acid can be obtained that includes only the entire coding sequence (ie, the sequence encoding the signal peptide and the mature protein remaining after excision of the signal peptide). Alternatively, conventional techniques can be used to obtain a nucleic acid that contains only the coding sequence for the mature protein that remains after excision of the signal peptide, or a nucleic acid that contains only the coding sequence for the signal peptide.

Similarly, it is possible to obtain a nucleic acid comprising any other desired part of the coding sequence for a secreted protein. For example, a nucleic acid may comprise at least 10 contiguous bases of an extended cDNA, such as one of the extended cDNAs described below. In another embodiment, the nucleic acid may comprise at least 15 contiguous bases of the extended cDNA, such as one of the extended cDNAs described below. Alternatively, the nucleic acid has the following extended cD
It may comprise at least 20 contiguous bases of the extended cDNA, such as one of the NAs. In another embodiment, the nucleic acid is an extended cDN, such as one of the following extended cDNAs:
It may comprise at least 25 consecutive bases of A. In yet another embodiment,
The nucleic acid may comprise at least 40 contiguous bases of the extended cDNA, such as one of the extended cDNAs described below.

[0166] Once the extended cDNA is obtained, its sequence is determined and the amino acid sequence encoded by the cDNA is determined. Once the encoded amino acid sequence has been determined,
Using only the degeneracy of the genetic code, many possible cDNAs encoding that protein can be created and identified. For example, allelic variants or other homologous nucleic acids can be identified as described below. Alternatively, a nucleic acid encoding the desired amino acid sequence can be synthesized in vitro.

In a preferred embodiment, the coding sequence can be selected using known codons or codon-pair dominance with respect to the host organism in which the cDNA is to be expressed.

As described in Example 28 below, the extended cD from the 5 ′ ESTS of the present invention
NA was obtained. Example 28 Characterization of Cloned Extended cDNA Obtained Using 5 'EST Using the procedure described in Example 27 above, the extended cDNA derived from the 5' EST of the present invention was transformed into various cDNAs. Obtained in tissue. The slightly elongated cD thus obtained
Specific examples of NA are shown in the following list.

Using this approach, SEQ ID NO: 17 (internal ID number 48-19-3-G
1-FL1) was obtained. This cDNA belongs to the above-mentioned “EST-ext” category and encodes a signal peptide MKKVLLLITAILAVAVG (SEQ ID NO: 18) with a von Heijne score of 8.2.

Using this procedure, SEQ ID NO: 19 (internal ID number 58-34-2-E7-F
L2) full-length cDNA was also obtained. This cDNA belongs to the above-mentioned “EST-ext” category, and has a von Heijne score of 5.5.
Encodes WWFQQGLSFLPSALVIWTSA (SEQ ID NO: 20).

Another full length cDNA obtained using the procedure described above is SEQ ID NO: 21 (internal ID
No. 51-27-1-E8-FL1). This cDNA belongs to the above-mentioned “EST-ext” category and has a von Heijne score of 5.9.
Encodes ASSALSPCLT (SEQ ID NO: 22).

Using the procedure described above, SEQ ID NO: 23 (internal ID number 76-4-1-G5-F
A full-length cDNA having the sequence of L1) was also obtained. This cDNA was obtained from the “ES
It belongs to the “T-ext” category and encodes the signal peptide ILSTVTALTFAXA (SEQ ID NO: 24) with a von Heijne score of 5.5.

Using this procedure, SEQ ID NO: 25 (internal ID number 51-3-3-B10-F
The full-length cDNA of L3) was also obtained. This cDNA belongs to the above-mentioned “new” category and has a von Heijne score of 10.1 and a signal peptide LVLTLC.
Encodes TLPLAVA (SEQ ID NO: 26).

Using this procedure, SEQ ID NO: 27 (internal ID number 58-35-2-F10-
The full length cDNA of FL2) was also obtained. This cDNA falls into the “new” category described above and has a von Heijne score of 10.7, a signal peptide LWLLF.
Encodes FLVTAIHA (SEQ ID NO: 28).

Currently, bacterial clones containing plasmids containing the full-length cDNAs described above are stored in the inventor's laboratory under the above-mentioned internal ID numbers. By growing an aliquot of the appropriate bacterial clone in an appropriate medium, it is possible to recover the insert from the stored material. Plasmid DNA can then be isolated using plasmid isolation procedures well known to those skilled in the art, such as alkaline lysis minipreps and large scale alkaline lysis plasmid isolation procedures. If necessary,
Plasmid DNA can be further enriched by centrifugation using a cesium chloride gradient, size exclusion chromatography, or anion exchange chromatography. The plasmid DNA obtained using these procedures is then
It is possible to operate using standard cloning techniques well known to those skilled in the art. Alternatively, PCR can be performed using primers designed at both ends of the cDNA insert. The PCR product corresponding to the cDNA can then be manipulated using cloning techniques well known to those skilled in the art.

[0176] The polypeptide encoded by the extended cDNA is converted to a signature that is a small amino acid sequence that is well conserved for the presence of known structural or functional motifs or among members of the protein family.
es) can be screened for the presence of The saved area is stored in the PROSITE databank, especially the file “prosite. dat (Release 13.0, November 1995, http: //expasy.hcug)
e. ch / sprot / prosite. html) is used to derive consensus patterns or matrices. Prosite_
convert and the site_scan program (http: // u
lrec3. unil. ch / ftpservur / prosite_sca
Using n), it is possible to find the signature on the extended cDNA.

The site. Assess the accuracy of sequence detection for new proteins by evaluating the frequency of inappropriate hits for the population of human secreted proteins contained in the databank SWISSPROT for each pattern obtained using the site_convert program in the dat file It is possible. The ratio between the number of hits for the shuffled protein (20 amino acid window size) and the number of hits for the native (non-shuffled) protein can be used as an index. place_
During a search using scan, any pattern with a ratio greater than 20% (one hit for shuffled protein versus five hits for native protein) can be omitted. A program used to shuffle protein sequences (db_shuffled) and a program used to determine statistics for each pattern in the protein databank (site_statis)
tics) is the ftp site HYPERLINK http://ulrec3.unil.ch/ftpserveur/pr
osite # scan http: // ulrec3. unil. ch / ftpservur / produce_scan.

In addition to PCR-based methods for obtaining extended cDNA, traditional hybridization-based methods can also be used. Using such a method, it is also possible to obtain mRNA that is the origin of 5 ′ EST, mRNA corresponding to the extended cDNA, or genomic DNA encoding a nucleic acid homologous to the extended cDNA or 5 ′ EST. Example 29 below provides an example of such a method. Example 29 Method for Obtaining True 5 ′ End of cDNA Containing the Entire Coding Region and Corresponding mRNA By replacing with the -dT primer, a full-length cDNA library can be prepared. For example, it is possible to use the oligonucleotide of SEQ ID NO: 14.

Alternatively, cDNA or genomic DNA libraries may be obtained from commercial sources or may be made using techniques well known to those skilled in the art. As described below, such a cDNA library or a genomic DNA library can be used to isolate an extended cDNA obtained from 5 ′ EST or a nucleic acid homologous to the extended cDNA or 5 ′ EST. Using conventional techniques, a cDNA library or genomic DNA library is hybridized to a detectable probe containing at least 10 contiguous nucleotides from the 5 'EST or extended cDNA. Preferably, the probe comprises at least 12, 15 or 17 contiguous nucleotides from the 5 'EST or extended cDNA. More preferably, the probe comprises at least 20 to 30 contiguous nucleotides from 5 'EST or extended cDNA. In some embodiments, the probe comprises more than 30 nucleotides from the 5 'EST or extended cDNA.

[0180] Sambrook et al., Molecular Cloning: A Labo.
rattro Manual 2d Ed. , Cold Spring Ha
rbor Laboratory Press, 1989 (the disclosure of
(Incorporated herein by reference) discloses techniques for identifying cDNA clones in a cDNA library that hybridize to a given probe sequence.
Genomic DNA can be isolated using the same technique.

Briefly, cDNA or genomic DNA clones that hybridize with detectable probes for further manipulation are identified and isolated as described below. Probes containing at least 10 contiguous nucleotides from the 5 'EST or extended cDNA are labeled with a detectable label, eg, a radioisotope or a fluorescent molecule. Preferably, the probe comprises at least 12, 15, or 17 contiguous nucleotides from the 5 'EST or extended cDNA. More preferably, the probe comprises 20 to 30 contiguous nucleotides from the 5 'EST or extended cDNA. In some embodiments, the probe comprises more than 30 nucleotides from the 5 'EST or extended cDNA.

Techniques for labeling probes are well known and include phosphorylation with polynucleotide kinase, nick translation, in vitro transcription, and non-radioactive techniques.
The cDNA or genomic DNA in the library is transferred to a nitrocellulose filter or a nylon filter and denatured. After blocking non-specific sites,
CDNA or genomic DNA containing a sequence capable of hybridizing to the probe
The filter is incubated with the labeled probe for a time sufficient to allow the probe to bind to the NA.

By changing the stringency of the hybridization conditions used to identify the extended or genomic DNA that hybridizes to the detectable probe, the extended cDNA with different levels of homology to the probe, as described below.
Can be identified and isolated. 1. Extended cDNA sequence or genomic cDN with high homology to labeled probe
Identification of the A Sequence To identify extended cDNA or genomic DNA with a high degree of homology to the probe sequence, the melting temperature of the probe can be calculated using the following equation:

For a probe 14-70 nucleotides long, the following formula: Tm = 81.5 + 16.6 (log [Na +]) + 0.41 (fraction G + C) −
(600 / N), where N is the length of the probe, and the melting temperature (T
m) is calculated.

When performing hybridization in a solution containing formamide, the formula: Tm = 81.5 + 16.6 (log [Na +]) + 0.41 (fraction G + C
)-(0.63% formamide)-(600 / N), where N is the length of the probe, can be used to calculate the melting temperature.

Prehybridization was performed in 6 × SSC, 5 × Denhardt reagent, 0.5% SDS, 100 μg of denatured and fragmented salmon sperm DNA, or 6 × SSC, 5 × Denhardt reagent, 0.5% SDS, 100 μg. Can be performed in denatured and fragmented salmon sperm DNA, 50% formamide. Sambrook et al. (Supra) include SSC and Denhardt.
A formulation for the solution is described.

Hybridization is performed by adding a detectable probe to the prehybridization solution. If the probe contains double-stranded DNA, it is denatured before adding to the hybridization solution. The filter is contacted with the hybridization solution for a time sufficient to allow the probe to hybridize to the extended cDNA or genomic DNA containing a sequence complementary to or homologous to the probe. In the case of a probe having a length of more than 200 nucleotides, hybridization can be performed at a temperature 15 to 25 ° C lower than Tm. For shorter probes, such as oligonucleotide probes, the Tm
Hybridization can be performed at a temperature lower by 15 to 25 ° C. When hybridizing in 6X SSC, it is preferred to hybridize at about 68 ° C. When hybridizing in a solution containing 50% formamide, it is preferred to hybridize at about 42 ° C.

All the above hybridizations would be considered to be under “stringent” conditions.

After hybridization, the filters are washed for 15 minutes at room temperature in 2X SSC, 0.1% SDS. The filter was then filtered with 0.1X SSC, 0
. Wash with 5% SDS at room temperature for 30 minutes to 1 hour. Thereafter, the solution was
Wash at hybridization temperature in X SSC, 0.5% SDS. A final wash is performed in 0.1X SSC at room temperature.

The extended cDNA, nucleic acid homologous to the extended cDNA or 5 ′ EST, or genomic DNA hybridized to the probe, is identified by autoradiography or other conventional techniques. 2. Extended cDNA sequence or genomic cDN with low homology to labeled probe
Acquisition of Sequence A In order to identify an extended cDNA, a nucleic acid homologous to the extended cDNA, or a genomic DNA having a low level of homology with the probe sequence, the above procedure can be modified. For example, an extended cDNA with low homology to the detectable probe sequence, an extended cD
Lower stringency conditions may be used to obtain nucleic acids homologous to NA, or genomic DNA. For example, in a hybridization buffer having a sodium concentration of about 1 M, the hybridization temperature may be lowered from 68 ° C. to 42 ° C. in 5 ° C. steps. After hybridization, the filter may be washed with 2X SSC, 0.5% SDS at the hybridization temperature. These conditions are considered to be “moderate” conditions above 50 ° C. and “low” conditions below 50 ° C.

Alternatively, hybridization may be performed in a buffer such as 6X SSC containing formamide at a temperature of 42 ° C. In this case, the formamide concentration in the hybridization buffer may be reduced by 5% from 50% to 0% in order to identify a clone having a low homology level with the probe. After hybridization, the filters may be washed with 50X 6X SSC, 0.5% SDS. These conditions are considered "moderate" conditions with more than 25% formamide and "low" conditions with less than 25% formamide.

The extended cDNA, nucleic acids homologous to the extended cDNA, or genomic DNA hybridized to the probe are identified by autoradiography. 3. Determining the degree of homology between the obtained extended cDNA and the labeled probe
If it is desired to obtain a nucleic acid encoding a protein associated with the protein encoded by A, BLAST2N can be used to bind between the hybridized nucleic acid and the extended cDNA or 5 'EST used as a probe. The level of homology can be further determined, and parameters may be modified depending on the length of the sequence being tested and the degree of homology. To determine the level of homology between the hybridized nucleic acid and the extended cDNA or 5 'EST from which the probe originated, the nucleotide sequence of the hybridized nucleic acid and the extended cDNA or 5''Compare with EST nucleotide sequence. For example, using the method described above, the extended cDNA from which the probe originated
Alternatively, a nucleic acid having at least 95% nucleic acid homology with 5 ′ EST can be obtained and identified. Similarly, using lower non-stringent hybridization conditions, the extended cDNA or 5 '
It is possible to obtain and identify nucleic acids having at least 90%, at least 85%, at least 80% or at least 75% homology with the EST.

To determine whether a clone encodes a protein with a certain amount of homology to the extended cDNA or the protein encoded by the 5 ′ EST,
The amino acid sequence encoded by the extended cDNA or 5 'EST is compared to the amino acid sequence encoded by the hybridizing nucleic acid. Homology is determined to exist when the amino acid sequence of the extended cDNA or 5 'EST is closely related to the amino acid sequence of the hybridizing nucleic acid. When the sequence is the same as the sequence of the extended cDNA or 5 'EST, or when the sequence contains one or more amino acid substitutions in which amino acids with similar characteristics have replaced each other,
The sequence is closely related. Using an algorithm such as FASTA, using the methods described above, and parameters depending on the length and degree of homology of the sequence being tested, the extended cDNA or 5 'EST from which the probe originated is coded. At least 95%, at least 90%, at least 85% with protein
, Nucleic acids encoding proteins having at least 80% or at least 75% homology.

In addition to the methods described above, other protocols are used to obtain extended cDNA using 5 ′ EST, as outlined in the next paragraph.

Extended cDNA can be prepared by obtaining mRNA from a tissue, cell, or organism of interest using poly A selection methods or mRNA preparation methods utilizing techniques well known to those skilled in the art. It is. A first primer capable of hybridizing to the polyA tail of the mRNA is hybridized to the mRNA and a reverse transcription reaction is performed to generate a first cDNA strand.

The first cDNA strand has at least 10 cDNAs derived from the sequence of SEQ ID NOs: 38-184.
Hybridize to a second primer containing consecutive nucleotides. This primer has at least 12, 15, from the sequence of SEQ ID NOs: 38-184,
Alternatively, it preferably contains 17 consecutive nucleotides. More preferably, the primer contains 20 to 30 consecutive nucleotides derived from the sequence of SEQ ID NOs: 38 to 184. In some embodiments, the primer comprises SEQ ID NOs: 38-18.
4 contains more than 30 nucleotides from the sequence. If it is desired to obtain an extended cDNA containing the entire protein coding sequence, including the true translation start site, the second primer used will include a sequence located upstream of the translation start site. The second primer is extended to generate a second cDNA strand that is complementary to the first cDNA strand. Alternatively, RT-PCR may be performed as described above using primers derived from both ends of the cDNA to be obtained.

An mRNA comprising the sequence of the 5 ′ EST, for which the extended cDNA is desired, is hybridized with a primer comprising at least 10 contiguous nucleotides of a sequence complementary to the 5 ′ EST, and the hybridized primer is reverse transcribed to obtain mRNA. By preparing the first cDNA strand from the above, it is possible to prepare an extended cDNA containing the 5 ′ fragment of mRNA. Preferably, the primer contains at least 12, 15, or 17 contiguous nucleotides from the 5 'EST. More preferably, the primer has 20 to 30 consecutive nucleotides from the 5 'EST.

Thereafter, a second cDNA strand complementary to the first cDNA strand is synthesized. The first c
By hybridizing a primer complementary to the sequence of the DNA strand to the first cDNA strand and extending the primer to produce a second cDNA strand, a second cD
It is possible to create NA chains.

[0199] The double-stranded extended cDNA generated using the methods described above is isolated and cloned. The extended cDNA may be cloned into a vector such as a plasmid or a viral vector capable of replicating in a suitable host cell. For example, a host cell
It can be a bacterial, mammalian, bird, or insect cell.

Isolating the mRNA, reverse transcribing the primer hybridized to the mRNA to produce a first cDNA strand, extending the primer to create a second cDNA strand complementary to the first cDNA strand, Techniques for isolating double-stranded cDNA and cloning the double-stranded cDNA are well known to those skilled in the art and are described in Current Protocols.
in Molecular Biology, John Wiley an
d Sons, Inc. 1997 and Sambrook et al. ,
Molecular Cloning: A Laboratory Man
ual, Second Edition, Cold Spring Har
Bor Laboratory Press, 1989, the entire disclosure of which is hereby incorporated by reference.
(Herein incorporated by reference).

