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WO2002048368A2 - Proteines associees a la croissance, a la differenciation et a la mort cellulaire - Google Patents

Proteines associees a la croissance, a la differenciation et a la mort cellulaire Download PDF

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WO2002048368A2
WO2002048368A2 PCT/US2001/047871 US0147871W WO0248368A2 WO 2002048368 A2 WO2002048368 A2 WO 2002048368A2 US 0147871 W US0147871 W US 0147871W WO 0248368 A2 WO0248368 A2 WO 0248368A2
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polypeptide
polynucleotide
cgdd
seq
amino acid
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WO2002048368A3 (fr
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Henry Yue
Li Ding
Catherine M. Tribouley
Bao Tran
Brendan M. Duggan
Cynthia D. Honchell
Mariah R. Baughn
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Incyte Corp
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Incyte Genomics Inc
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Priority to JP2002550083A priority Critical patent/JP2005502305A/ja
Priority to CA002430906A priority patent/CA2430906A1/fr
Priority to AU2002227373A priority patent/AU2002227373A1/en
Priority to EP01996229A priority patent/EP1385954A2/fr
Publication of WO2002048368A2 publication Critical patent/WO2002048368A2/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to nucleic acid and amino acid sequences of proteins associated with cell growth, differentiation, and death and to the use of these sequences in the diagnosis, treatment, and prevention of cell pro-iterative, autoimmune, developmental, and reproductive disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of proteins associated with cell growth, differentiation, and death.
  • Cell division is the fundamental process by which all living things grow and reproduce. In unicellular organisms such as yeast and bacteria, each cell division doubles the number of organisms. In multicellular species many rounds of cell division are required to replace cells lost by wear or by programmed cell death, and for cell differentiation to produce a new tissue or organ. Details of the cell division cycle may vary, but the basic process consists of three principle events. The first event, interphase, involves preparations for cell division, replication of the DNA, and production of essential proteins. In the second event, mitosis, the nuclear material is divided and separates to opposite sides of the cell. The final event, cytokinesis, is division and fission of the cell cytoplasm. The sequence and timing of cell cycle transitions is under the control of the cell cycle regulation system which controls the process by positive or negative regulatory circuits at various check points.
  • Progression through the cell cycle is governed by the intricate interactions of protein complexes. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including cyclins, cyclin-dependent protein kinases, growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, and tumor-suppressor proteins.
  • Cyclins act by binding to and activating a group of cyclin-dependent protein kinases (Cd s) which then phosphoiylate and activate selected proteins involved in the mitotic process. Cyclins are characterized by a large region of shared homology that is approximately 180 amino acids in length and referred to as the "cyclin box" (Chapman, D.L. and Wolgemuth, DJ. (1993) Development 118:229-40). In addition, cyclins contain a conserved 9 amino acid sequence in the N-terminal region of the molecule called the "destruction box".
  • Cyclins are degraded through the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaroytic ceUs and in some bacteria.
  • UCS ubiquitin conjugation system
  • the UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control ceUular processes such as gene transcription and ceU cycle progression.
  • the UCS is implicated in the degradation of mitotic cyclin kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, ceU surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, supra).
  • ubiquitin conjugation and protein degradation occurs in five principle steps (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207).
  • First ubiquitin (Ub), a smaU, heat stable protein is activated by a ubiquitin-activating enzyme (El) in an ATP dependent reaction which binds the C- terminus of Ub to the thiol group of an internal cysteine residue in El .
  • activated Ub is transferred to one of several Ub-conjugating enzymes (E2).
  • E2 Ub-conjugating enzymes
  • Different ubiquitin-dependent proteolytic pathways employ structuraUy similar, but distinct ubiquitin-conjugating enzymes that are associated with recognition subunits which direct them to proteins carrying a particular degradation signal.
  • E2 transfers the Ub molecule through its C-terminal glycine to a member of the ubiquitin-protein ligase family, E3.
  • E3 transfers the Ub molecule to the target protein. Additional Ub molecules may be added to the target protein forming a multi-Ub chain structure.
  • Fifth, the ubiquinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and Ub is released for re-utilization.
  • Ub-conjugating enzymes are important for substrate specificity in different UCS pathways.
  • AU E2s have a conserved domain of approximately 16 kDa caUed the UBC domain that is at least 35% identical in aU E2s and contains a centraUy located cysteine residue required for ubiquitin- enzyme thiolester formation (Jentsch, supra).
  • a weU conserved proline-rich element is located N- terminal to the active cysteine residue. Structural variations beyond this conserved domain are used to classify the E2 enzymes.
  • Class I E2s consist almost exclusively of the conserved UBC domain.
  • Class II E2s have various unrelated C-terminal extensions that contribute to substrate specificity and ceUular localization.
  • Class HI E2s have unique N-terminal extensions which are believed to be involved in enzyme regulation or substrate specificity.
  • a mitotic cyclin-specific E2 (E2-C) is characterized by the conserved UBC domain, an N- terminal extension of 30 amino acids not found in other E2s, and a 7 amino acid unique sequence adjacent to this extension. These characteristics together with the high affinity of E2-C for cyclin identify it as a new class of E2 (Aristarkhov, A. et al. (1996) Proc. Natl. Acad. Sci. 93:4294-99).
  • Ubiquitin-protein ligases (E3s) catalyze the last step in the ubiquitin conjugation process, covalent attachment of ubiquitin to the substrate.
  • E3 plays a key role in determining the specificity of the process. Only a few E3s have been identified so far.
  • One type of E3 ligases is the HECT (homologous to E6-AP C-terminus) domain protein family.
  • E6-AP E6-associated protein
  • HPN human papiUomavirus
  • the C-terminal domain of the HECT proteins contains the highly conserved ubiquitin-binding cysteine residue.
  • the ⁇ -terminal region of the various HECT proteins is variable and is believed to be involved in specific substrate recognition (Huibregtse, J.M. et al. (1997) Proc. ⁇ atl Acad. Sci. USA 94:3656-3661).
  • ceU proliferation disorders can be identified by changes in the protein complexes that normaUy control progression through the ceU cycle.
  • a primary treatment strategy involves reestabUshing control over ceU cycle progression by manipulation of the proteins involved in ceU cycle regulation ( ⁇ igg, E.A. (1995) BioEssays 17:471-480).
  • Apoptosis regulators ⁇ igg, E.A. (1995) BioEssays 17:471-480.
  • Apoptosis is the geneticaUy controUed process by which unneeded or defective ceUs undergo programmed ceU death. Selective elimination of ceUs is as important for morphogenesis and tissue remodeUng as is ceU proliferation and differentiation. Lack of apoptosis may result in hyperplasia and other disorders associated with increased ceU proliferation. Apoptosis is also a critical component of the immune response. Immune ceUs such as cytotoxic T-ceUs and natural kiUer ceUs prevent the spread of disease by inducing apoptosis in tumor ceUs and virus-infected ceUs. In addition, immune ceUs that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response.
  • apoptosis undergo distinct morphological changes. Hallmarks of apoptosis include ceU shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology. BiochemicaUy, apoptotic ceUs are characterized by increased intraceUular calcium concentration, fragmentation of chromosomal DNA, and expression of novel ceU surface components.
  • Apoptosis generaUy proceeds in response to a signal which is transduced intraceUularly and results in altered patterns of gene expression and protein activity.
  • Signaling molecules such as hormones and cytokines are known both to stimulate and to inhibit apoptosis through interactions with ceU surface receptors.
  • the Bcl-2 family of proteins are key regulators of apoptosis. There are at least 15 Bcl-2 family members within 3 subfamilies. These proteins have been identified in mammahan ceUs and in viruses, and each possesses at least one of four Bcl-2 homology domains (BH1 to BH4), which are highly conserved. Bcl-2 family proteins contain the BH1 and BH2 domains, which are found in members of the pro-survival subfamily, while those proteins which are most similar to Bcl-2 have aU four conserved domains, enabling inhibition of apoptosis foUowing encounters with a variety of cytotoxic chaUenges.