Alternatively, a procedure as described in Example 29 may be used for obtaining full-length or extended cDNA. In this approach, full-length or extended cDNA is prepared from mRNA and cloned into double-stranded phagemid as described below. The double-stranded phagemid cDNA library is then made single-stranded by treatment with an endonuclease, such as the Gene II product of phage F1, and exonuclease (Chang et al., Gene 127: 95-8, 1993). A biotinylated oligonucleotide comprising the sequence of the 5 'EST, or a fragment comprising at least 10 nucleotides thereof, is hybridized to a single-stranded phagemid. This fragment preferably contains at least 12, 15, or 17 contiguous nucleotides from the 5 'EST. More preferably, the fragment comprises 20-30 contiguous nucleotides from the 5 'EST. In some methods, fragments may contain more than 30 contiguous nucleotides from the 5 'EST.

The hybrid between the biotinylated oligonucleotide and the phagemid with an insert containing a 5 ′ EST sequence was isolated by incubating the hybrid with streptavidin-coated paramagnetic beads and collecting the beads using a magnet. (Fry et al., Biotechniques, 13: 124-131, 1992). Thereafter, the phagemid containing the 5 'EST sequence thus obtained is released from the beads, and converted into double-stranded DNA using a primer specific to the 5' EST sequence. Alternatively, a protocol such as the Gene Trapper kit (Gibco BRL) may be used. The double-stranded DNA thus obtained is transformed into bacteria. The extended cDNA containing the 5 'EST sequence is identified by colony PCR or colony hybridization.

Using any of the methods described above in Section III, a plurality of extended cDNAs containing full-length protein coding sequences or sequences encoding only the mature protein remaining after removal of the signal peptide were prepared as follows. Can be provided as a cDNA library for subsequent evaluation of the encoded protein or for diagnostic assays. IV. Expression of the protein encoded by the extended cDNA isolated using 5 'EST As described in Example 30 below, an extended cDNA comprising the entire protein coding sequence of the corresponding mRNA or a portion thereof, for example, The cDNA encoding the mature protein can be used to express the encoded secreted protein or a portion thereof. If desired, the extended cDNA may include a sequence encoding a signal peptide to facilitate secretion of the expressed protein. As described below, it is sufficient to be able to simultaneously clone multiple extended cDNAs, including the entire protein coding sequence or a portion thereof, into an expression vector to create an expression library for analyzing the encoded protein. Will be appreciated. Example 30 Expression of a protein encoded by a gene corresponding to 5 ′ EST or a part thereof In order to express a protein encoded by a gene corresponding to 5 ′ EST (or a part thereof), the whole protein was expressed. Full-length cDNAs containing the coding region or extended cDNAs containing sequences adjacent to the 5 'EST (or a portion thereof) are obtained as described in Examples 27-29 and cloned into an appropriate expression vector. Optionally, the nucleic acid may include a sequence encoding a signal peptide to facilitate secretion of the expressed protein. The nucleic acid inserted into the expression vector may also include a sequence upstream of the sequence encoding the signal peptide, for example, a sequence that regulates the expression level or a sequence that confers tissue-specific expression.

Using conventional cloning techniques, a nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector. The expression vector can be any of mammalian, yeast, insect or bacterial expression systems well known to those skilled in the art. Commercial vectors and expression systems are available from Gen.
etics Institute (Cambridge, MA), Strat
agene (La Jolla, California), Promega (
Madison, Wisconsin) and Invitrogen (Sa
n Diego, California). An expression vector, if necessary, to enhance expression and promote proper protein folding, as described by Hatfield et al., US Pat. No. 5,082,767, which is incorporated herein by reference. The codon context and codon pairing of the sequence can be optimized for the particular expression organism into which is introduced.

The cDNA clone cloned into the expression vector contains the total protein (ie, signal peptide and mature protein), the mature protein (ie,
A protein produced by excision of the signal peptide), the signal peptide or any other portion thereof.

[0206] Representative methods for expressing the protein encoded by the extended cDNA corresponding to the 5 'ESTs or nucleic acids described above are provided below. First, the methionine start codon of the gene and the polyA signal of the gene are identified. If the nucleic acid encoding the expressed polypeptide lacks methionine serving as a start site, the starting methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. it can. Similarly, if the extended cDNA lacks a polyA signal,
For example, this sequence can be added to the construct by splicing the polyA signal from pSG5 (Stratagene) using the BglII and SalI restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene). pXT1 is LTR and Moloney M
Contains part of the gag gene available from urine Leukemia Virus. The location of the LTR in the construct allows for efficient and stable transfection. The vector contains the Herpes Simplex thymidine kinase promoter and the selectable neomycin gene. Using an oligonucleotide primer that is complementary to the extended cDNA or a portion thereof and contains a restriction endonuclease sequence for Pst I incorporated into the 5 'primer and BglII at the 5' end of the corresponding cDNA 3 'primer, the extended cD
Care is taken to ensure that the NA is co-located with the polyA signal, and an extended cDNA or a portion thereof encoding the polypeptide to be expressed is obtained from the bacterial vector by PCR. The purified fragment obtained from the PCR reaction thus obtained is digested with PstI, blunt-ended with exonuclease, digested with BglII, purified, and further containing the polyA signal and containing the blunt / blunt B
glII).

Lipofectin (Life Technologies, Inc.,
The ligation product is transfected into mouse NIH 3T3 cells under the conditions outlined in the product specifications using a Grand Island, New York). 600 μg / ml G418 (Sigma, St. Louis)
After growing the transduced cells in S. Missouri), positive transductants are selected. Preferably, the expressed protein is released into the medium, which results in easy purification.

Alternatively, the extended cDNA may be cloned into pED6dpc2 as described above. The pED6dpc2 construct thus obtained may be transfected into a suitable host cell, for example, COS 1 cells. Select and grow methotrexate resistant cells. It is preferred that the protein expressed from the extended cDNA be released into the medium, resulting in easier purification.

The proteins in the medium are separated by gel electrophoresis. If necessary, proteins can be ammonium sulfate precipitated before electrophoresis, or separated based on size or charge.

[0210] As a control, an expression vector lacking the cDNA insert is introduced into a host cell or organism and the protein in the medium is harvested. The secreted protein present in the medium is detected using techniques well known to those skilled in the art, such as Coomassie blue or silver staining, or using antibodies against the protein encoded by the extended cDNA.

[0211] The synthetic 15-mer peptide having the sequence encoded by the appropriate 5 'EST, extended cDNA, or a portion thereof, is used to generate antibodies capable of specifically recognizing the protein of interest. It is possible. The synthetic peptide is injected into mice to generate antibodies to the polypeptide encoded by the 5 'EST, the extended cDNA, or a portion thereof.

A secreted protein from a host cell or organism containing an expression vector containing an extended cDNA derived from the 5 ′ EST or a portion thereof is compared to that of a control cell or organism. The presence of a band in the medium of cells containing the expression vector that is not present in the medium of control cells indicates that the extended cDNA encodes a secreted protein. Generally, the band corresponding to the protein encoded by the extended cDNA has a mobility close to that expected based on the number of amino acids in the open reading frame of the extended cDNA. However, bands may have different mobilities than expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

Alternatively, when the protein expressed from the expression vector does not contain a sequence directing its secretion, a protein expressed from a host cell containing an expression vector containing an insert encoding the secreted protein or a part thereof Can be compared to a protein expressed in a control host cell containing an expression vector without the insert. The presence of bands not present in samples from cells containing the expression vector without the insert in samples from cells containing the expression vector with the insert indicates that the desired protein or a portion thereof was expressed. . Generally, a band has the expected mobility for a secreted protein or a portion thereof. However, bands may have different mobilities than expected as a result of modifications such as glycosylation, ubiquitination, or enzymatic cleavage.

[0214] Standard immunochromatographic techniques can be used to purify the protein encoded by the extended cDNA. In such a procedure, a solution containing a secreted protein, such as a medium or cell extract, is applied to a column having an antibody against the secreted protein attached to a chromatography matrix. The secreted protein is bound to an immunochromatographic column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound secreted protein is then released from the column and recovered using standard techniques.

[0215] If antibody production is not possible, the extended cDNA sequence, or a portion thereof, may be incorporated, using a chimeric polypeptide, into an expression vector designed for use in a purification mechanism. In this manner, the extended cDNA or a portion thereof coding sequence is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera may be β-globin or a nickel binding polypeptide. The chimeric protein is purified using a chromatography matrix with antibodies to β-globin or with nickel attached. It is possible to manipulate the protease cleavage site between the β-globin gene or nickel binding polypeptide and the extended cDNA or a portion thereof. Thus, it is possible to separate the two chimeric polypeptides from each other by protease digestion.

One expression vector useful for generating β-globin chimeras is pSG5 (Stratagene), which encodes rabbit β-globin. Intron II of the rabbit β-globin gene promotes splicing of the expressed transcript, and a polyadenylation signal integrated into the construct increases expression levels. These techniques described are well known to those skilled in molecular biology. The standard method is Dav
is et al. , (Basic Methods in Molecula
r Biology, Davis, Divner, and Battery
, Ed. , Elsevier Press, NY, 1986), and many of the methods are described in Stratagene, Life.
Technologies, Inc. Or available from Promega.
in vitro Express ^™ Translation Kit (S
Additional polypeptides can be made from the construct using an in vitro translation system such as Tratagene.

After expressing and purifying the secreted protein encoded by the 5 ′ EST, the extended cDNA, or fragments thereof, the purified protein is deposited on the surface of various cell types as described in Example 31 below. The ability to bind can be tested. The protein groups to be evaluated simultaneously for the effects specifically described below, and other biological roles for which assays to determine the effects are available, include multiple proteins expressed from these cDNAs. It will be appreciated that this may be the case. Example 31 Analysis of Secreted Proteins to Determine Whether the Secreted Protein Binds to the Cell Surface The protein encoded by the 5 ' EST, the extended cDNA, or a fragment thereof, was prepared as described in Example 30 Clone into expression vector. The protein is purified by size, charge, immunochromatography or other techniques well known to those skilled in the art. After purification, the protein is labeled using techniques well known to those skilled in the art. The labeled protein is incubated with cells or cell lines derived from various organs or tissues, and the protein is bound to any receptors present on the cell surface. After incubation, cells are washed to remove non-specifically bound proteins. The labeled protein is detected by autoradiography. Alternatively, the unlabeled protein may be incubated with the cells and detected using a detectable label, for example, an antibody to which a fluorescent molecule has been attached.

The specificity of cell surface binding is analyzed by performing competition assays in which various amounts of unlabeled protein are incubated with the labeled protein. As the amount of competitive unlabeled protein increases, the amount of labeled protein bound to the cell surface decreases. As a control, various amounts of unlabeled protein unrelated to the labeled protein are included in some binding reactions. In the binding reaction, increasing the content of unrelated unlabeled protein does not decrease the amount of labeled protein bound to the cell surface, suggesting that the protein encoded by the cDNA specifically binds to the cell surface Is done.

As described above, secreted proteins have a number of important physiological effects and, as a result, have been shown to be valuable sources of therapy. As described below, it is possible to evaluate the secreted protein encoded by the extended cDNA or a portion thereof, produced by Examples 27-29, to determine its physiological activity. Of proteins expressed from Example 32 extended cDNA or a portion thereof, a cytokine, as assayed above for cell proliferation or cell differentiation activity, secreted proteins may act as cytokines or can influence cell growth or differentiation, There is. Many protein factors discovered to date have shown activity in one or more factor-dependent cell proliferation assays, including all known cytokines, and thus these assays
It is a convenient way to confirm cytokine activity. 32D, DA2, DA1G, T10
, B9, B9 / 11, BaF3, MC9 / G, M ⁺ (preB M ⁺ ), 2E8,
RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7
In cell lines, including but not limited to c and CMK, any one of a number of routine factor-dependent cell proliferation assays will demonstrate the activity of the protein encoded by the extended cDNA. The protein encoded by the extended cDNA or a portion thereof modulates T cell or thymocyte proliferation in the assays described above or as described in the following references, which are incorporated herein by reference. Ability to be assessed (Curren
t Protocols in Immunology, Ed. by Col
igan et al. , Green Publishing Assoc
Iates and Wiley-Interscience; Takaie
t al. , J. et al. Immunol. 137: 3494-3500, 1986
. Bertagnolli et al. , J. et al. Immunol. 145
: 1706-1712, 1990; Bertagnolli et al. , C
ell. Immunol. 133: 327-341, 1991; Berta
gnolli, et al. , J. et al. Immunol. 149: 3778-
3783, 1992; Bowman et al. , J. et al. Immunol.
152: 1756-1761, 1994).

In addition, many assays are known for cytokine production and / or proliferation of splenocytes, lymph node cells and thymocytes. Such as C
current Protocols in Immunology, supra 1:
3.12.1-3.12.14, and Schreiber In Curre
nt Protocols in Immunology, supra 1: 6.8.
The technique disclosed in 1-6.8.8 is mentioned.

[0221] The protein encoded by the cDNA can also be assayed for its ability to regulate the growth and differentiation of hematopoietic cells or lymphocyte producing cells.
Many assays for such activity are well known to those skilled in the art, including the assays in the following references, which are incorporated herein by reference: Bottomly
et al. , In Current Protocols in Immu
nology. 1: 6.3.1-6.3.12; deVries et.
al. , J. et al. Exp. Med. 173: 1205-1121,1991
Moreau et al .; , Nature 36: 690-692,198.
8; Greenberger et al. Proc. Natl. Aca
d. Sci. U. S. A. 80: 2931- 2938, 1983; Nor
Dan, R .; , In Current Protocols in Immu
nology. 1: supra, 6.6.1-6.6.5; Smith et al.
. Proc. Natl. Acad. Sci. U. S. A. 83: 1
857-1861, 1986; Bennett et al. , In Curr
ent Protocols in Immunology, supra 1: 6.1
5.1; Ciarretta et al. , In Current Prot
ocols in Immunology. Ibid 1: 6.13.1.

[0222] The protein encoded by the cDNA can be assayed for its ability to modulate a T cell response to an antigen. Many assays for such activity are well known to those skilled in the art, including the assays in the following references, which are incorporated herein by reference: Current Protocols in
Immunology, supra, Chapter 3 (Introduction on Mouse Lymphocyte Function)
Vitro assay), Chapter 6 (cytokines and their cellular receptors) and Chapter 7 (immunological studies in humans); Weinberger et al. , P
rc. Natl. Acad. Sci. USA 77: 6091-60
95, 1980; Weinberger et al. , Eur. J. Im
mun. 11: 405-411, 1981; Takai et al. , J. et al. Immunol. 137: 3494-3500, 1986; Takaie
t al. , J. et al. Immunol. 140: 508-512, 1988.

The protein exhibiting cytokine, cell proliferation, or cell differentiation activity can then be formulated as a medicament and used in the treatment of a clinical condition where induction of cell proliferation or differentiation is beneficial. Alternatively, as detailed below, the genes encoding these proteins or nucleic acids that regulate the expression of these proteins are introduced into a suitable host cell to increase or decrease the expression of these proteins as desired. It is also possible to make it. Example 33 Assay of Protein Expressed from Extended cDNA or Part thereof for Activity as an Immune Modulator The proteins encoded by the above cDNAs can be evaluated for their effects as immunomodulators. . For example, the proteins can be evaluated for their activity that affects thymocyte or spleen cell cytotoxicity. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Coligan
Ed., Current Protocols in Immunology, G
reene Publishing Associates and Wheel
y-Interscience, Chapter 3 (in terms of mouse lymphocyte function)
in vitro assays 3.1-3.19) and Chapter 7 (immunological studies in humans); Hermann et al., Proc. Natl. Acad. Sci. U
SA 78: 2488-2492, 1981; Hermann et al. Imm
unol. 128: 1968-1974, 1982; Handa et al. I
mmunol. 135: 1564-1572, 1985; Takai et al.
Immunol. 137: 3494-3500, 1986; Takai et al.
J. Immunol. 140: 508-512, 1988; Bowman et al. Virology 61: 1992-1998; Bertagnoll
i et al., Cell. Immunol. 133: 327-341, 1991; B
lown et al. Immunol. 153: 3079-3092, 1994
The assay described in the above.

The proteins encoded by the cDNAs can be evaluated for their effect on T cell-dependent immunoglobulin responses and isotype switching. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Mal
iszewski, J .; Immunol. 144: 3028-3033,1
990; Mond et al., Current Protocols in Immun.
, J., J., 1: 3.8.1-3.8.16, supra;

The proteins encoded by the above cDNAs can be evaluated for effects on immune effector cells, such as on Th1 cells and cytotoxic lymphocytes. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference:
: Current Protocols in Immunology (supra)
Chapter 3 (In Vitro Assays for Mouse Lymphocytes 3.1-3.1)
9) and Chapter 7 (Immunological studies in humans); Takai et al. Imm
unol. 137: 3494-3500, 1986; Takai et al. I
mmunol. 140: 508-512, 1988; Bertagnolli
J. et al. Immunol. 149: 3778-3783, 1992;

The proteins encoded by the above cDNAs can be evaluated for their effect on dendritic cell-mediated activation of inactive T cells. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Guery et al. Immu
nol. 134: 536-544, 1995; Inaba et al. Exp.
Med. 173: 549-559, 1991; Macatonia et al.
Immunol. 154: 5071-5079, 1995; Porgado
r et al. Exp. Med. 182: 255-260, 1995; Nai.
r et al. Virol. 67: 4062-4069, 1993; Huang
Et al., Science 264: 961-965, 1994; Macatonia.
J. et al. Exp. Med 169: 1255-1264, 1989; Bha
rdwaj et al., Journal of Clinical Investiga.
tion 94: 797-807, 1994; and Inaba et al. Ex
p. Med 172: 631-640, 1990;

The proteins encoded by the cDNAs can be evaluated for their effect on lymphocyte longevity. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Darzynkiewicz et al., Cytometry.
13: 785-808, 1992; Gorczyca et al., Leukemia 7
: 659-670, 1993; Gorczyca et al., Cancer Res.
53: 1945-1951, 1993; Itoh et al., Cell 66: 233-.
243, 1991; Zacharchuk, J .; Immunol. 145:
4037-4045, 1990; Zamai et al., Cytometry 14: 8.
91-897, 1993; Gorczyca et al., Int. J. Oncol.
1: 639-648, 1992.

The proteins encoded by the cDNAs can be evaluated for their effect on the early stages of T cell differentiation and cell development. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Antica et al., Blood 84: 111-117, 1994. ; Fine
Et al., Cell. Immunol. 155: 111-122, 1994; Ga
ly et al., Blood 85: 2770-2778, 1995; Toki et al., Pr.
oc. Nat. Acad Sci. USA 88: 7548-7551,
1991; but not limited thereto.

Proteins that exhibit activity as immune system modulators may then be formulated as medicaments and used to treat clinical conditions where modulation of immune activity is beneficial. For example, the protein is useful in treating a variety of immunodeficiencies and disorders (such as severe combined immunodeficiency), such as regulating the growth and proliferation of T and / or B lymphocytes (up or down) and NK cells and other Is useful for inducing the cytolytic activity of the cell population. These immunodeficiencies are either genetic or viral (eg, HIV)
And may be caused by bacterial or fungal infections or from autoimmune disorders. More specifically, infectious diseases caused by viral, bacterial, fungal or other infections can be treated using the protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention. Include HIV, hepatitis virus, herpes virus, mycobacteria, genus Leishmania, Pl
Various fungal infections, such as infections by the genus amodium and candidiasis. In this regard, of course, when it is desired to generally promote the immune system, for example, in the treatment of cancer, the protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention is useful. .

Alternatively, the protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention may be used, for example, for connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pneumonia, gyan- It may be used for the treatment of autoimmune diseases such as Barre's syndrome, autoimmune thyroiditis, insulin-dependent diabetes, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye diseases. 5'ES of the present invention
Such proteins encoded by extended cDNAs derived from T are also useful for treating allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory disorders. Other conditions in which immunosuppression is desired (eg, organ transplantation) can also be treated using the protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention.

Using the proteins of the present invention, it is also possible to modulate the immune response either upwards or downwards.

Down regulation may involve inhibiting or blocking an immune response that is already in progress, or may involve preventing the induction of an immune response. The function of activated T cells can be inhibited by suppressing the T cell response and / or inducing specific tolerance of the T cells. Immunosuppression of T cell responses
Generally, it is an aggressive non-antigen specific process that requires continuous exposure of T cells to the inhibitor. Tolerance involves inducing T cell unresponsiveness or anergy, but differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. In fact, in the absence of a tolerizing agent, re-exposure to a specific antigen does not show a T cell response, demonstrating tolerance.

[0233] Down-regulating or preventing one or more antigen functions (eg, including but not limited to B lymphocyte antigen functions such as B7 costimulation), eg, activated T Preventing high levels of lymphokine synthesis by cells has been demonstrated in the context of tissue, skin and organ transplantation, as well as in graft versus host disease (
GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplants, the graft is T
Rejection of the graft is initiated by its recognition as foreign by the cells, followed by destruction of the graft by an immune response. B7 lymphocyte antigen and its natural ligand on immune cells (eg, only a soluble monomeric form of a peptide having B7-2 activity or a soluble monomeric form of the peptide and another B lymphocyte antigen (
For example, when a molecule that inhibits or inhibits the interaction with B7-1, B7-3) or a monomeric form of a peptide that blocks antibodies is administered prior to transplantation. Can bind the molecule to the natural ligand on the immune cell without transmitting the corresponding costimulatory signal. In this regard, blocking B lymphocyte antigen function prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Further, the lack of costimulation may also be sufficient to anerize the T cells, thereby inducing tolerance in the subject. Inducing long-term tolerance with B lymphocyte antigen blocking reagents may avoid the need for repeated administration of these blocking reagents.
To achieve adequate immunosuppression or tolerance in a subject, it may be necessary to block the function of the B lymphocyte antigen combination.

Using animal models that are expected to be evaluated in humans, organ transplant rejection or G
The effect of a particular blocking reagent in preventing VHD can be evaluated. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogenic Langerhans islet cell grafts in mice, both CVs in vivo.
It has been used to study the immunosuppressive effects of TLA4Ig fusion proteins. For these, see Lenschow et al., Science 257: 789-.
792, 1992 and Turka et al., Proc. Natl. Acad.
Sci USA, 89: 11102-11105, 1992.
Furthermore, a GVHD mouse model (Paul, edited by Fundamental Imm
unology, Raven Press, New York, 1989, 84
6-847) using in vivo for the progression of the disease.
Can be used to determine the effect of blocking B lymphocyte antigen function.

[0235] Blocking antigen function is also therapeutically effective for treating autoimmune diseases. Many autoimmune disorders are due to inappropriate activation of T cells, where T cells are reactive to their own tissues and are exposed to cytokines and autoantibodies that are involved in the pathogenesis of the disease. Promotes production. Preventing the activation of self-reactive T cells can reduce or eliminate the symptoms of the disease. Auto-antibodies that inhibit T cell activation by administering reagents that block T cell costimulation by disrupting the receptor / ligand interaction of B lymphocyte antigens and potentially participate in disease processes Alternatively, the production of T cell-induced cytokines can be prevented. In addition, blocking reagents can induce antigen-specific tolerance of autoreactive T cells, which can lead to long-term relief from disease. Many animal models of a well-characterized human autoimmune disease can be used to determine the effect of a blocking reagent in preventing or alleviating an autoimmune disorder. Examples include mouse experimental autoimmune encephalitis, MRL
/ Pr / pr mice or systemic lupus erythematosus in NZB cross mice, autoimmune antigenic arthritis in mice (murine autoimmunocolla)
gen arthritis, diabetes in OD mice and BB rats, and experimental myasthenia gravis in mice (Ed. Paul, supra, 840-85).
See page 6).

As a means of up-regulating the immune response, up-regulating antigen function (preferably B lymphocyte antigen function) may also be therapeutically effective. Up-regulation of the immune response may involve either enhancing an existing immune response or eliciting an early immune response as shown in the examples below. For example, enhancing the immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection. In addition, administration of a systemic stimulated form of B lymphocyte antigen may alleviate systemic viral diseases such as influenza, common colds, and encephalitis.

Alternatively, T cells are removed from an infected patient and express in vitro the peptide encoded by the extended cDNA derived from the 5 ′ EST of the invention, or together with the peptide, derived from the 5 ′ EST of the invention. By co-stimulating the T cells with APC raised with a viral antigen that expresses a stimulated form of the soluble peptide encoded by the extended cDNA, and reintroducing the T cells sensitized in vitro into the patient, The patient may have an enhanced antiviral immune response. The infected cells can now deliver the costimulatory signal to the T cells in vivo and thus activate the T cells.

In another application, up-regulation or enhancement of antigen function, preferably of B lymphocytes, may be beneficial in inducing tumor immunity. 5 of the present invention
Administer to a subject a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide encoded by an extended cDNA derived from 'EST. Thus, the tumor-specific tolerance of the subject can be overcome. If desired, the tumor cells can be transfected to express the peptides in combination. For example, tumor cells obtained from a patient may be obtained by ex vivo ex vivo peptide alone or a combination of the peptide and a peptide having a B7-1-like activity and / or a B7-3-like activity. It can be transfected with an expression vector that directs expression. The transfected tumor cells are returned to the patient,
As a result, these peptides are expressed on the surface of the transfected cells. Alternatively, gene therapy techniques can be used to target tumor cells for transfection in vivo.

The presence of a peptide encoded by the extended cDNA derived from the 5 ′ EST of the present invention, having the activity of a B lymphocyte antigen, on the surface of the tumor cells provides the necessary costimulatory signal to the T cells. , T against transfected tumor cells
A cell-mediated immune response is induced. In addition, a sufficient amount of MHC class I or MH
Tumor cells that are unable to re-express or completely impair C-Class II molecules are treated with all or a portion of MHC Class I α and β ₂ microglobulins or MHC Class II α and MHC Class II β chains (eg, The cytoplasmic domain) can be transfected with a nucleic acid that encodes the MHC class I or class II protein on the cell surface, respectively. When suitable MHC class I or class II molecules are expressed in conjunction with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3), T cell mediated to transfected tumor cells A sexual immune response is induced. Optionally, a gene encoding an antisense construct that blocks MHC class II expression, such as the invariant chain, may also be co-transfected with DNA encoding a peptide having B lymphocyte antigen activity to present tumor-associated antigens To induce tumor-specific immunity. Thus, inducing a T cell-mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject. Alternatively, as described in detail below, a gene encoding a protein of these immune system modulators or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell and the protein, if desired, May be increased or decreased. The protein encoded by the assay described above extended cDNA or a portion thereof of proteins expressed from the extended cDNA or a portion thereof directed to Example 34 hematopoiesis regulatory activity may be evaluated for their hematopoiesis regulating activity. For example, the effect of the protein on the differentiation of embryonic stem cells can be evaluated. Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Johansson et al., Cell. Biol.
15: 141-151, 1995; Keller et al., Mol. Cell.
Biol. 13: 473-486, 1993; McClanahan et al., Bl.
wood, 81: 2903-2915, 1993;

The proteins encoded by the above-described extended cDNAs or portions thereof can be evaluated for their effect on stem cell longevity and stem cell differentiation.
Many assays for such activity are well known to those of skill in the art and include the following references, which are incorporated herein by reference: Freshney
, Methylcellulose Colony Forming Asa
ys, in Culture of Hematopoietic Cells
. Ed., Freshney et al., Pages 265-268, Wiley-Liss, In.
c. , New York, NY. 1994; Hirayama et al., Proc.
Natl. Acad. Sci. USA 89: 5907-5911,1.
992; McNiece and Bridell, in Culture of
Hematopoietic Cells, supra; Neben et al., Exp.
Hematol. 22: 353-359, 1994; Ploemacher and Cobblestone In Culture of Hematopo.
ieic Cells, supra, 1-21; Spooncer et al., in Cul.
cure of Hematopoietic Cells, supra, 163-1.
79 and Sutherland in Culture of Hemat
opoietic Cells, supra, 139-162;

The protein exhibiting hematopoietic regulatory activity can then be formulated as a medicament and used to treat a clinical condition in which regulating hematopoiesis is beneficial, such as treating bone marrow or lymphocyte cell deficiency. Improved hematopoietic regulation is also noted by the surrounding biological activity that supports the colony forming cells or factor-dependent cell lines. For example, proteins alone or in combination with other cytokines that support the growth or proliferation of erythroid progenitors may be used, for example, in the treatment of various anemias or to stimulate the production of erythroid progenitors and / or erythroid cells. Useful in combination with irradiation / chemotherapy. Proteins that support the growth or proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, conventional CSF activity) can be used, for example, with chemotherapy to prevent or treat the resulting myelosuppression. May be useful in combinations of Proteins that support megakaryocyte growth and proliferation, i.e., platelet growth and proliferation, can prevent or treat various platelet abnormalities, such as thrombocytopenia, and can generally be used to replace or assist platelet infusion . Thus, proteins that support the growth and proliferation of hematopoietic stem cells, which can mature into some and all of the above hematopoietic cells, are susceptible to a variety of stem cell abnormalities (such as those normally treated with transplantation, eg, aplastic anemia) And paroxysmal nocturnal hemoglobinuria), as well as normal cells or cells engineered for gene therapy, either in vivo or ex vivo (in combination with bone marrow transplantation or peripheral progenitor). It is therapeutically useful for the expansion of stem cell fractions after irradiation / chemotherapy, either by cell transplantation (allogeneic or xenogeneic). Alternatively, as described in detail below, a gene encoding a protein having hematopoietic regulatory activity or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell and expressed as desired. Can be increased or decreased. Protein encoded by Example 35 tissue extended cDNA or protein expressed from a portion thereof intended for regulation of growth assays extension cDNA or portions thereof, may be evaluated for their effect on tissue growth be able to. Many assays for such activity are well known to those of skill in the art and are described in International Patent Publication WO 95/1603.
5, International Patent Publication WO95 / 05846, and International Patent Publication WO91 / 0
No. 7491, which are incorporated herein by reference.

[0242] Assays for wound healing activity include those described by Winter, Epiderma.
l Wound Healing, pp. 71-112, Maibach and Ro.
ed., Year Book Medical Publishers, I
nc. , Chicago, Eaglstein and Me
rtz, J. et al. Invest. Dermatol. 71: 382-84, 1978, which is incorporated by reference herein, but is not limited thereto.

The proteins involved in regulating tissue growth can then be formulated as medicaments and used to treat clinical conditions where regulating tissue growth is beneficial. For example, the proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention may also be used to grow or regenerate bone, cartilage, tendons, ligaments and / or nervous tissue, and to heal wounds and repair tissues. Compositions used for replacement, as well as burns,
It may be useful in treating incisions and ulcers.

The protein encoded by the extended cDNA derived from the 5 ′ ESTs of the present invention may be used to induce cartilage and / or bone growth in an environment where bone is not normally formed, and thus may be used in humans and other animals. Applicable for healing fractures and cartilage damage or defects in Such preparations using the proteins of the invention may have prophylactic use in reducing closed and open fractures and improving fixation of artificial joints. De novo osteosynthesis induced by osteogenic agents helps repair congenital, trauma-induced or oncological resection-induced craniofacial defects and is also useful in cosmetic surgery.

The proteins of the present invention can also be used in the treatment of periodontal disease and other dental restoration processes. Such agents can provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells, or induce osteogenic progenitor cell differentiation. The proteins of the invention may also be used, for example, by stimulating bone and / or cartilage repair or by inhibiting the process of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory or inflammatory processes. It is also useful for treating osteoporosis or osteoarthritis.

Another category of tissue regeneration activity that can be attributed to the protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention is tendon / ligament formation. The protein encoded by the extended cDNA derived from the 5 'ESTs of the present invention induces the formation of such tissues in environments where tendon / ligament-like or other tissues are not normally formed, and humans and other It has applicability in healing tendon or ligament rupture, deformation and other tendon or ligament defects in animals. Such preparations using proteins that induce tendon / ligament-like tissue prevent damage to tendon or ligament tissue, as well as improve tendon or ligament fixation to bone or other tissue, and repair defects to tendon or ligament tissue May have prophylactic uses. The de novo tendon / ligament-like tissue formation induced by the composition encoded by the extended cDNA derived from the 5 ′ ESTs of the present invention is capable of producing congenital, traumatic or otherwise derived tendons or ligaments. It is useful for repairing ligament defects and is also useful in cosmetic surgery for attaching or repairing tendons or ligaments. Extended cDNA derived from 5 'EST of the present invention
A composition that attracts tendon or ligament forming cells, stimulates proliferation of tendon or ligament forming cells, induces differentiation of tendon or ligament forming cell precursor cells, or causes tissue repair Ex vivo to get back in vivo
To provide an environment for inducing the growth of tendon / ligament cells or progenitor cells in the cell. The compositions of the present invention are also useful for treating tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The composition may include a suitable matrix and / or sequestering agent well known in the art as a carrier.

The proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention may be used for the growth of nerve cells and regeneration of nerve and brain tissue, ie, diseases and gangrene of the central nervous system and peripheral nervous system, and nerves. It is also useful for treating mechanical and traumatic disorders involving cell or neural tissue degeneration, death or trauma. More specifically, proteins are expressed in peripheral nervous system disorders such as peripheral nerve damage, peripheral and localized neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager. It may be used to treat central nervous system disorders such as the syndrome. In addition, conditions that can be treated according to the present invention include mechanical and traumatic disorders such as spinal cord disorders, head trauma, and cerebrovascular diseases such as stroke. Peripheral neuropathy caused by chemotherapy or other medical therapy can also be treated using the proteins of the present invention.

The proteins of the present invention are also useful for promoting better or faster closure of non-healing wounds, such as pressure ulcers, ulcers with vascular insufficiency , Surgical and traumatic wounds, and the like.

The proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention also include organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (
Be capable of exhibiting the development or regeneration of other tissues, such as smooth muscle, skeletal muscle or myocardium) and vascular (including vascular endothelial) tissues, or activity to promote the growth of cells comprising such tissues Is expected. One of the desired effects is to allow normal tissue development by inhibiting or modulating fibrotic scarring. The proteins of the present invention may also exhibit angiogenic activity.

The proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention may be used to protect or regenerate gut and lung or liver fibrosis, reperfusion injury in various tissues, and conditions caused by systemic cytokine injury. It is also useful for the treatment of

The protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention may also promote or inhibit the differentiation of said tissue from progenitor tissues or cells, or inhibit the growth of said tissue. Useful.

Alternatively, as described in detail below, a gene encoding a protein having activity to regulate tissue growth or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell, and As described above, the expression of the protein can be increased or decreased. Protein encoded by the assay extended cDNA or a portion thereof of protein expressed from the extended cDNA or a portion thereof and directed to modulation of embodiment 36 reproductive hormones, they that regulate reproductive hormones, such as follicle stimulating hormone You can also evaluate your ability. Many assays for such activity are well known to those of skill in the art and include those disclosed in the following references, which are incorporated herein by reference: Vale et al., Endocrinol. 91: 562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Nature 321: 776-779, 1986; Mason et al., Nature 318: 659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:30
91-3095, 1986, Current Protocols in Immunology Section 6.12, edited by Colligan et al., Greene Pu
Blistering Associates and Wiley-Interscience; Taub et al. Clin. Invest. 95: 137
0-1376, 1995; Lind et al., APMIS 103: 140-146.
1995; Muller et al., Eur. J. Immunol. 25:17
Gruber et al. Immunol. 152: 5860-
5867, 1994; Johnston et al. Immunol. Fifteen
3: 1762-1768, 1994.