  • pro-survival subfamily include Bcl-2, Bcl-x L , Bcl-w, McH, and Al in mammals; NF-13 (chicken); CED-9 (Caenorhabditis elegans); and viral proteins BHRF1, LMW5-HL, O F16, KS-Bcl-2, and E1B-19K.
  • the BH3 domain is essential for the function of pro-apoptosis subfamily proteins.
  • the two pro-apoptosis subfamilies, Bax and BH3, include Bax, Bak, and Bok (also called Mtd); and Bik, Blk, Hrk, BNIP3, Bim L , Bad, Bid, and Egl-1 (C.
  • the proteins of the two pro-apoptosis subfamilies may be the antagonists of pro-survival subfamily proteins. This is iUustrated in C. elegans where Egl-1, which is required for apoptosis, binds to and acts via CED-9 (for review, see Adams, J.M. and S. Cory (1998) Science 281:1322-1326).
  • Heterodimerization between pro-apoptosis and anti-apoptosis subfamily proteins seems to have a titrating effect on the functions of these protein subfamihes, which suggests that relative concentrations of the members of each subfamily may act to regulate apoptosis.
  • Heterodimerization is not required for a pro-survival protein; however, it is essential in the BH3 subfamily, and less so in the Bax subfamily.
  • the Bcl-2 protein has 2 isoforms, alpha and beta, which are formed by alternative splicing. It forms homodimers and heterodimers with Bax and Bak proteins and the Bcl-X isoform Bcl-x s . Heterodimerization with Bax requires intact BH1 and BH2 domains, and is necessary for pro-survival activity. The BH4 domain seems to be involved in pro-survival activity as weU. Bcl-2 is located within the inner and outer mitochondrial membranes, as weU as within the nuclear envelope and endoplasmic reticulum, and is expressed in a variety of tissues. Its involvement in foUicular lymphoma (type II chronic lymphatic leukemia) is seen in a chromosomal translocation T(14;18) (q32;q21) and involves i_---munoglobulin gene regions.
  • foUicular lymphoma type II chronic lymphatic leukemia
  • the Bcl-x protein is a dominant regulator of apoptotic ceU death.
  • Alternative splicing results in three isoforms, Bcl-xB, a long isoform, and a short isoform.
  • the long isoform exhibits ceU death repressor activity, while the short isoform promotes apoptosis.
  • Bcl-xL forms heterodimers with Bax and Bak, although heterodimerization with Bax does not seem to be necessary for pro-survival (anti- apoptosis) activity.
  • Bcl-xS forms heterodimers with Bcl-2.
  • Bcl-x is found in mitochondrial membranes and the perinuclear envelope.
  • Bcl-xS is expressed at high levels in developing lymphocytes and other ceUs undergoing a high rate of turnover.
  • Bcl-xL is found in adult brain and in other tissues' long-Uved post-mitotic ceUs.
  • the BH1, BH2, and BH4 domains are involved in pro-survival activity.
  • the Bcl-w protein is found within the cytoplasm of almost aU myeloid ceU lines and in numerous tissues, with the highest levels of expression in brain, colon, and saUvary gland. This protein is expressed in low levels in testis, liver, heart, stomach, skeletal muscle, and placenta, and a few lymphoid ceU Unes.
  • Bcl-w contains the BH1 , BH2, and BH4 domains, aU of which are needed for its ceU survival promotion activity.
  • mice in which Bcl-w gene function was disrupted by homologous recombination were viable, healthy, and normal in appearance, and adult females had normal reproductive function, the adult males were infertile. In these males, the initial, prepuberty stage of spermatogenesis was largely unaffected and the testes developed normaUy. However, the seminiferous tubules were disorganized, contained numerous apoptotic ceUs, and were incapable of producing mature sperm.