The reproductive hormones or those proteins that exhibit activity as modulators of cell motility may then be formulated as medicaments and used to treat clinical conditions where modulation of reproductive hormones would be beneficial. For example, the extended cDN derived from the 5 ′ EST of the present invention
The protein encoded by A may also exhibit activin or inhibin related activity. Inhibin is characterized by its ability to inhibit follicle stimulating hormone (FSH) release, and activin is characterized by its ability to stimulate FSH release. Therefore,
The protein encoded by the extended cDNA derived from the 5 ′ EST of the present invention, alone or in the form of a heterodimer with a member of the inhibin α family, reduces fertility in female mammals and reduces male fertility. It may be useful as a contraceptive based on the ability to reduce spermatogenesis in mammals. Administration of sufficient amounts of other inhibins can induce infertility in these animals. Alternatively, the protein of the present invention exists as a homodimer or a heterodimer with other protein subunits of the Inhibin B group, and FSH from cells of the anterior pituitary gland.
It may be useful as a fertility induction therapy based on the ability of the activin molecule to stimulate release. See, for example, U.S. Pat. No. 4,798,885. The disclosure content of this reference is incorporated herein by reference. The proteins of the present invention are also useful in advancing the development of fertility in sexually immature mammals so as to enhance the fertility of livestock such as cattle, sheep and pigs during their lifetime.

Alternatively, as described in detail below, a gene encoding a protein having reproductive hormone regulatory activity or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell and, as desired, Protein expression can be increased or decreased. Example 37 Assay of Protein Expressed from Extended cDNA or Part thereof for Chemotactic / Chemokinetic Activity The protein encoded by the extended cDNA or part thereof can also be evaluated for chemotactic / chemokinetic activity. it can. For example, proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention include, for example, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial and / or endothelial cells. May have chemotactic or chemokinetic activity (eg, acting as a chemokine) on mammalian cells containing. Using chemotactic and chemokinetic proteins,
The desired cell population can be moved or attracted to the desired site of action. Chemotactic and chemokinetic proteins offer particular advantages in the treatment of wounds and other trauma to tissues, as well as in the treatment of localized infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent.

If a protein or peptide can directly or indirectly stimulate the directed orientation or movement of a particular cell population, the protein or peptide will have chemotactic activity on that particular cell population. Have. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a population of cells can be readily determined by using such a protein or peptide in any known assay for cell chemotaxis.

The activity of the protein of the present invention can be measured, inter alia, by the following method.

Assays for chemotactic activity (identifying proteins that induce or prevent chemotaxis) are based on the ability of the protein to induce cell migration across membranes, and the ability of one cell population to It consists of an assay that measures the ability of the protein to induce adhesion to the population. Suitable assays for motility and attachment include Curr
ent Protocols in Immunology, Coligan
, Kruisbeek, Margulies, Shevach and Strob
er, Pub. Green Publishing Associate
s and Wiley-Interscience, Chapter 6.12: 6.12. 1-6.12.28; Taub et al. Clin. Invest. 95: 1 370-1376, 1995; Lind et al., APMIS 103: 140-14.
6, 1995; Mueller et al., Eur. J. Immunol. 25
Gruber et al., J. Am. Immunol. 152: 5 860-5867, 1994; Johnston et al. Immunol. 153: 1762-1768, 1994, but are not limited thereto. Protein encoded by the assay extended cDNA or a portion thereof of protein expressed from Example 38 extended cDNA or portion thereof intended for regulation of blood clotting may also be evaluated for their effects on blood clotting it can. Many assays for such activity are well known to those of skill in the art, including those disclosed in the following references, which are incorporated herein by reference: Lin
et al. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al., Thrombosis Res. 45: 413
-419, 1987; Humphrey et al., Fibrinolysis 5: 71-79, 1991; Schaub, Prostaglandins 35: 467-474, 1988.

Then, those proteins involved in the regulation of blood coagulation are formulated as medicaments,
Modulation of blood coagulation may be used to treat clinical conditions where beneficial. For example, the proteins of the present invention may also exhibit hemostatic or thrombolytic activity.
As a result, such proteins may inhibit coagulation and other hemostatic events in the treatment of various coagulation disorders, including genetic disorders such as hemophilia, or in the treatment of wounds resulting from trauma, surgery or other causes. It is expected to be useful for promoting.
The proteins of the present invention may also be useful in preventing and treating thrombolysis or inhibition of thrombus formation and conditions resulting from thrombus formation, such as cardiovascular and central nervous system infarction (eg, stroke). Alternatively, as described in detail below, a gene encoding a protein having blood clotting activity or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell and the expression of the protein is increased as desired. Can be increased or decreased. Protein encoded by Example 39 Receptor / Ligand involvement assay extended cDNA or a portion thereof of a protein that is extended cDNA or a portion thereof or we expressed targeting of the interaction to the receptor / ligand interactions Can also be assessed for their involvement. Many assays for such involvement are well known to those of skill in the art, including those disclosed in the following references, which are incorporated herein by reference:
current Protocols in Immunology, 7.7.2
Chapter 8.1-7.28.22, edited by Colligan et al., Greene Publishing Associates and Wiley-Interscience; Takai et al., Pro. Natl. Acad. Sci. USA
84: 6864-6868, 1987; Bierer et al. Exp. M ed. 168: 1145-1156, 1988; Rosenstein et al. Exp. Med. 169: 149-160, 1989; Stoltemborg et al. Immunol. Methods 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995; Gyuris et al., Cell 75: 791-803, 1993.

For example, the proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention exhibit activity as receptors, receptor ligands or inhibitors or agonists of receptor / ligand interactions. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their (Including but not limited to cell adhesion molecules such as selectins, integrins and their ligands) and receptor / ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses But not limited to them. Receptors and ligands are also useful for screening for potential peptide or small molecule inhibitors of the relevant receptor / ligand interaction. The proteins encoded by the extended cDNAs derived from the 5 ′ ESTs of the present invention, including but not limited to receptor and ligand fragments, are useful as inhibitors of receptor / ligand interactions per se. possible. Alternatively, as described in detail below, a gene encoding a protein involved in receptor / ligand interaction or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell and, as desired, Protein expression can be increased or decreased. Example 40 Assay of Protein Expressed from Extended cDNA or Part thereof for Anti-inflammatory Activity The protein encoded by the extended cDNA or a part thereof can also be evaluated for anti-inflammatory activity. Anti-inflammatory activity stimulates cells involved in the inflammatory response, thereby inhibiting or promoting cell-cell interactions (eg, cell attachment), thereby chemotaxis of cells involved in the inflammatory process. It can be achieved by inhibiting or promoting sex, by inhibiting or promoting cell extravasation, or by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Proteins exhibiting such activity can be used to treat inflammatory conditions, including chronic or acute conditions. The inflammatory symptoms include inflammation associated with infection (sepsis shock, sepsis or systemic inflammatory response syndrome), ischemic-reperfusion injury (ischemia-reperfusioninury), endotoxin mortality, arthritis, complement-mediated hyperacute rejection, Examples include, but are not limited to, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease, or diseases caused by excessive production of cytokines such as TNF or IL-1. The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials. Alternatively, as described in detail below, a gene encoding a protein having anti-inflammatory activity or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell and the expression of the protein is increased as desired. Can be increased or decreased. Protein encoded by the assay extended cDNA or a portion thereof of proteins expressed from the extended cDNA or a portion thereof directed to Example 41 Tumor Inhibition activity can also be evaluated for tumor inhibition activity. In addition to the activities described above for immunological treatment or prevention of tumors, the proteins of the invention may exhibit other anti-tumor activities. The protein can directly or indirectly inhibit tumor growth (eg, via ADCC). Proteins inhibit tumor growth by acting on tumor tissue or tumor precursor tissue, thereby inhibiting the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis). Tumor-inhibiting activity of the protein by causing the production of other factors, agents or cell types that inhibit it, or by suppressing, eliminating or inhibiting factors, agents or cell types that promote tumor growth Can be indicated. Alternatively, as described in detail below, a gene encoding a protein having tumor inhibitory activity or a nucleic acid that regulates the expression of such a protein is introduced into a suitable host cell, and the expression of the protein is increased as desired. Can be increased or decreased.

A protein of the invention may also exhibit one or more of the following additional activities or effects: increasing, inhibiting, or killing, an infectious agent, or killing the infectious agent, such as the infectious agent. Factors include, but are not limited to, bacteria, viruses, fungi and other parasites); affecting (suppressing or enhancing) bodily characteristics, such as height , Weight, hair color,
Eye color, skin, fat to lean ratio or other tissue pigmentation, or the size or shape of organs or body parts (eg,
Such as, but not limited to, changes in bone morphology or shape); affecting biorhythms or circadian rhythms or rhythms; affecting fertility of male or female subjects; Affect the metabolism, catabolism, anabolism, processing, utilization, storage or excretion of fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional factors or components; That (the behavioral characteristics include appetite,
Libido, stress, cognition (including cognitive impairment), depression (including but not limited to depression) and violent behavior); producing analgesic or other pain-suppressing effects; non-hematopoietic lineage Promoting differentiation and expansion of embryonic stem cells in the humoral or endocrine activity; in the case of enzymes, correcting enzyme deficiency and treating deficiency-related diseases; treating hyperproliferative abnormalities such as psoriasis; Immunoglobulin-like activity (eg, the ability to bind to an antigen or complement); and which act as an antigen in a vaccine composition and exhibit cross-reactivity with such a protein or another material or such a protein Ability to elicit an immune response against Alternatively, as described in detail below, a gene encoding a protein involved in any of the above activities, or a nucleic acid that regulates the expression of such a protein, is introduced into a suitable host cell and, as desired, The expression of the protein can be increased or decreased. Example 42 Identification of a Protein That Interacts with a Polypeptide Encoded by an Extended cDNA A protein (eg, a receptor protein) that interacts with a polypeptide encoded by a cDNA derived from 5 ′ EST or a fragment thereof is a Matchmaker Two protein. Identification can be performed using a two-hybrid system such as Hybrid System 2 (catalog number K1604-1, Clontech). As described in the instructions accompanying the kit (incorporated herein by reference), the cDNAs derived from the 5 'ESTs or fragments thereof were replaced with the DNA of the yeast transcriptional activator GAL4.
It is inserted into an expression vector so that it is in frame with the DNA encoding the binding domain. CDNA in a cDNA library encoding a protein capable of interacting with the polypeptide encoded by the extended cDNA or a portion thereof
Are inserted into the second expression vector such that they are in frame with the DNA encoding the activation domain of GAL4. The two expression plasmids are transformed into yeast, and the yeast is plated on a selection medium that selects for expression of the selectable marker in each expression vector and GAL4-dependent expression of the HIS3 gene. A transformant capable of growing on a medium lacking histidine was identified as GA
Screen for L4-dependent lacZ expression. Histidine selection and l
Those cells that are positive in both acZ assays contain a plasmid that encodes a protein that interacts with the polypeptide encoded by the extended cDNA or a portion thereof.

Alternatively, to identify molecules that interact with the polypeptide encoded by the extended cDNA, see Lustig et al., Methods in Enzymolo.
gy 283: 83-99, 1997 and U.S. Patent No. 5,654,150, the disclosure of which is incorporated herein by reference.
In such a system, in a pool of vectors containing an extended cDNA insert cloned downstream of a promoter that drives transcription in vitro,
Perform a transcription reaction in vitro. The resulting mRNA pool was
Introduce into opus laevis oocytes. The oocytes are then assayed for the desired activity.

Alternatively, pooled in vitro transcripts produced and pooled as described above may be translated in vitro. The pooled in vitro translation products can be assayed for the desired activity or interaction with a known polypeptide.

A variety of additional techniques can be used to find proteins or other molecules that interact with the polypeptide encoded by the extended cDNA. Otherwise,
An affinity column containing the polypeptide encoded by the extended cDNA or a portion thereof can be constructed. Optionally, in this method, the affinity column contains a chimeric protein in which a protein encoded by the extended cDNA or a part thereof is fused to glutathione S-transferase. The mixture of cellular proteins or the pool of expressed proteins described above is applied to an affinity column. The protein that interacts with the polypeptide attached to the column is then isolated and isolated from Ramunsen et al., Electrophoresis.
18: 588-598, 1997, and can be analyzed on a 2-D electrophoresis gel. The disclosure of that reference is incorporated herein by reference. Alternatively, the proteins retained on the affinity column can be purified by electrophoresis-based methods and sequenced. The same method can be used to isolate antibodies, screen phage display products, or screen phage display human antibodies.

Edwards and Leatherbarrow, Analytical
As described in Biochemistry 246: 1-6, 1997, optical biosensors can also be used to screen for proteins that interact with the polypeptide encoded by the extended cDNA or a portion thereof.
The disclosure of that reference is incorporated herein by reference. The main advantage of this method is that the association ratio between the protein and the other interacting molecule can be determined. Thus, it is possible to specifically select interacting molecules with a high or low association ratio. Typically, a target molecule is bound to the sensor surface (via a carboxymethyl dextran matrix) and a sample of the test molecule is placed in contact with the target molecule. When the test molecule binds to the target molecule, a change in refractive index and / or concentration occurs. This change is detected by a biosensor. However, this change occurs in a temporary field (extending a few hundred nanometers from the sensor surface). In these screening assays, the target molecule can be one of the polypeptides encoded by the extended cDNA or a portion thereof, and the test sample comprises:
It can be a collection of proteins extracted from a tissue or cell, a pool of expressed proteins, a combinatorial peptide and / or chemical library, or a group of peptides displayed by phage. The tissue or cell from which the test protein is extracted can be from any species.

In other methods, the target protein is immobilized and the test population is a collection of unique polypeptides encoded by the extended cDNA or a portion thereof.

To study the interaction between the protein encoded by the extended cDNA or a portion thereof and the drug, microdialysis was performed using Wang et al., Chromatogra.
pia 44: 205-208, 1997 or the Bus method.
ch et al. Chromatogr. 777: 311-328, 1997.
Can be used in combination with the affinity capillary electrophoresis described in (1). The disclosure of that reference is incorporated herein by reference.

It will be appreciated by those skilled in the art that proteins expressed from the extended cDNA or portions thereof can be assayed for many activities in addition to those specifically enumerated above. For example, the expressed protein can be evaluated for applications involving the modulation and control of inflammation, tumor growth or metastasis, infection, or other clinical symptoms. Furthermore, proteins expressed from the extended cDNA or a part thereof may be useful as nutritional or cosmetic substances.

[0268] Proteins expressed from the cDNA or portions thereof can be used to generate antibodies capable of specifically binding to the expressed protein or fragment thereof, as described in Example 40 below. . The antibody is c-derived from 5 'EST
Full length protein encoded by DNA, cDNA derived from 5 'EST
May be capable of binding to the mature protein encoded by (i.e., the protein resulting from cleavage of the signal peptide), or the signal peptide encoded by the cDNA derived from 5'EST. Alternatively, the antibody is the cDNA described above.
May be capable of binding to a fragment of at least 10 amino acids of the protein encoded by In some embodiments, the antibody may be capable of binding a fragment of at least 15 amino acids of the protein encoded by the above cDNA. In still other embodiments, the antibody may be capable of binding a fragment of at least 25 amino acids of a protein expressed from the extended cDNA,
The fragment contains at least 25 amino acids of the protein encoded by the above cDNA. In a further embodiment, the antibody may be capable of binding to a fragment of at least 40 amino acids of the protein encoded by the cDNA described above. Example 43 Production of Antibodies Against Human Proteins Substantially pure proteins or polypeptides are isolated from cells transfected or transformed as described in Example 30. For example, Am
Adjust the protein concentration of the final preparation by concentrating to a level of a few μg / ml on an icon filter. Then, monoclonal or polyclonal antibodies to the protein can be prepared as follows. 1. Production of Monoclonal Antibodies by Hybridoma Fusions Monoclonal antibodies directed against the epitope of any peptide identified and isolated as described by Kohler and Milstein, Nature 25
6: 495, 1975, or can be prepared from mouse hybridomas according to methods derived therefrom. Briefly, for several weeks, mice are repeatedly inoculated with several milligrams of the selected protein or peptides derived from the protein. The mice are then sacrificed and the antibody-producing spleen cells are isolated. The spleen cells are fused with polyethylene glycol with mouse myeloma cells and growth of the system on selective media containing aminopterin (HAT media) destroys excess unfused cells. Dilute the successfully fused cells and place aliquots of the dilutions into the wells of a microtiter plate, where culture growth is continued. Originally Engval, Meth. Enzy
mol. 70: 419, 1980 (the disclosure of which is incorporated herein by reference) by detecting antibodies in well supernatants by immunoassay procedures such as ELISA and methods derived therefrom. Identify the clone. The selected positive clones can be cultured and their monoclonal antibody products recovered for use. For detailed procedures for monoclonal antibody production, see Davis et al., Basic Methods in Molec.
ullar Biology Elsevier, New York, 21-2
Section. The disclosure of that reference is incorporated herein by reference. 2. Production of polyclonal antibodies by immunizationA suitable animal is immunized with an expressed protein or a peptide derived therefrom that is unmodified or that can be modified to enhance immunogenicity. A polyclonal antiserum containing antibodies to the heterologous epitopes of the protein can be prepared. Many factors associated with both antigen and host species affect the production of effective polyclonal antibodies. For example, small molecules tend to be less immunogenic than other molecules and may require the use of carriers and adjuvants. In addition, the response of the host animal will vary depending on the site of inoculation or dose, and inappropriate or excessive amounts of antigen will reduce antiserum titers. Small doses (ng levels) of antigen administered to multiple intradermal sites appear to be the most reliable. An effective immunization protocol for rabbits
tukaitis et al. Clin. Endocrinol. Metab
. 33: 988-991 (1971). The disclosure of that reference is incorporated herein by reference.