  • This mouse model may be applicable to some cases of human male sterility and suggests that alteration of programmed ceU death in the testes may be useful in modulating fertihty (Print, CG. et al. (1998) Proc. Natl. Acad. Sci. USA 95:12424-12431).
  • Bcl-w Studies in rat ischemic brain found Bcl-w to be overexpressed relative to its normal low constitutive level of expression in nonischemic brain. Furthermore, in vitro studies to examine the mechanism of action of Bcl-w revealed that isolated rat brain mitochondria were unable to respond to an addition of recombinant Bax or high concentrations of calcium when Bcl-w was also present. The normal response would be the release of cytochrome c from the mitochondria. AdditionaUy, recombinant Bcl-w protein was found to inhibit calcium-induced loss of mitochondrial transmembrane potential, which is indicative of permeability transition.
  • Bcl-w may be a neuro-protectant against ischemic neuronal death and may achieve this protection via the mitochondrial death-regulatory pathway (Yan, C. et al. (2000) J. Cereb. Blood Flow Metab. 20:620- 630).
  • the bfl-1 gene is an additional member of the Bcl-2 family, and is also a suppressor of apoptosis.
  • the Bfl-1 protein has 175 amino acids, and contains the BHl, BH2, and BH3 conserved domains found in Bcl-2 family members. It also contains a Gin-rich NH 2 -terminal region and lacks an NH domain 1, unlike other Bcl-2 family members.
  • the mouse Al protein shares high sequence homology with Bfl-1 and has the 3 conserved domains found in Bfl-1.
  • Bfl-1 Apoptosis induced by the p53 tumor suppressor protein is suppressed by Bfl-1, similar to the action of Bcl-2, Bcl-xL, and EBN- BHRFl (D'Sa-Eipper, C. et al. (1996) Cancer Res. 56:3879-3882).
  • Bfl-1 is found intiaceUularly, with the highest expression in the hematopoietic compartment, i.e. blood, spleen, and bone marrow; moderate expression in lung, smaU intestine, and testis; and minimal expression in other tissues. It is also found in vascular smooth muscle ceUs and hematopoietic malignancies.
  • Cancers are characterized by continuous or uncontroUed cell proliferation. Some cancers are associated with suppression of normal apoptotic ceU death. Strategies for treatment may involve either reestablishing control over ceU cycle progression, or selectively stimulating apoptosis in cancerous ceUs ( ⁇ igg, E.A. (1995) BioEssays 17:471-480). Immunological defenses against cancer include induction of apoptosis in mutant ceUs by tumor suppressors, and the recognition of tumor antigens by T lymphocytes. Response to mitogenic stresses is frequently controUed at the level of transcription and is coordinated by various transcription factors. For example, the Rel/ ⁇ F-kappa B family of vertebrate transcription factors plays a pivotal role in inflammatory and immune responses to radiation.
  • the ⁇ F-kappa B family includes p50, p52, RelA, RelB, cRel, and other D ⁇ A-binding proteins.
  • the p52 protein induces apoptosis, upregulates the transcription factor c-Jun, and activates c-Jun ⁇ -terminal kinase 1 (J ⁇ K1) (Sun, L. et al. (1998) Gene 208:157-166).
  • Most ⁇ F-kappa B proteins form D ⁇ A-binding homodimers or heterodimers. Dimerization of many transcription factors is mediated by a conserved sequence known as the bZIP domain, characterized by a basic region foUowed by a leucine zipper.
  • the Fas/Apo-1 receptor is a member of the tumor necrosis factor (T ⁇ F) receptor family. Upon binding its ligand (Fas ligand), the membrane-spanning FAS induces apoptosis by recruiting several cytoplasmic proteins that transmit the death signal.
  • FAS-associated protein factor 1 FAF1
  • FAS-associated factors have been isolated from numerous other species, including quail and fly (Frohlich, T. et al. (1998) J. CeU Sci. 111:2353-2363).
  • Another cytoplasmic protein that functions in the tiansmittal of the death signal from Fas is the Fas-associated death domain protein, also known as FADD.
  • FADD transmits the death signal in both FAS-mediated and TNF receptor-mediated apoptotic pathways by activating caspase-8 (Bang, S. et al. (2000) J. Biol. Chem. 275:36217-36222).
  • DFF DNA fragmentation factor
  • CAD DNA fragmentation factor
  • ICAD ICAD
  • CEDE- A and CDDE-B Two mouse homologs of DFF45/TCAD, termed CEDE- A and CDDE-B, have recently been described (Inohara, N. et al. (1998) EMBO J. 17:2526-2533).
  • CIDE-A and CIDE-B expression in mammalian ceUs activated apoptosis, while expression of CIDE-A alone induced DNA fragmentation.
  • FAS-mediated apoptosis was enhanced by CIDE-A and QDE-B, further implicating these proteins as effectors that mediate apoptosis.
  • caspases A number of downstream effector molecules, particularly proteases such as the cysteine proteases called caspases, are involved in the initiation and execution phases of apoptosis. The activation of the caspases results from the competitive action of the pro-survival and pro-apoptosis Bcl-2-related proteins (Print, CG. et al. (1998) Proc. Natl. Acad. Sci. USA 95:12424-12431).
  • a pro-apoptotic signal can activate initiator caspases that trigger a proteolytic caspase cascade, leading to the hydrolysis of target proteins and the classic apoptotic death of the ceU.
  • Caspases are synthesized as inactive zymogens consisting of one large (p20) and one smaU (plO) subunit separated by a smaU spacer region, and a variable N-terminal prodomain. This prodomain interacts with cofactors that can positively or negatively affect apoptosis.
  • An activating signal causes autoproteolytic cleavage of a specific aspartate residue (D297 in the caspase-1 numbering convention) and removal of the spacer and prodomain, leaving a pl0/p20 heterodimer. Two of these heterodimers interact via their smaU subunits to form the catalyticaUy active tetramer.
  • caspase family members have been shown to promote dimerization and auto-processing of procaspases.
  • Some caspases contain a "death effector domain" in their prodomain by which they can be recruited into self-activating complexes with other caspases and FADD protein- associated death receptors or the TNF receptor complex.
  • two dimers from different caspase family members can associate, changing the substrate specificity of the resultant tetramer.
  • a caspase recruitment domain (CARD) is found within the prodomain of several apical caspases and is conserved in several apoptosis regulatory molecules such as Apaf-2, RAEDD, and ceUular inhibitors of apoptosis proteins (IAPs) (Hofmann, K. et al. (1997) Trends Biochem. Sci. 22:155-157).
  • the regulatory role of CARD in apoptosis may be to aUow proteins such as Apaf-1 to associate with caspase-9 (Li, P. et al. (1997) CeU 91:479-489).
  • ARC apoptosis repressor with a CARD
  • ARC functions as an inhibitor of apoptosis and interacts selectively with caspases
  • caspases Koseki, T. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5156-5160.
  • AU of these interactions have clear effects on the control of apoptosis (reviewed in Chan S.L. and M.P. Mattson (1999) J. Neurosci. Res. 58:167-190; Salveson, G.S. and NM. Dixit (1999) Proc. ⁇ atl. Acad. Sci. USA 96:10964-10967).
  • Tumor suppressor genes are generaUy defined as genetic elements whose loss or inactivation contributes to the deregulation of ceU proliferation and the pathogenesis and progression of cancer. Tumor suppressor genes normaUy function to control or inhibit ceU growth in response to stress and to limit the proliferative life span of the ceU. When tumorigenic and non-tumorigenic cells are fused in culture, the resulting hybrid ceUs are usuaUy non-tumorigenic. Loss of tumorigenicity is attributed to heritable factors within the non-tumorigenic ceU which suppress tumor development. Several tumor suppressor genes have been identified and are of great interest to researchers and clinicians seeking to investigate and control cancer growth.