[0269] Periodic boosts can be made and the antiserum is harvested when its titer begins to drop. Antibody titers are measured semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of antibody. For example, Ouchterlony et al., H
andbook of Experimental Immunology 1
Chapter 9, D.C. See Wier (eds), Blackwell (1973). The disclosure of that reference is incorporated herein by reference. The plateau concentration of the antibody is usually in the range of 0.1-0.2 mg / ml serum (about 12 μM). See, for example, Fisher, D.A. Manual of Clinical Immun
technology, 2nd edition (edited by Rose and Friedman), Amer. Soc.
For Microbiol. , Washington, D.C. C. (19
By preparing a competitive binding curve as described in 80), the affinity of the antiserum for the antigen is determined. The disclosure of that reference is incorporated herein by reference.

Antibody preparations prepared according to any protocol are useful in quantitative immunoassays for determining the concentration of a substance having an antigen in a biological sample. They are used semi-quantitatively or quantitatively to identify the presence of an antigen in a biological sample. The antibodies may be used in therapeutic compositions to kill cells that express the protein or reduce the level of the protein in the body. V. 5 'ESTs or 5' ESTs or 5 'ESTs (or derived therefrom cDNA or genomic DNA uses the present invention as a reagent sequences from a portion thereof
) Can be used as reagents in isolation procedures, diagnostic assays, and forensic procedures. For example, a sequence derived from 5 ′ EST (or cDNA or genomic DNA obtained therefrom) may be detectably labeled and used as a probe to isolate other sequences that can hybridize to the sequence. In addition, 5 'EST (
Alternatively, sequences derived from cDNA or genomic DNA) can be used to design PCR primers for use in isolation, diagnostic, and forensic procedures. 1.5 Use of the sequence derived from 5 'EST or 5' EST or a part thereof in isolation, diagnostic and forensic procedures Example 44 Preparation of PCR primers and amplification of DNA 5 ' EST sequence (or cDNA obtained therefrom) Or genomic DNA) can be used to prepare PCR primers for a variety of applications, including isolation procedures for cloning nucleic acids capable of hybridizing to such sequences, diagnostic techniques and forensic techniques. PCR primers are at least 10 bases, preferably at least 12, 15, or 17 bases in length. More preferably, P
CR primers are at least 20-30 bases in length. In some embodiments, PCR primers may be longer than 30 bases. The primer pairs have approximately the same G / C ratio so that the melting points are approximately the same. Various P
CR technology is well known to those skilled in the art. For an overview of PCR technology, see Mole
cultural Cloning to Genetic Engineering
, White eds, Methods in Molecular Bilog
y 67: Humana Press, Totowa 1997. The disclosure of that reference is incorporated herein by reference. These PCR
In each of the procedures, a P for each side of the nucleic acid sequence to be amplified
The CR primer was used for dNTP and Taq polymerase, Pfu polymerase,
Alternatively, it is added to a suitably prepared nucleic acid sample together with a thermostable polymerase such as Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers specifically hybridize to the complementary nucleic acid sequence in the sample. The hybridized primer is extended. Thereafter, another cycle of denaturation, hybridization and extension begins. The cycle is repeated multiple times to generate an amplified fragment containing the nucleic acid sequence between the primer sites. Example 45 Use of 5 'EST as a Probe Probes derived from 5' EST sequences, including full length cDNA or genomic sequences (or cDNA or genomic DNA obtained therefrom), include radioisotopes and non-radioactive labels. Labeled with a detectable label well known to those skilled in the art,
A detectable probe can also be provided. Detectable probes may be single-stranded or double-stranded, and may be made using techniques known to those of skill in the art, including in vitro transcription, nick translation, or kinase reactions. A nucleic acid sample containing a sequence capable of hybridizing to the labeled probe is contacted with the labeled probe. When the nucleic acid in the sample is double-stranded,
The nucleic acid may be denatured before contacting the probe. For some applications, the nucleic acid sample may be immobilized on a surface such as a nitrocellulose or nylon membrane.
Nucleic acid samples can include nucleic acids obtained from various sources, including genomic DNA, cDNA libraries, RNA, or tissue samples.

The procedures used to detect the presence of a nucleic acid capable of hybridizing to a detectable probe include Southern blotting, Northern blotting,
Well-known techniques such as dot blotting, colony hybridization, and plaque hybridization are included. For some applications, the nucleic acid capable of hybridizing to the labeled probe is cloned into a vector, such as an expression vector, a sequencing vector, or an in vitro transcription vector, to characterize and express the hybridized nucleic acid in the sample. Can be easier. For example, using such a technique, sequences in a genomic or cDNA library that can hybridize to the detectable probes described in Example 30 above may be isolated and cloned.

The PCR primers generated as described in Example 44 above can also be used for forensic analysis, such as the DNA fingerprinting techniques described in Examples 46-50 below. Such analysis may utilize a detectable probe or primer based on the sequence of the 5 ′ EST or cDNA or genomic DNA isolated using the 5 ′ EST. Example 46 Forensic Matching by DNA Sequencing In one exemplary method, a DNA sample is isolated by conventional methods, eg, from a forensic specimen of hair, semen, blood or skin cells. Then, according to Example 44, a panel of PCR primers based on the many 5 'ESTs of Example 25 or cDNA or genomic DNA isolated from the 5' ESTs as described above was used to obtain approximately 5 minutes in length from forensic specimens. Amplify DNA of 100-200 bases. The corresponding sequence is obtained from the test subject. The sequence is then determined for each of these identified DNAs using standard techniques, and by comparing the sample database, if there is a difference between the sequence from the subject and the sequence from the sample, Determine this. The presence of a statistically significant difference between the subject's DNA sequence and the DNA sequence from the sample indicates a lack of identity. Such a lack of identity can be demonstrated, for example, by a single sequence. On the other hand, identity should be demonstrated by the fact that they all match in multiple sequences. Preferably, at least 50 statistically identical sequences of 100 bases in length are used to prove identity between the suspect and the sample. Example 47 Positive Identification by DNA Sequencing The techniques outlined in the previous example can be used on a larger scale to provide unique fingerprint-type identification of any individual. In the present technology, many 5 ′ EST sequences derived from Example 25 or c
Prepare primers from DNA or genomic DNA sequences. Preferably, 20 to
Use 50 different primers. Using these primers, a corresponding number of PCR-produced DNA segments are obtained from the individual in question according to Example 44. The nucleotide sequence of each of these DNA segments is determined using the method described in Example 46. A database of sequences created through this procedure uniquely identifies the individual from whom the sequence was obtained. The same panel of primers can then later be used to absolutely correlate the individual with tissue or other biological specimens. Example 48 Forensic Identification by Southern Blot The procedure of Example 47 is repeated to obtain at least 10 amplified sequences from individuals and specimens. Preferably, the panel contains at least 50 amplified sequences. More preferably, the panel contains 100 amplified sequences. In some embodiments, the panel contains 200 amplified sequences. Then, this P
The DNA produced by the CR is digested, preferably with one or a combination of four base-specific restriction enzymes. Such enzymes are commercially available and known to those skilled in the art. After digestion, the resulting gene fragments are size-separated in duplicate wells on an agarose gel and transferred to nitrocellulose using Southern blotting techniques well known to those skilled in the art. For an overview of Southern blotting, see Dav
is et al. (Basic Methods in Molecular Biolo)
gy, 1986, Elsevier Press 62-65). The disclosure of that reference is incorporated herein by reference.

The sequence of 5 ′ EST (or cDNA or genomic DNA obtained therefrom) or a fragment thereof of at least 10 bases may be radioactively analyzed using methods known in the art, such as nick translation, end labeling, etc. Alternatively, chromogenic labeling is performed and a method known in the art (Davis et al., Supra).
To hybridize to the Southern blot. Preferably, the probe is
It comprises at least 12, 15, 17 contiguous nucleotides from the 5 'EST (or cDNA or genomic DNA obtained therefrom). More preferably,
The probe was 5 'EST (or cDNA or genomic DNA obtained therefrom).
)) From at least 20-30 contiguous nucleotides. In some embodiments, the probe comprises more than 30 contiguous nucleotides from the 5 'EST (or cDNA or genomic DNA obtained therefrom).

Preferably, at least 5 to 10 of these labeled probes are used,
More preferably, at least about 20 or 30 are used to provide a unique pattern. The resulting band emerging from hybridization of a large sample of 5 'EST (or cDNA or genomic DNA obtained therefrom) is a unique identifier. Since restriction enzyme cleavage is different for all individuals, the pattern of bands on the Southern blot is also unique. 5 'EST (or c obtained therefrom)
An increase in the number of DNA or genomic DNA) provides a statistically higher level of confidence in the identification. This is because the number of band sets used for identification increases. Example 49 Dot Blot Identification Method Another technique for identifying individuals using the 5 'EST sequences disclosed herein utilizes dot hybridization techniques.

Genomic DNA is isolated from the nucleus of the subject to be identified. An oligonucleotide probe of about 30 bp in length corresponding to at least 10, preferably 50 DNA sequences from the 5 'EST or cDNA or the genome obtained therefrom is synthesized. The probe is used to hybridize to genomic DNA via conditions known to those skilled in the art. Use polynucleotide kinase (Pharmacia), end-labeled oligonucleotide with P ^32. Dot blots are made by spotting genomic DNA onto nitrocellulose or the like using a vacuum dot blot manifold (BioRad, Richmond California). Using techniques known in the art (Davis et al., Supra), bake or UV-filter nitrocellulose filters containing genomic sequences.
, Prehybridize, and hybridize with the labeled probe. ^{The 32} P-labeled DNA fragments are sequentially hybridized under stringent conditions with high success rates to detect minimal differences between the 30 bp sequence and the DNA. Tetramethylammonium chloride is useful for identifying clones containing a small number of nucleotide mismatches (Wood et al., Proc. N.
atl. Acad. Sci. USA 82 (6): 1585-1588,
1985). The disclosure of that reference is incorporated herein by reference. A unique pattern of dots distinguishes one individual from another.

The following alternative fingerprinting techniques use 5 ′ EST sequences (or cDNA or genomic DNA obtained therefrom) or oligonucleotides containing at least 10 contiguous bases from these sequences as probes. be able to. Preferably, the probe comprises at least 12, 15, 17 contiguous nucleotides from the 5 'EST sequence (or cDNA or genomic DNA obtained therefrom). More preferably, the probe is a 5 'EST sequence (
Or cDNA or genomic DNA obtained therefrom).
Contains ~ 30 contiguous nucleotides. In some embodiments, the probe comprises 5
It contains at least more than 30 contiguous nucleotides from the 'EST (or cDNA or genomic DNA obtained therefrom).

Preferably, multiple probes with sequences from different genes are used in alternative fingerprinting techniques. In Example 50 below, the probe was 5'ES
An exemplary alternative fingerprint procedure derived from T is provided. Example 50 Alternative "Fingerprint" Identification Methods Using commercially available oligonucleotide services such as Genset, Paris, France, a large number, e.g., 50, 100, or 200 5'ES
Prepare a 20-mer oligonucleotide from T. Using techniques well known to those skilled in the art,
A DNA sample is treated with a cell sample from a test subject. EcoR
Digest with restriction enzymes such as I and XbaI. After digestion, the samples are applied to electrophoresis wells. Although procedures known in the art may be modified to accommodate polyacrylamide electrophoresis, in this example, a sample containing 5 μg of DNA is loaded into wells and separated on a 0.8% agarose gel. . The gel is transferred to nitrocellulose using standard Southern blotting techniques.

Pool 10 ng of each oligonucleotide and end label with ³² P. Nitrocellulose is pre-hybridized with a blocking solution and hybridized with a labeled probe. After hybridization and washing, the nitrocellulose filters are exposed to X-Omat AR X-ray film. The resulting hybridization pattern is unique for each individual.

In this example, it is further contemplated that the number of probe sequences used for further accuracy or clarity can be varied.

Using the protein encoded by the extended cDNA to identify the tissue type or cell type from which the sample was derived as described in Example 51,
And 43 can also be prepared. Example 51 Identification of Tissue Type or Cell Type by Labeled Tissue-Specific Antibodies Examples 30 and 43 Bound to a Directly or Indirectly Detectable Marker
Specific tissue identification is achieved by visualizing the tissue-specific antigen with an antibody preparation according to Id. The selected labeled antibody species binds to their specific antigen-binding partners in a soluble protein extract from a tissue section, cell suspension, or tissue sample, forming a pattern for quantitative or semi-quantitative interpretation. provide.

The antisera for these procedures must have potency that exceeds that of the natural preparation. Because antibodies are isolated by isolating the gamma globulin fraction.
For example, it is concentrated to a mg / ml level by ion exchange chromatography or ammonium sulfate fractionation. Also, to provide the most specific antiserum, the gamma globulin fraction must be freed of, e.g., undesired antibodies against common proteins, e.g., by insoluble immunosorption, before labeling the antibody with a marker. No. Either monoclonal or heterologous antisera is suitable for either procedure. A. Immunohistochemistry Technique Purified high titer antibodies prepared as described above can be used, for example, in Fudenber.
g, Basic and Clinical Immunology, Chapter 26,
Third Edition, Lange, Los Altos, California, 198
0, or Rose et al. Methods in Immunodiagnosis
Chapter 12, Second Edition, John Wiley and Sons, New York (1
980) (the disclosures of which are incorporated herein by reference).

Either the fluorescent marker fluorescein or rhodamine is preferred, but the antibody can be labeled with an enzyme such as horseradish peroxidase that supports the color reaction with the substrate. As described below, a marker can also be added to the tissue binding antibody in the second step. Alternatively, the specific antiserum antibody can be labeled with ferritin or other high-density electronic particles and examined by electron microscopy for localization of the ferritin bound to the antigen-antibody complex. In another approach, the antibody is detected, for example, by labeling the antibody with ¹²⁵ I and covering the antibody treatment preparation with a photographic emulsion.

Preparations for performing the procedure can include monoclonal or polyclonal antibodies against a single protein or peptide identified as specific for a tissue type, eg, brain tissue, or Several tissue-specific antigens with different antigenicities can be used in the panel, independently if necessary or in mixtures.

Prepare tissue sections and cell suspensions for immunohistochemical testing by common histological techniques. Multiple cryostat sections of unknown tissue and controls (approximately 4
μm, unfixed) and each slide was covered with a different dilution of the antibody preparation. Sections of known and unknown tissues must also be treated with preparations to provide positive controls, negative controls, eg, pre-immune serum, and controls for non-specific staining, eg, buffer.

The treated sections are incubated in a wet chamber for 30 minutes at room temperature, rinsed, and then washed with buffer for 30-40 minutes. Remove the excess liquid and spread the marker.

If the tissue-specific antibody was not labeled in the first incubation, the antibody at this point would be a secondary antibody-antibody reaction, such as fluorescein or enzyme-linked antibodies to the immunoglobulin class of antiserum producing species, For example, it can be labeled by adding a fluorescein-labeled antibody against mouse IgG. Such labeled sera are commercially available.

[0287] Antigens found in tissues by the above procedure can be quantified by measuring the intensity of color or fluorescence on tissue sections and by calibrating the signal using appropriate standards. B. Identification of tissue-specific soluble proteins Visualization of tissue-specific proteins and identification of unknown tissue from the procedure
Performed using labeled antibody reagents and detection strategies described for immunohistochemistry, but the sample is prepared according to electrophoretic techniques and the proteins extracted from the tissue are distributed in an order based on molecular weight for detection.

The tissue sample is homogenized using a Virtis apparatus. The cell suspension is disrupted by Dounce homogenization or osmotic lysis, in each case using surfactants necessary to disrupt cell membranes and conventional in the art. Insoluble cellular components such as nuclei, microsomes, and membrane fragments are removed by ultracentrifugation and, if necessary, the fraction containing the soluble protein is concentrated and stored for analysis.

See, eg, Davis et al., Basic Methods in Molecular.
ar Biology, verses 19-2, edited by Leder, Elsevier, Ne
w. York, 1986, the disclosure of which is incorporated herein by reference, by a conventional SDS polyacrylamide gel electrophoresis, wherein the amount of polysaccharide is such that the entire molecular weight range of the protein to be detected in the sample can be divided. Acrylamide gel is used for one set of gels to separate samples of soluble proteins into individual protein species. In order to estimate the molecular weight of the constituent proteins, size markers are run in parallel. The sample size for the analysis is in a convenient volume of 5-55 μl and contains about 1-100 μg of protein. Aliquots of each of the separated proteins were transferred by blotting on nitrocellulose filter paper, maintaining the pattern of degradation during this process. Prepare multiple copies. For a procedure known as Western blot analysis, see Davi
s, L .; Et al., Supra, section 19-3. One set of nitrocellulose blots is stained with Coomassie blue dye to visualize all sets of proteins for comparison to the proteins bound to the antibody. The remaining nitrocellulose filters are then incubated with a solution of one or more specific antisera to tissue-specific proteins prepared as described in Examples 30 and 43. In this procedure, the appropriate positive and negative samples and reagent controls are run as in Procedure A above.

In both procedures A and B, a detectable label can be attached to the primary tissue antigen-primary antibody complex according to various strategies and permutations thereof. In a simple approach, the primary specific antibody can be labeled. Alternatively, the unlabeled conjugate can be conjugated to a labeled secondary anti-IgG antibody. In another approach, either a primary or secondary antibody is conjugated to a biotin molecule. The biotin molecule binds to the avidin binding marker in a subsequent step. According to another strategy, the antibody label or radioactive protein A can be any Ig
It has the property of binding to G and in the final step binds to either the primary or secondary antibody.

The visualization of tissue-specific antigens that bind to one or more tissue-specific antibodies prepared from a gene sequence identified from an extended cDNA sequence at levels above those found in control tissues can be of unknown origin. Tissue can be identified, eg, a forensic sample, or a differentiated tumor tissue that has metastasized to a foreign body site.