  • p53 The role of p53 in the pathogenesis of cancer has been extensively studied. (Reviewed in Aggarwal, M. L. et al. (1998) J. Biol. Chem. 273:1-4; Levine, A. (1997) CeU 88:323-331.) About 50% of aU human cancers contain mutations in the p53 gene. These mutations result in either the absence of functional p53 or, more commonly, a defective form of p53 which is overexpressed. p53 is a transcription factor that contains a central core domain required for D ⁇ A binding. Most cancer- associated mutations in p53 localize to this domain. In normal proliferating ceUs, p53 is expressed at low levels and is rapidly degraded.
  • p53 expression and activity is induced in response to DNA damage, abortive mitosis, and other stressful stimuli. In these instances, p53 induces apoptosis or arrests ceU growth until the stress is removed. Downstream effectors of p53 activity include apoptosis-specific proteins and ceU cycle regulatory proteins, including Rb, oncogene products, cyclins, and ceU cycle-dependent kinases.
  • KAI1 The metastasis-suppressor gene KAI1 (CD82) has been reported to be related to the tumor suppressor gene p53.
  • KAI1 is involved in the progression of human prostatic cancer and possibly lung and breast cancers when expression is decreased.
  • KAI1 encodes a member of a structuraUy distinct family of leukocyte surface glycoproteins.
  • the family is known as either the tetraspan transmembrane protein family or transmembrane 4 superfamily (TM4) as they span the plasma membrane four times.
  • TM4 transmembrane 4 superfamily
  • the family is composed of integral membrane proteins having a N-terminal membrane-anchoring domain which functions as both a membrane anchor and a translocation signal during protein biosynthesis. The N-terminal membrane-anchoring domain is not cleaved during biosynthesis.
  • the TM4 family has three additional transmembrane regions, seven or more conserved cysteine residues, aU are similar in size (218 to 284 residues), and aUhave a large extiaceUular hydrophilic domain with three potential N-glycosylation sites.
  • the promoter region contains many putative binding motifs for various transcription factors, including five AP2 sites and nine Spl sites.
  • Gene structure comparisons of KAI1 and seven other members of the TM4 superfamily indicate that the spUcing sites relative to the different structural domains of the predicted proteins are conserved. This suggests that these genes are related evolutionarily and arose through gene duplication and divergent evolution (Levy, S. et al. (1991) J. Biol. Chem. 266:14597-14602; Dong, J.T. et al. (1995) Science 268:884-886; Dong, J.T. et al., (1997) Genomics 41:25-32).
  • LGU1 Leucine-rich gene-GUoma Inactivated (LGI1) protein shares homology with a number of transmembrane and extraceUular proteins which function as receptors and adhesion proteins.
  • LGU is encoded by an LLR (leucine-rich, repeat-containing) gene and maps to 10q24. LGU has four LLRs which are flanked by cysteine-rich regions and one transmembrane domain (SomerviUe, R.P., et al. (2000) Mamm. Genome 11:622-627). LGU expression is seen predominantly in neural tissues, especiaUy brain.
  • the invention features purified polypeptides, proteins associated with cell growth, differentiation, and death, referred to coUectively as "CGDD” and individuaUy as “CGDD-1,” “CGDD-2,” “CGDD-3,” “CGDD-4,” and “CGDD-5.”
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-5.
  • the polynucleotide is selected from the group consisting of SEQ ED NO: 6- 10.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably Unked to a polynucleotide encoding a polypeptide selected fromthe group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l -5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group
  • the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-5.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:6-10, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:6-10, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:6-10, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:6-10, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionaUy, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:6-10, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:6-10, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionaUy, if present, the amount thereof.
  • the invention -further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO:l -5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-5, and a pharmaceutically acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5.
  • the invention additionaUy provides a method of treating a disease or condition associated with decreased expression of functional CGDD, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from tlie group consisting of SEQ ED NO: 1-5.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional CGDD, comprising administering to a patient in need of such treatment the composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ED NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with overexpression of functional CGDD, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-5.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specificaUy binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-5, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-5, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-5, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-5.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ED NO:6-10, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6- 10, n) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected fromthe group consisting of SEQ ID NO:6-10, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of n), and v) an RNA equivalent of i)-iv).