In addition to their application and identification in forensic medicine, 5 ′ ESTs (or cDNA or genomic DNA obtained therefrom) can be mapped to chromosomal locations. Example 52 below describes radioactive hybrid (RH) mapping of human chromosomal regions using 5 ′ ESTs. Example 53 below describes a representative procedure for mapping the position of the 5 'EST on the human chromosome. In Example 54 below, 5 ′ on metaphase chromosomes by fluorescence in situ hybridization (FISH)
The EST mapping will be described. One skilled in the art will appreciate that the methods of Examples 52-54 can be used to map cDNA or genomic DNA from 5 'ESTs for their chromosomal location. 2. Use of 5 'ESTs or sequences derived therefrom or portions thereof in chromosome mapping Example 52 Radioactive Hybrid Mapping of 5' ESTs to the Human Genome Radioactive hybrid (RH) mapping is a somatic genetic approach, It can be used for resolution mapping. In this approach, a cell line containing one or more human chromosomes is irradiated with lethal radiation and each chromosome is cut into fragments having a size that depends on the radiation dose. These fragments are recovered in cultured rodent cells to obtain subclones containing different parts of the human genome. The technology is described in Benham et al., Genom.
ics 4: 509-517, 1989; and Cox et al., Science.
250: 245-250, 1990, the contents of all of which are incorporated herein by reference. Subclones have random and independent properties, allowing efficient mapping of any human genomic marker. Human DNA isolated from 80-100 cell lines provides a mapping reagent for sequencing the 5'EST. With this approach, it is possible to use the frequency of cuts between markers to measure distances and build maps with as fine a resolution as is done using conventional ESTs (Schuler). S
science 274: 540-546, 1996, incorporated herein).

Using RH mapping, growth hormone (GH) and thymidine kinase (
TK) gene across human chromosomes 17q22-q25.3 (Foster et al.,
Genomics 33: 185-192, 1996), a region surrounding the Gorlin syndrome gene (Obermayr et al., Eur. J. Hum. Genet.
4: 242-245, 1996), 60 loci spanning the entire short arm of chromosome 12 (Raeymaekers et al., Genomics 29: 170-178).
, 1995) Human chromosome 22 containing the locus of type 2 neurofibromatosis as well as 13 loci on the long arm of chromosome 5 (Warrington et al., Gen.
omics 11: 701-708, 1991). Example 53 Mapping of 5 'ESTs to human chromosomes using PCR technology Using a PCR-based methodology, 5' ESTs (or cDs obtained therefrom)
NA or genomic DNA) can be assigned to the human chromosome. In such an approach, oligonucleotide primer pairs are designed from the 5 'EST (or cDNA or genomic DNA obtained therefrom) to minimize the potential for amplification via introns. Preferably, the length of the oligonucleotide primer is 18-23 bp and is designed for PCR amplification. Creating PCR primers from known sequences is well known to those skilled in the art. For an overview of PCR technology, see Erlich, PCR Technology, Princ.
iples and Applications for DNA Ampli
fiction, Freeman and Co. , New York,
See 1992. The disclosure of that reference is incorporated herein.

[0294] Primers are used in the polymerase chain reaction (PCR) to amplify a template from total human genomic DNA. The PCR conditions are as follows: 60 ng
Of genomic DNA as a template for PCR, using 80 ng of each oligonucleotide primer, 0.6 units of Taq polymerase, and 1 μC
u with ³² P-labeled deoxycytidine triphosphate. PCR is performed in a microplate thermocycler (Techne) under the following conditions: 94 ° C, 1.4.
30 min at 55 ° C, 2 min; and 72 ° C for 2 min;
Final extension for 0 minutes. Amplification products were analyzed on a 6% polyacrylamide sequencing gel and visualized by autoradiography. If the length of the resulting PCR product is the same as the distance between the ends of the primer sequence in the extended cDNA induced by the primer, two panels of human-rodent somatic cell hybrids,
PCR with DNA templates from BIOS PCRable DNA (BIOS Corporation) and NIGMS human-rodent somatic cell hybrid mapping panel number 1 (NIGMS, Gamden, NJ)
Repeat the reaction.

Using PCR, a series of somatic hybrid cell lines containing a defined set of human chromosomes was cloned into a predetermined 5 ′ EST (or cDNA or genomic DNA obtained therefrom).
Screening for the presence of NA). DNA is isolated from somatic cell hybrids and used as a starting template for a PCR reaction using a primer pair from 5 ′ EST (or cDNA or genomic DNA obtained therefrom). Only those somatic hybrids that have a chromosome containing the human gene corresponding to the 5 'EST (or cDNA or genomic DNA obtained therefrom) will yield an amplified fragment. The 5 'EST (or cDNA or genomic DNA obtained therefrom) is assigned to chromosomes by analyzing the separation pattern of the PCR product from the somatic cell hybrid DNA template.
The single human genome present in all cell hybrids giving rise to the amplified fragment is 5 'EST (or cDNA or genomic DNA obtained therefrom).
). For an overview of techniques and analysis resulting from somatic gene mapping experiments, see Ledbetter et al., Genomics 6:47.
See 5-481, 1990. The disclosure of that reference is incorporated herein. Example 54 Mapping of Extended 5 ' EST to Chromosome Using Fluorescent In Situ Hybridization Fluorescent in situ hybridization allows 5' EST (or cDNA or genomic DNA obtained therefrom) to a specific location on a given chromosome.
) Can be mapped. Chromosomes to be used for fluorescence in situ hybridization techniques can be obtained from a variety of sources including cell cultures, tissues, or whole blood.

In a preferred embodiment, the chromosomal location of the 5 ′ EST (or cDNA or genomic DNA obtained therefrom) is determined by the method of Cherif et al. (Proc. Natl.
. Acad. Sci. U. S. A. , 87: 6639-6643, 1
990), the disclosure of which is incorporated herein. Metaphase chromosomes are prepared from phytohemagglutinin (PHA) -stimulated blood cell donors. PHA-stimulated lymphocytes from healthy males were isolated from RPMI-164.
Incubate in medium 0 for 72 hours. For synchrony, methotrexate (10 μM)
For 5 hours, followed by 5-bromodeoxyuridine (5-BrdU, 0.1 mM
) Is added for 6 hours. Before harvesting the cells, colcemide (1 μg / ml) is added for the last 15 minutes. Cells are harvested, washed with RPMI, incubated with a hypotonic solution of KCl (75 mM) at 37 ° C. for 15 minutes, and mixed with methanol: acetic acid (3: 1).
) Is replaced three times and fixed. The cell suspension is dropped on a glass slide and air dried. 5 'EST (or cDNA or genomic DNA obtained therefrom)
From the manufacturer (Bethesda Research Laboratories)
, Bethesda, MD) and labeled with biotin-16dUTP by nick translation, using a Sephadex G-50 column (P
and purified using S. harcia, Upsala, Sweden. Immediately prior to hybridization, pellet the DNA with hybridization buffer (50% formamide, 2 × SSC, 10% dextran sulfate, 1%).
mg / ml sonicated salmon sperm DNA, pH 7) and denature the probe at 70 ° C for 5-10 minutes.

Slides kept at −20 ° C. were treated with RNase A (100 μg / ml) for 3 hours.
Treat at 7 ° C. for 1 hour, rinse 3 × with 2 × SSC and dehydrate with ethanol series. The chromosome preparation is denatured in 70% formamide, 2 × SSC at 70 ° C. for 2 minutes, and then dehydrated at 4 ° C. Slide the slide to Proteinase K (20 mM Tris-HC)
(10 μg / 100 ml in ₂ mM CaCl ₂ ) at 37 ° C. for 8 minutes and dehydrate. The hybridization mixture containing the probe is placed on a slide, covered with a coverslip, sealed with rubber glue and incubated overnight at 37 ° C. in a wet chamber. After hybridization and post-hybridization washes, the biotinylated probe is detected with avidin-FITC and amplified with a further layer of biotinylated goat anti-avidin and avidin-FITC. Obtain fluorescent R bands as described above for chromosome localization (Cherif et al., Supra).
. Observe the slides under a LEICA fluorescence microscope (DMRXA). The chromosome is counterstained with propidium iodide and the probe's fluorescent signal appears as two symmetric yellow-green spots on both chromatids (red) of the fluorescent R-band chromosome. Thus, a particular 5 'EST (or cDNA or genomic DNA obtained therefrom) may be located in the R-band of a particular cellular gene on a given chromosome.

Once the 5 ′ ESTs (or cDNA or genomic DNA obtained therefrom) have been assigned to particular chromosomes, using the techniques described in Examples 52-54 above, One can build a high resolution map of the chromosomes in which they are located or identify chromosomes in a sample. Example 55 Use of 5 'EST to Build or Expand a Chromosome Map As described above, chromosome mapping involves assigning certain specific sequences to specific chromosomes. Once a unique sequence has been mapped to a given chromosome, the order of the unique sequence relative to other unique sequences located on the same chromosome is determined. One approach to chromosome mapping is to use the extended cDNA (
Or genomic DNA obtained therefrom) utilizing a series of yeast artificial chromosomes (YACs) having thousands of long inserts from the chromosome of the organism from which they are obtained. For this approach, see Nagaraja et al., Genome Research 7:21.
0-222, 1997. The disclosure of that reference is incorporated herein. Briefly, in this approach, each chromosome is cut into overlapping pieces and inserted into a YAC vector. The YAC inserts are screened using PCR or other methods and they are
'EST (or cDNA or genomic DNA obtained therefrom). Once an insert containing the 5 'EST (or cDNA or genomic DNA obtained therefrom) is found, the insert is analyzed using PCR or other methods, and the insert is located on the chromosome or 5'. It is determined whether the EST (or the cDNA or genomic DNA obtained therefrom) contains other sequences known to be present in the region from which it was derived. This process is repeated for each insert in the YAC library to determine the relative position of each extended cDNA (or genomic DNA obtained therefrom) with respect to another known chromosomal marker and other known chromosomal markers. Can be determined. In this way, a high resolution map of the distribution of many unique markers along each chromosome of an organism can be obtained.

As described in Example 56 below, extended cDNA (or genomic DNA obtained therefrom) can also be used to identify genes associated with a particular phenotype, such as a genetic disease or drug response. it can. 3. Use of 5 'ESTs or Sequences Derived Therefrom or Fragments thereof in Gene Identification Example 56 Identification of Genes Associated with Hereditary Diseases or Drug Responses
NA) illustrates a useful approach for the association of a particular phenotypic trait. In this example, a specific 5 ′ EST (or cDNA or genomic DNA obtained therefrom) is used as a test probe, and the specific phenotypic trait between 5 ′ EST (or cDNA or genomic DNA obtained therefrom) and Check for relevance.

Using the techniques described in Examples 52 and 53 or other techniques known in the art, 5 ′ ESTs (or cDNA or genomic DNA obtained therefrom)
) Is mapped to a specific location on the human chromosome. A search o
fMendelian Inheritance in Man (McKus
Ick in Mendelian Inheritance in Man)
(Johns Hopkins University Welch Medi
(available online via calLibrary) is a region of the human chromosome containing the 5 'EST (or cDNA or genomic DNA obtained therefrom) that contains several known genes and some unidentified genes. This indicates a highly gene-rich region containing a disease or phenotype. Thus, genes corresponding to 5 ′ ESTs (or cDNA or genomic DNA obtained therefrom) are direct candidates for each of these genetic disorders.

[0301] Cells from patients with these diseases or phenotypes are isolated and expanded in culture. Using PCR primers from 5 'EST (or cDNA or genomic DNA obtained therefrom), genomic DNA, mRNA or cD
Screen for NA. Patient's 5 'EST (or cDN obtained therefrom)
If A (or genomic DNA) does not amplify, the sequence can be considered relevant for the particular disease by further analysis. Alternatively, PCR analysis produces fragments of various lengths when the sample is derived from an individual with a phenotype associated with the disease, rather than when the sample is derived from a healthy individual, which results in a 5 ′ fragment. This shows that genes containing EST may be responsible for genetic diseases. VI. 5 'EST for construction of the vector (or cDN obtained therefrom)
Use of the 5 ′ EST of the present invention (or cDNA or genomic DNA obtained therefrom)
) Can also be used to construct secretion vectors that can direct the secretion of the protein encoded by the gene in the vector. Such secretion vectors facilitate the purification or enrichment of the protein encoded by the gene inserted into the vector by reducing the number of background proteins that must purify or enrich the desired protein. be able to. An exemplary secretion vector is described in Example 57 below. 1. Construction secretion vector Construction Example 57 Secretion vectors secretion vector contains a host cell of interest, tissue, or a promoter capable of directing the expression of genes in an organism. Such promoters include:
Examples include the Rous sarcoma virus promoter, the SV40 promoter, the human cytomegalovirus promoter, and other promoters well known to those skilled in the art.

[0302] The signal sequence from the 5 'EST (or cDNA or genomic DNA obtained therefrom) is operably linked to the promoter such that the mRNA transcribed from the promoter directs the translation of the signal peptide. The host cell, tissue, or organism can be any cell, tissue, or organism that recognizes the signal peptide encoded by the signal sequence of 5 ′ EST (or cDNA or genomic DNA obtained therefrom). Suitable hosts include mammalian cells, tissues or organisms, avian cells, tissues or organisms, insect cells, tissues or organisms, or yeast.

Further, secretion vectors contain a cloning site for inserting a gene encoding the protein to be secreted. The cloning site is used for cloning of the inserted gene that matches the signal sequence so that the fusion protein in which the signal peptide is fused to the protein encoded by the inserted gene is expressed from mRNA transcribed from the promoter. To facilitate. The signal peptide directs extracellular secretion of the fusion protein.

The secretion vector may be DNA or RNA and may be integrated into the host chromosome, may be stably maintained as an extrachromosomal replicon of the host, or may be an artificial chromosome. Or may be transiently present in the host. Many nucleic acid backbones suitable for use in secretion vectors are known to those of skill in the art, and include retroviral vectors, SV40 vectors, bovine papillomavirus vectors, yeast integrated plasmids, yeast episomal plasmids, yeast artificial chromosomes, human artificial chromosomes, P elements Vectors, baculovirus vectors, or bacterial plasmids that can be transiently introduced into a host.

The secretion vector may also include a polyA signal so that polyA is located downstream of the gene inserted into the secretion vector.

After inserting the gene encoding the protein desired to be secreted into the secretion vector, the secretion vector can be prepared using calcium phosphate precipitation, DEAE-dextran, electroporation, liposome-mediated transfection, virus particles, Alternatively, it is introduced into the host cell, tissue or organism as bare DNA. The protein encoded by the inserted gene is then purified from the supernatant using conventional techniques such as ammonium sulfate precipitation, immunoprecipitation, immunochromatography, size exclusion chromatography, ion exchange chromatography, and HPLC. Or concentrate. Alternatively, the secreted protein is present in the supernatant or host growth medium in a sufficiently concentrated or pure state so that it can be used for the intended purpose without further enrichment. May be.

[0307] The signal sequence may also be inserted into a vector designed for gene therapy. In such a vector, the signal sequence is
The RNA is operably linked to the promoter such that the RNA encodes a signal peptide. The cloning site is located downstream of the signal peptide so that the gene encoding the protein desired to be secreted is easily inserted into the vector and fused to the signal peptide. The vector is introduced into a suitable host cell. The protein expressed from the promoter is secreted extracellularly, thereby exerting a therapeutic effect.

The 5 'EST may be used to clone sequences located upstream of the 5' EST that can regulate gene expression, including promoter sequences, enhancer sequences, and Other upstream sequences that can affect the level of transcription or translation are included. Once identified and cloned, these upstream regulatory sequences can be used in expression vectors designed to direct the expression of the inserted gene in the desired local, temporal, developmental or quantitative manner. . Example 58 describes a method for cloning an extended cDNA or a sequence upstream of the 5 'EST. 2. With extension cDNA or 5 'EST of using chromosome walking techniques for cloning identification Example 58 upstream sequences from genomic DNA upstream sequences having promoter activity or modulating activity, sequences derived from the extended cDNA or 5'EST Can be used to isolate the promoter of the corresponding gene. Clonte
In one chromosome walking technique utilizing the Genome Walker ^™ kit sold by Ch., each of the five complete genomic DNA samples is digested with a different restriction enzyme that has a six base recognition site and generates blunt ends. I do. After digestion,
Oligonucleotide adapters are ligated to both ends of the obtained genomic DNA fragment.

For each of the five genomic DNA libraries, use the outer adapter primers and outer gene-specific primers provided with the kit and the first according to the manufacturer's instructions (incorporated herein by reference). Is performed. The gene-specific primer should be selected to be specific for the extended cDNA or 5 'EST of interest and has a melting temperature, length, and extended cDNA compatible with the use of the primer in a PCR reaction. Or it should have a position within the primer at the 5 'EST. Each first PCR reaction contained 5 ng of genomic DNA, 5 μl of 10 × Tth reaction buffer, 0.2 mM of various dNTPs, outer adapter primers and outer gene-specific primers in a total volume of 50 μl. Contains ₂ μM, 1.1 mM Mg (OAc) ₂ , and 1 μl Tth polymerase 50 × mix. The reaction cycle of the first PCR reaction is as follows: 1 minute -94 ° C / 2 seconds -94 ° C, 3 minutes-
72 ° C (7 cycles) / 2-94 ° C, 3 minutes-67 ° C (32 cycles) / 5
-67 ° C for minutes.

The product of the first PCR reaction is diluted and a template for the second PCR reaction is used according to the manufacturer's instructions, using a pair of nested primers located inside the amplicon resulting from the first PCR reaction. Use as For example, 5 of the first PCR reaction mixture
μl of the reaction product may be diluted 180-fold. The reaction is performed in a volume of 50 μl having the same composition as that of the first PCR reaction except that nested primers are used. The first nested primer was specific for the adapter and was
Included with me Walker ^™ kit. The second nested primer is
The specific extended cDNA or 5'EST to which the primer is to be cloned
And have a melting temperature, length, and location in the extended cDNA or 5 'EST that is compatible with use in PCR reactions. 2nd P
The reaction parameters of the CR reaction are as follows: 1 minute -94 ° C / 2 seconds -94
C, 3 minutes -72C (6 cycles) / 2 seconds -94C, 3 minutes -67C (25 cycles) / 5 minutes -67C. The product of the second PCR reaction is purified, cloned, and sequenced using standard techniques.

Alternatively, two or more human genomic DNA libraries can be constructed by using two or more restriction enzymes. The digested genomic DNA is cloned into a vector that can be converted into single-stranded, circular, or linear DNA. Extension c
DNA or a biotinylated oligonucleotide containing at least 15 nucleotides from the 5 'EST sequence is hybridized to the single-stranded DNA. Hybrids of biotinylated oligonucleotides with single-stranded DNA containing extended cDNA or EST sequences are isolated as described in Example 29 above. Thereafter, the single-stranded DNA containing the extended cDNA or EST sequence is released from the beads, and a primer specific to the extended cDNA or 5'EST sequence or a primer corresponding to the sequence contained in the cloning vector is used. Convert to single-stranded DNA.
The resulting double-stranded DNA is transformed into bacteria. DNA containing the 5 'EST or extended cDNA sequence is identified by colony PCR or colony hybridization.

Once the upstream genomic sequence has been cloned and sequenced as described above, the expected promoter and transcription start site in the upstream sequence may be extended cDNA or 5 ′ ES
Sequences upstream of T can be identified by comparing to a database containing known transcription initiation sites, transcription factor binding sites, or promoter sequences.

In addition, promoters in upstream sequences can be identified using a promoter reporter vector, as described in the Examples. Example 59 Identification of the Promoter in the Cloned Upstream Sequence The genomic sequence upstream of the extended cDNA or 5 'EST was
asic, pSEAP-Enhancer, pβgal-Basic, pβga
pEGFP commercially available from l-Enhancer or Clontech
Clone into a suitable promoter reporter vector, such as a -1 promoter reporter vector. Briefly, each of these promoter reporter vectors contains secreted alkaline phosphatase, β-galactosidase,
Or a multiple cloning site located upstream of a reporter gene encoding a protein that can be easily assayed, such as a green fluorescent protein. The sequence upstream of the extended cDNA or 5'EST is inserted in both directions into the cloning site upstream of the reporter gene and introduced into a suitable host cell. The level of the reporter gene is assayed and compared to the levels obtained from a vector without an insert at the cloning site. An increase in the expression level of the vector containing the insert, as compared to the control vector, indicates that the promoter is present in the insert. If necessary, the upstream sequence can be cloned into a vector containing an enhancer to increase the level of transcription from a weak promoter sequence. The significant level of expression observed for the vector without insert is:
Indicates that the promoter sequence is present in the inserted upstream sequence.

[0314] Suitable host cells for the promoter reporter vector can be selected based on the above determination of the expression pattern of the extended cDNA or EST. For example, if expression pattern analysis indicates that mRNA corresponding to a particular extended cDNA or 5'EST is expressed in fibroblasts, a promoter reporter vector can be introduced into a human fibroblast cell line. .

[0315] The promoter sequence in the upstream genomic DNA can be further defined by constructing a nested deletion in the upstream DNA using conventional techniques, such as exonuclease III digestion. The resulting deletion fragment can be inserted into a promoter reporter vector to determine whether the deletion reduces or eliminates promoter activity. In this way, the boundaries of the promoter can be defined. If desired, use site-directed mutagenesis or linker scanning alone or in combination to remove potential transcription factor binding sites within the promoter to identify potential individual regulatory sites within the promoter May be. By inserting mutations at the cloning site of the promoter reporter vector, the effect of these mutations on the level of transcription can be determined. Example 60 Cloning and Identification of Promoter Using the method described in Example 58 above using 5'EST, sequences upstream of several genes were obtained. Using primer pair GGG AAG ATG GAG ATA GTA TTG CCTG (SEQ ID NO: 29) and CTG CCA TGT ACA TGA TAG AGA GAT TC (SEQ ID NO: 30), P13H2 (named by the inventors) (SEQ ID NO: 31) Was obtained.

Primer Pair GTA CCA GGGG ACT GTG ACC ATT
GC (SEQ ID NO: 32) and CTG TGA CCA TTG CTC CCA
Using AGA GAG (SEQ ID NO: 33), a promoter with P15B4 (named by the inventors) (SEQ ID NO: 34) was obtained.

Using primer pairs CTG GGA TGG AAG GCA CGG TA (SEQ ID NO: 35) and GAG ACC ACA CAG CTA GAC AA (SEQ ID NO: 36), P29B6 (named by the inventors) (SEQ ID NO: 37)
) Was obtained.

FIG. 4 shows a schematic diagram of the isolated promoters and how to assemble them with the corresponding 5 ′ tags. Computer program MatInspector
Using Release 2.0 (August 1996), upstream sequences were screened based on the presence of motifs similar to transcription factor binding sites or known transcription start sites.

[0319] Table VII describes the transcription factor binding sites present on each of these promoters. The column marked "Matrix" is the MatInsp used
Indicates the name of the vector matrix. The column labeled "position" indicates the 5 'position of the promoter site. Sequence numbering starts from the transcription site determined by matching the genomic sequence to the 5 'EST sequence. The column labeled "orientation" indicates the DNA strand at which the site was found, and the + strand indicates the genomic sequence and 5
'Coding strand determined by matching with EST sequence. The column labeled "Score" indicates the MatInspector score for this site. The column labeled "Length" indicates the site length (in nucleotides). The column labeled "Sequence" indicates the sequence of the found site.

Bacterial clones containing plasmids containing the above promoter sequences are currently stored in our laboratory with the internal identification numbers indicated above. The insert can be removed from the precipitate by growing an aliquot of the appropriate bacterial clone in the appropriate medium. Plasmid DNA can then be isolated using plasmid isolation procedures well known to those skilled in the art, such as alkaline lysis miniprep or bulk alkaline lysis plasmid isolation methods. If desired, the plasmid DNA may be further concentrated by centrifugation on a cesium chloride gradient, size exclusion chromatography, or anion exchange chromatography. The resulting plasmid DNA may then be manipulated using these procedures using standard cloning techniques well known to those skilled in the art. Or 5'EST
PCR can be performed using primers designed at both ends of the insert. The PCR product corresponding to the 5 'EST can then be manipulated using standard cloning techniques well known to those skilled in the art.

Using an extended cDNA or promoter and other regulatory sequences located upstream of the 5 ′ EST, an expression vector is designed that can direct expression in a desired local, temporal, developmental or quantitative manner. be able to. Using the results of the expression analysis described in Example 26 above, a promoter can be selected that can be directed in a desired local, temporal, developmental or quantitative pattern. For example, if a promoter that results in high levels of expression in muscle is desired, an extended cDNA derived from mRNA that is expressed at high levels in muscle or a promoter sequence upstream of 5 ′ EST can be obtained by the method described in Example 26. As determined, it can be used in expression vectors.

Preferably, the desired promoter is located near the multiple restriction site to facilitate cloning of the desired insert upstream of the promoter, so that the promoter can drive the expression of the inserted gene. I do. The promoter can be inserted into a conventional nucleic acid backbone designed for extrachromosomal replication, integration into the host chromosome, or transient expression. Suitable scaffolds for the expression vectors of the present invention include scaffolds of retroviruses, scaffolds from eukaryotic episomes such as SV40 or bovine papilloma virus, scaffolds from bacterial episomes, or artificial chromosomes.

Preferably, the expression vector also contains a polyA signal downstream of a multiple restriction site to direct polyadenylation of mRNA transcribed from the gene inserted into the expression vector.

After identifying the promoter sequence using the procedures of Examples 58-60, proteins that interact with the promoter can be identified as described in Example 61 below. EXAMPLE 61 Identification of a protein that interacts with a promoter sequence, upstream regulatory sequence, or mRNA. Sequences within the promoter region that are likely to bind to the factor can be identified. For example, a deletion can be made in a reporter plasmid containing a promoter sequence of interest operably linked to an assayable reporter gene. Reporter plasmids carrying various deletions in the promoter region are transfected into appropriate host cells and the effect of the deletion on expression levels is assayed. Using site-directed mutagenesis, linker linker scanning analysis, or other techniques well known to those skilled in the art, the transcription factor binding site can be further localized in regions where the deletion reduces expression levels. it can.

One-hybrid such as the system described in the instructions accompanying the Matchmaker One-Hybrid System kit (Cat. No. K1603-1), commercially available from Clontech, the disclosure of which is incorporated herein by reference. A one-hybrid system can be used to identify nucleic acids that encode proteins that interact with sequences in the promoter. Briefly, the Matchmaker One-Hybrid System is used as follows. The target sequence for which it is desired to identify a binding protein is cloned upstream of the selection reporter gene and integrated into the yeast genome. Preferably, multiple copies of the target sequence are inserted in tandem into the reporter plasmid. A library containing a fusion of the cDNA to be evaluated for its ability to bind to a promoter and the activation domain of a yeast transcription factor such as GAL4 is transformed into a yeast strain containing the integrated reporter gene sequence. Sow this yeast on a selective medium,
Cells expressing the selectable marker linked to the promoter sequence are selected. Colonies growing on the selection medium contain genes encoding proteins that bind to the target sequence. By determining the sequence, the insert in the gene encoding the fusion protein is further characterized. Further, the insert may be an expression vector or
It may be inserted into an in vitro transcription vector. Binding of the polypeptide encoded by the insert to the promoter DNA can be confirmed by techniques well known to those skilled in the art, such as gel shift analysis or DNAase protection analysis. VII. USE OF 5 'ESTs (or cDNA may properly genomic DNA derived therefrom) in gene therapy also, 5' ESTs in gene therapy including antisense and triple helix strategies as described in the following Examples 62 and 63 ( Or the cD obtained from it
NA or genomic DNA). In the antisense approach,
By hybridizing a nucleic acid sequence complementary to the mRNA to the mRNA in a cell, expression of the protein encoded by the mRNA is prevented. Antisense sequences can prevent the expression of a gene through various mechanisms. For example,
Antisense sequences can inhibit the ability of the ribosome to translate mRNA. Alternatively, antisense sequences can block the transport of mRNA from the nucleus to the cytoplasm, limiting the amount of mRNA available for translation. Another mechanism by which antisense sequences can suppress gene expression is by interfering with mRNA splicing. In yet another strategy, the antisense nucleic acid can be incorporated into a ribozyme that can specifically cleave the target mRNA. Example 62 Preparation and Use of Antisense Oligonucleotides The antisense nucleic acid molecule to be used in gene therapy can be either a DNA or RNA sequence, and the sequence of 5'EST (or cDNA or genomic DNA obtained therefrom). May be included. The antisense nucleic acid should have sufficient length and melting temperature to form an intracellular duplex that is sufficiently stable to inhibit expression of the mRNA in double stranded form. For methods for designing antisense nucleic acids suitable for gene therapy applications, see Green et al., Ann. Rev .. Biochem. 55: 569-597, 1986;
And Izant and Weintraub, Cell 36: 1007-101.
No. 5,1984. These references are incorporated herein by reference.

In some methods, the nucleotides encoding the protein are reversed by reversing the orientation of the coding region relative to the promoter to transcribe the opposite strand from the one normally transcribed in the cell. An antisense nucleic acid is obtained from the sequence. In vitro, such as systems that produce transcripts using T7 or SP6 polymerase
oAn antisense molecule can be transcribed using a transcription system. Another approach involves the transcription of an antisense nucleic acid in vivo by operably linking a DNA containing the antisense sequence to the promoter of an expression vector.

Alternatively, an oligonucleotide complementary to the strand normally transcribed in the cell is inserted in
It may be synthesized in vitro. Therefore, the antisense nucleic acid is
And can hybridize to mRNA to create a duplex. In some embodiments, the antisense sequence contains an altered sugar phosphate backbone to increase stability and decrease the sensitivity of the antisense sequence to RNase activity. For examples of modifications suitable for use in antisense methods, see Rossi
Et al., Pharmacol. Ther. 50 (2): 245-254,199
1 and is incorporated herein by reference.

Various types of antisense oligonucleotides complementary to the sequence of the 5 ′ EST (or cDNA or genomic DNA obtained therefrom) can be used. In one preferred embodiment, the international application PCT WO 94/23026
Use the stable and semi-stable antisense oligonucleotides described in Ref. Said references are incorporated herein by reference. In these molecules, the 3 'end or both 3' and 5 'ends are connected by intramolecular hydrogen bonds between complementary base pairs. These molecules are more resistant to exonuclease attack and have improved stability compared to conventional antisense oligonucleotides.

In another preferred embodiment, antisense oligonucleotides against herpes simplex virus types I and II described in International Application WO 95/04141 are used. Said references are incorporated herein by reference.

In yet another preferred embodiment, the covalently crosslinked antisense oligonucleotides described in International Application WO 96/31523 are used. Said references are incorporated herein by reference. These double- or single-stranded oligonucleotides each have a covalent cross-link between one or more oligonucleotides or within the oligonucleotide, wherein the bond is, respectively, the primary amino acid of one strand. Consisting of an amide bond between the group and a carboxyl group on the other chain, or between a primary amino group and a carboxyl group on the same chain, wherein the primary amino group is
Substituted directly at the 2 'position of the monosaccharide ring of a chain nucleotide, the carboxyl group is carried by an aliphatic spacer group that is substituted on the other chain or on a nucleotide or nucleotide analog of the same chain.

[0331] The antisense oligonucleotides and oligonucleotides described in International Application WO 92/18522 can be used. Said references are incorporated herein by reference. These molecules are stable as degradation and contain at least one transcriptional regulatory recognition sequence that binds to regulatory proteins and are therefore effective as decoys. These molecules may contain "hairpin" structures, "dumbbell" structures, "modified dumbbell" structures, "crosslinked" decoy structures, and "loop" structures.

In another preferred embodiment, a circular double stranded oligonucleotide as described in European Patent Application 0 572 287 A2 is used. Said references are incorporated herein by reference. These linked oligonucleotides "dumbbells" contain a binding site for the transcription factor and inhibit gene expression under the control of the transcription factor by sequestering the transcription factor.

The use of closed antisense oligonucleotides as disclosed in International Application WO 92/19732 is also contemplated. Said references are incorporated herein by reference. Because these molecules do not have free ends, they are more resistant to degradation by exonucleases than conventional oligonucleotides. These oligonucleotides are multifunctional and interact with multiple regions that are not adjacent to the target mRNA.

[0334] The appropriate level of antisense nucleic acid required to inhibit expression of a gene can be determined using in vitro expression analysis. Antisense molecules can be introduced into cells by diffusion, injection, infection, transfection, or h-region mediated transfer using procedures known in the art. For example, an antisense nucleic acid may be a naked oligonucleotide, an oligonucleotide encapsulated in a lipid, an oligonucleotide encapsulated in a viral protein, or an oligonucleotide operably linked to a promoter contained in an expression vector. Nucleic acids can be introduced into the body. The expression vector may be any of a variety of expression vectors known in the art, including a retroviral or viral vector, a vector capable of extrachromosomal replication, or an integration vector. The vector may be DNA or RNA.

The antisense molecule is introduced into the cell sample, preferably at many different concentrations between 1 × 10 ⁻¹⁰ M and 1 × 10 ⁻⁴ M. Once the minimum concentration that can properly regulate gene expression is identified, the optimized dose is interpreted as a dose appropriate for use in vivo. For example, the inhibitory concentration of 1 × 10 ^{−7 in} the case of culture is about 0.6 m
Interpreted as a dose of g / kg body weight. Oligonucleotide levels reaching 100 mg / kg body weight or more are possible if the toxicity of the oligonucleotide is tested in laboratory animals. It is further contemplated that cells may be removed from the vertebrate, treated with the antisense oligonucleotide, and reintroduced into the vertebrate.

It is further contemplated that the antisense oligonucleotide sequence may be incorporated into a ribozyme sequence so that the antisense can specifically bind to and cleave the target mRNA. For technical applications of ribozymes and antisense oligonucleotides, see Rossi et al., Supra.

In a preferred application of the invention, the polypeptide encoded by the gene is first identified so that the effectiveness of the antisense inhibition on translation can be monitored. Monitoring uses techniques including, but not limited to, RIA and ELISA, functional assays, or antibody-mediated tests such as radiolabelling.

The 5 ′ ESTs of the present invention (or cDNA or genomic DNA obtained therefrom)
) Can also be used for gene therapy based on intracellular triple helix formation. Triple helix oligonucleotides are used to inhibit transcription from the genome. They are,
It is particularly useful for studying changes in cell activity. This is because it relates to a particular gene. 5 ′ EST of the present invention (or cDNA obtained therefrom)
Or, more preferably, a portion of those sequences can be used to inhibit gene expression in an individual with a disease associated with expression of a particular gene. Similarly, the 5 'EST sequence (or cDNA obtained therefrom)
Or genomic DNA) can be used to study the effect of inhibiting the transcription of a particular gene in a cell. Previously, homopurine sequences were considered to be most useful in triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove of the homopurine: homopyrimidine sequence. Therefore, 5 '
Both types of sequences from the EST or from the gene corresponding to the 5 'EST are intended to be within the scope of the present invention. Example 63 Preparation and Use of Triple Helix Probes The sequence of the 5 ' EST (or cDNA or genomic DNA obtained therefrom) is scanned to obtain a 10-mer that can be used in a triple helix-based strategy to inhibit gene expression. Identify the 20-mer homopyrimidine or homopurine stretch. After identification of a candidate homopyrimidine or homopurine stretch, the efficiency of gene expression inhibition is assessed by introducing various amounts of the oligonucleotide containing the candidate sequence into tissue culture cells that normally express the target gene. Oligonucleotides can be prepared on an oligonucleotide synthesizer or GENSET
, Paris, France, etc. can be purchased from specialists in oligonucleotide synthesis.

Oligonucleotides can be introduced into cells using a variety of methods known to those of skill in the art, including calcium phosphate precipitation, DEAE-dextran, electroporation, liposome-mediated trans Examples include, but are not limited to, the efection method or the natural uptake method.

The treated cells are monitored for changes in cell function or reduced gene expression using techniques such as Northern blotting, RNase protection assays, or PCR-based strategies to target cells being treated with the oligonucleotide. Monitor gene transcript levels. The cell function to be monitored is predicted based on the homology of a target gene corresponding to the extended cDNA from which the oligonucleotide is derived to a known gene sequence associated with a specific function. In particular, if the extended cDNA is associated with a disease using the technique described in Example 56, the cell may be expressed based on the presence of abnormal physiological activity in the cell derived from an individual having the particular genetic disease. You can also predict the function.

The gene expression in tissue culture cells was then determined using the techniques described above and described in Example 62 at the dose calculated based on the in vitro results, as described in Example 62. Oligonucleotides that are effective to inhibit
It can be introduced in vo.

In some embodiments, the natural (β) anomer of the oligonucleotide unit can be replaced with an α anomer, making the oligonucleotide more resistant to nucleases. Further, an intercalating agent such as ethidium bromide (interca) is used.
A binding agent or the like can be attached to the 3 ′ end of the α oligonucleotide to stabilize the triple helix. For information on making oligonucleotides suitable for triple helix formation, see Griffin et al., Science.
245: 967-971, 1989, which is incorporated herein by reference. Example 64 for expression of the proteins encoded in the host organism, using the cDNA obtained as described above using the 5 'ESTs of the use the invention the resulting cDNA by have use the 5' ESTs,
The encoded protein can be expressed in the host organism to produce a beneficial effect. In such procedures, the encoded protein may be transiently expressed in the host organism or may be stably expressed in the host organism.
The encoded protein can have any of the activities described above. The encoded protein may be a protein that lacks the host organism, or may increase an existing level of the protein in the host organism.

[0343] A full-length extended cDNA encoding a signal peptide and mature protein, or an extended cDNA encoding only the mature protein, can be introduced into a host organism. The extended cDNA can be introduced into a host organism using various techniques known to those skilled in the art. For example, an extended cDNA can be injected as bare DNA into a host such that the encoded protein is expressed in the host organism, thereby producing a beneficial effect.

Alternatively, the extended cDNA can be cloned into an expression vector downstream of a promoter that is active in the host organism. The expression vector can be any expression vector designed for use in gene therapy, such as a viral or retroviral vector. The expression vector can be introduced directly into the host organism so that the encoded protein is expressed in the host organism, thereby producing a beneficial effect. Otherwise,
The expression vector can be introduced into cells in vitro. Thereafter, cells containing the expression vector are selected and introduced into the host organism, where they express the encoded protein and produce a beneficial effect. Signal peptide encoded by extended cDNA derived Example 65 protein from the use of the signal peptide 5'EST or SEQ ID NO: 38-184 are by Ri code 5'EST or from the resulting sequence to populate the cells Can be used as a carrier to transfer the peptide or protein of interest, the so-called cargo, into tissue culture cells (Lin et al., J. Biol. Chem. 270: 1422).
5-1258, 1995; Du et al. Peptide Res. , 5
1: 235-243, 1998; Rojas et al., Nature Biotec.
h. , 16: 370-375, 1998).

If a limited size (up to about 25 amino acids) of a cell-penetrating peptide is to be transported across the cell membrane, chemical synthesis can be used to either the C-terminal or the N-terminal of the cargo peptide of interest. The crab h region can be added. Alternatively, if a longer peptide or protein is to be introduced into a cell, the nucleic acid may be genetically engineered using techniques well known to those of skill in the art to extend the c region encoding the h region.
A DNA sequence can be attached to the 5 'or 3' end of the DNA sequence encoding the cargo polypeptide. The so engineered nucleic acid is then transfected into appropriate cells, and then translated, either in vivo or in vitro, using conventional techniques to produce the resulting cell permeable polypeptide. I do. The appropriate host cells are then simply incubated with the cell permeable polypeptide and then transported across the membrane.

The present method can be applied to study a variety of intracellular functions and cellular processes. For example, the method has been used to probe function-related domains of intracellular proteins and to investigate protein-protein interactions related to signaling pathways (Lin et al., Supra; Lin et al., J. Biol. Chem.
271: 5305-5308, 1996; Rojas et al. Biol.
Chem. , 271: 27456-27461, 1996; Liu et al., Pr.
oc. Natl. Acad. Sci. USA, 93: 11819-
11824, 1996; Rojas et al., Bioch. Biophys.
Res. Commun. 234: 675-680, 1997).

[0347] Such techniques can be used in cell medicine to transfer proteins that produce a therapeutic effect. For example, cells isolated from a patient can be treated with the transferred therapeutic protein and then re-introduced into the host organism.

Alternatively, the h region of the signal peptide of the invention can be used in combination with a nuclear localization signal to deliver nucleic acid to the nucleus of a cell. Such oligonucleotides may be antisense oligonucleotides or oligonucleotides designed to form triple helices, as described in Examples 62 and 63, to inhibit processing and / or maturation of target cell RNA. It may be a nucleotide.

As described above, the 5 ′ EST cDNA of the present invention or a part thereof can be used for various purposes. Using polynucleotides to make recombinant proteins suitable for analysis, characterization or therapeutic use; suitable proteins (either constitutively or at specific stages of tissue differentiation or development or disease states) As a marker for the tissue to be expressed; as a molecular weight marker on a Southern gel; as a chromosomal marker or tag (if labeled) to identify chromosomes or map the location of related genes; compare the patient's endogenous DNA To identify potential genetic abnormalities; as a probe to hybridize and thereby discover novel related DNA sequences; as a source of information to obtain PCR primers for gene fingerprinting; Select oligomers to attach to "gene chips" or other supports To produce (including examining expression patterns); to raise anti-protein antibodies using DNA immunization techniques; and to raise anti-DNA antibodies or elicit another immune response. It can be expressed as an antigen. If the polynucleotide encodes a protein that binds or potentially binds another protein (e.g., in a receptor-ligand interaction, etc.), the polynucleotide may be a polynucleotide encoding another protein for which binding occurs. To identify nucleotides or identify inhibitors of binding interactions, use an interaction trap assay (e.g., the assay described in Gyuris et al., Cell 75: 791-803, 1993, etc.), wherein the reference is disclosed. The contents are incorporated herein by reference).

The proteins or polypeptides provided by the present invention may be used in assays to determine biological activity involving a panel of multiple proteins for high-throughput screening; eliciting antibodies or other immune responses Reagents (including labeled reagents) in assays designed to quantitatively determine the level of the protein (or its receptor) in a biological fluid; the corresponding protein (either constitutive or As a marker for a tissue to be suitably expressed (either at a particular stage of tissue differentiation or development or at a disease state); and, of course, similarly used to isolate correlated receptors or ligands Can be. If a protein binds or potentially binds another protein, the protein can be used to identify other proteins where binding occurs or to identify inhibitors of binding interactions. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Some or all of these research utilities can be developed to commercial reagent levels or kit forms as research products.

The methods for achieving the uses listed above are well known to those skilled in the art. References disclosing such methods include Molecular Cloning.
A Laboratory Manual, 2nd edition, Cold Spring
Harbor Laboratory Press, Sambrook, F
Rich and Maniatis eds., 1989, and Methods i
n Enzymology; Guide to Molecular Clon
ing Technologies, Academic Press, Berg
er and Kimmel, 1987, but are not limited thereto.

The polynucleotides and proteins of the present invention can also be used as a nutritional source or supplement. Such uses include, but are not limited to, use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the protein or polynucleotide of the invention can be added to the food of the particular organism, or as a discrete solid or liquid preparation, in powder, pill, solution, suspension or capsule dosage form. Etc. can be administered. In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

Although the invention has been described in terms of certain preferred embodiments, other embodiments that are apparent to those skilled in the art from the disclosure herein are within the scope of the invention. Accordingly, the scope of the present invention is intended to be defined solely by reference to the appended claims. All documents cited herein are hereby incorporated by reference in their entirety.

[0355]

[Table 1]

[0356]

[Table 2]

[0357]

[Table 3]

[0358]

[Table 4]

[0359]

[Table 5]

[0360]

[Table 6]

[0361]

[Table 7]

[0362]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the procedure for obtaining a cDNA selected to contain the 5 ′ end of the mRNA from which the cDNA is derived.

FIG. 2. von Haijin at the 5 ′ EST in each of the categories described herein.
Distribution of scores (Von Heijne score) and their 5 ′ EST
Indicates the probability of encoding a signal peptide.

FIG. 3 shows the general method used to clone and sequence an extended cDNA having a sequence flanking the 5 ′ EST.

FIG. 4 (Description of promoter structure isolated from signal tag 5 ′ EST) shows a schematic description of the isolated promoters and how to assemble them with the corresponding 5 ′ tag.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/21 C12Q 1/68 A 5/10 C12N 15/00 ZNAA C12Q 1/68 5/00 A (81 ) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA ( AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, Z, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Lacroix-Brno F-69230 Saint-Genis-Laval, Rout de Bourlet, 93

Claims (37)

[Claims] 1. A purified or isolated nucleic acid comprising a sequence comprising or complementary to one of SEQ ID NOs: 38-184. 2. The nucleic acid according to claim 1, wherein the nucleic acid is a recombinant. 3. at least 10 of one of SEQ ID NOs: 38 to 184;
A purified or isolated nucleic acid comprising a sequence comprising or complementary to two consecutive bases. 4. A purified or isolated nucleic acid comprising a sequence comprising at least 15 consecutive bases of one of SEQ ID NOs: 38 to 184 or a sequence complementary thereto. 5. The nucleic acid according to claim 4, wherein said nucleic acid is a recombinant. 6. The sequence of one of SEQ ID NOs: 38 to 184 or SEQ ID NO: 3.
A purified or isolated nucleic acid consisting of at least 15 bases capable of hybridizing under stringent conditions to one of the sequences complementary to the sequences of 8-184. 7. The nucleic acid according to claim 6, wherein the nucleic acid is a recombinant. 8. A purified or isolated nucleic acid encoding a human gene product, wherein said portion of said human gene product has a sequence encoded by one of SEQ ID NOs: 38-184. . 9. A purified or isolated nucleic acid having the sequence of one of SEQ ID NOs: 38-184 or having a sequence complementary thereto. 10. A purified or isolated nucleic acid comprising one nucleotide of SEQ ID NOs: 38-184 encoding a signal peptide. 11. A purified or isolated polypeptide comprising a signal peptide encoded by one of the sequences of SEQ ID NOs: 38-184. 12. A vector encoding a fusion protein comprising a polypeptide and a signal peptide, comprising one of the sequences of SEQ ID NOs: 38 to 184 operably linked to a second nucleic acid encoding said polypeptide. The vector, comprising a first nucleic acid encoding a signal peptide encoded by the first nucleic acid. 13. A method for instructing extracellular secretion of a polypeptide or insertion of a polypeptide into a membrane, comprising obtaining the vector according to claim 12, and introducing the vector into a host cell to obtain a fusion protein. Wherein the E. coli is secreted into the extracellular environment of the host cell or inserted into the membrane of the host cell. 14. A method for transferring a polypeptide into a cell, comprising: providing the cell with a signal peptide encoded by one of SEQ ID NOs: 38-184 operably linked to the polypeptide. The above method comprising contacting a fusion protein comprising: 15. A method for producing a cDNA encoding a human secreted protein, a part of which is encoded by one of SEQ ID NOs: 38 to 184, comprising: a cDNA comprising one of the sequences of SEQ ID NOs: 38 to 184. Conditions under which the probe can hybridize to the cDNA with the cDNA and a detectable probe comprising at least 15 consecutive nucleotides of the sequences of SEQ ID NOs: 38 to 184 or a sequence complementary thereto. Contacting below, identifying a cDNA that hybridizes to the detectable probe, and isolating the cDNA that hybridizes to the detectable probe. 16. An isolated or purified cDNA encoding a human secreted protein, wherein said human secreted protein is a protein encoded by one of SEQ ID NOs: 38-184 or a fragment thereof comprising at least 10 amino acids. The above cDNA, wherein the cDNA is obtained by the method of claim 15. 17. The c of claim 16, wherein the cDNA comprises the entire protein coding sequence, a portion of which is included in one of the sequences of SEQ ID NOs: 38-184.
DNA. 18. A method for producing a cDNA comprising one of the sequences of SEQ ID NOs: 38 to 184, comprising a first cell hybridizable to an aggregate of human cell-derived mRNA molecules and a poly-A tail of the mRNA. A primer is contacted, the first primer is hybridized to the poly A tail, and the mRNA is reverse transcribed to produce a first cDNA strand, at least 15 nucleotides of one of SEQ ID NOs: Creating a second cDNA strand complementary to said first cDNA strand using at least one primer comprising: and isolating a cDNA comprising said first cDNA strand and said second cDNA strand The above method comprising the steps of: 19. An isolated or purified cDNA encoding a human secreted protein, wherein said human secreted protein is a protein encoded by one of SEQ ID NOs: 38-184, or a fragment thereof comprising at least 10 amino acids. The cDNA according to claim 18, wherein the cDNA is obtained by the method according to claim 18. 20. The cDNA of claim 19, wherein said cDNA comprises the entire protein coding sequence, a portion of which is included in one of the sequences of SEQ ID NOs: 38-184. 21. The method according to claim 21, wherein the second strand of cDNA contacts the first strand of cDNA with a first pair of primers;
The first pair of primers comprises a second primer comprising at least 15 contiguous nucleotides of one of SEQ ID NOs: 38-184 and a third primer having a sequence contained within the sequence of the first primer. Performing a first polymerase chain reaction with said first pair of nested primers,
Producing a first PCR product; contacting said first PCR product with a second pair of primers, wherein:
The second pair of primers includes a fourth primer and a fifth primer;
Here, the fourth primer includes at least 15 consecutive nucleotides of one of SEQ ID NOs: 38 to 184, and the fourth and fifth primers hybridize to a sequence in the first PCR product. 19. The method of claim 18, wherein the method is soyable and is made by performing a second polymerase chain reaction, thereby producing a second PCR product. 22. An isolated or purified cDNA encoding a human secreted protein, wherein said human secreted protein comprises a protein encoded by one of SEQ ID NOs: 38-184 or a fragment thereof comprising at least 10 amino acids. Wherein said cDNA is obtained by the method of claim 21;
DNA. 23. The c of claim 22, wherein the cDNA comprises the entire protein coding sequence, a portion of which is included in one of the sequences of SEQ ID NOs: 38-184.
DNA. 24. The second cDNA strand, the first cDNA strand and at least 15 of the sequences of SEQ ID NOs: 38 to 184.
Contacting a second primer comprising two consecutive nucleotides; hybridizing the second primer to the first strand cDNA;
19. The method of claim 18, wherein said method is made by extending said hybridized second primer to generate said second cDNA strand. 25. An isolated or purified cDNA encoding a human secreted protein, said human secreted protein comprising a protein partially encoded by one of SEQ ID NOs: 38-184, or at least 10%. 25. The cDNA as described above, comprising a fragment thereof consisting of amino acids, wherein said cDNA is obtained by the method of claim 24. 26. The c of claim 25, wherein the cDNA comprises the entire protein coding sequence, a portion of which is included in one of the sequences of SEQ ID NOs: 38-184.
DNA. 27. A method for producing a protein comprising one of the sequences of SEQ ID NOs: 185-331, comprising encoding the entire protein coding sequence partially contained in one of the sequences of SEQ ID NOs: 38-184. Obtaining said cDNA, inserting said cDNA into an expression vector such that it is operably linked to a promoter, introducing said expression vector into a host cell, whereby said cDN is transferred to said host cell.
The above method comprising producing the protein encoded by A and isolating said protein. 28. An isolated protein obtained by the method of claim 27. 29. A method for obtaining a promoter DNA, comprising obtaining DNA located upstream of the nucleic acid of SEQ ID NOs: 38 to 184 or a sequence complementary thereto, screening the upstream DNA, and instructing the initiation of transcription. Identifying the promoter to be obtained and isolating the DNA containing the identified promoter. 30. The method of claim 29, wherein said obtaining step comprises chromosomal walking from said nucleic acid of SEQ ID NOs: 38-184 or a sequence complementary thereto. 31. The method of claim 30, wherein said screening step comprises inserting said upstream sequence into a promoter reporter vector. 32. The method of claim 30, wherein said screening step comprises identifying a motif in said upstream DNA that is a transcription factor binding site or a transcription initiation site. 33. An isolated promoter obtained by the method according to claim 32. 34. An isolated or purified protein comprising one of the sequences of SEQ ID NOs: 185-331. 35. An array of individual ESTs or fragments thereof of at least 15 nucleotides in length, wherein the improvement comprises at least one of the sequences of SEQ ID NOs: 38-184 or the sequence of SEQ ID NOs: 38-184. Such an array, comprising adding one of the complementary sequences, or a fragment thereof of at least 15 nucleotides, to said array. 36. The method of claim 35, comprising at least two of the sequences of SEQ ID NOs: 38-184, a sequence complementary to the sequence of SEQ ID NOs: 38-184, or a fragment thereof of at least 15 contiguous nucleotides. Array of. 37. The method of claim 35, comprising at least five of the sequences of SEQ ID NOs: 38-184, a sequence complementary to the sequence of SEQ ID NOs: 38-184, or a fragment thereof of at least 15 contiguous nucleotides. Array of.