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:6-10, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 6- 10, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected fromthe group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amotmt of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptides of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNA Ubrary for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with appUcable descriptions, references, and threshold parameters.
  • a reference to "a host ceU” includes a pluraUty of such host ceUs
  • a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth.
  • aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skiU in the art to which this invention belongs.
  • CGDD refers to the amino acid sequences of substantiaUy purified CGDD obtained from any species, particularly a mammaUan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of CGDD.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of CGDD either by directly interacting with CGDD or by acting on components of the biological pathway in which CGDD participates.
  • aUeUc variant is an alternative form of the gene encoding CGDD.
  • AUelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
  • a gene may have none, one, or many aUeUc variants of its naturaUy occurring form.
  • Common mutational changes which give rise to aUelic variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding CGDD include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as CGDD or a polypeptide with at least one fiinctional characteristic of CGDD. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oUgonucleotide probe of the polynucleotide encoding CGDD, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CGDD.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent CGDD.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of CGDD is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophi-icity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophi-icity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine
  • amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturaUy occurring protein molecule, “amino acid sequence” and like terms are not meant to Umit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • AmpUfication relates to the production of additional copies of a nucleic acid sequence. AmpUfication is generaUy carried out using polymerase chain reaction (PCR) technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of CGDD.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of CGDD either by directly interacting with CGDD or by acting on components of the biological pathway in which CGDD participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind CGDD polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oUgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • chemicaUy e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specificaUy to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eUcit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Ubraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2-NH ), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specificaUy cross-Unked to their cognate Ugands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (BUnd, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-Uke molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oMgonucleotides having modified backbone Mnkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oMgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oMgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capabiUty of the natural, recombinant, or synthetic CGDD, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • compositions comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding CGDD or fragments of CGDD may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabiMzing agent such as a carbohydrate.
  • a stabiMzing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (AppMed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVTEW fragment assembly system (GCG, Madison WT) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • GELVTEW fragment assembly system GCG, Madison WT
  • Phrap Universality of Washington, Seattle WA
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with tlie properties of the original protein, i. e. , the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Trp Phe Tyr Tyr His, Phe, Trp Val He, Leu, Thr
  • Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha heUcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
  • a "fragment” is a unique portion of CGDD or the polynucleotide encoding CGDD which is identical in sequence to but shorter in length than the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentiaUy selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a -fragment of SEQ ID NO: 6- 10 comprises a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:6-10, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:6-10 is useful, for example, in hybridization and ampMfication technologies and in analogous methods that distinguish SEQ ID NO:6-10 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO: 6- 10 and the region of SEQ ID NO: 6- 10 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ED NO.1-5 is encoded by a fragment of SEQ ED NO.6-10.
  • a fragment of SEQ ED NO : 1 -5 comprises a region of unique amino acid sequence that specificaUy identifies SEQ ED NO: 1-5.
  • a fragment of SEQ ED NO: 1-5 is useful as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO: 1-5.
  • the precise length of a fragment of SEQ ID NO: 1-5 and the region of SEQ ED NO: 1-5 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a “fuU length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “fuU length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aUgned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize aMgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AMgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AMgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aMgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aMgned using a standardized algorithm.
  • Methods of polypeptide sequence aMgnment are weU-known. Some aMgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and iydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • Mnear ⁇ crochromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome repMcation, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abiUty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive anneaMng conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for anneaMng of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive anneaMng conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (T--) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Useful variations on these wash conditions wiU be readily apparent to those of ordinary skiU in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobiMzed on a soMd support (e.g., paper, membranes, filters, chips, pins or glass sMdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaUng molecules, which may affect ceMular and systemic defense systems.
  • An "immunogenic fragment” is a polypeptide or oMgopeptide fragment of CGDD which is capable of eMciting an immune response when introduced into a Mving organism, for example, a mammal.
  • immunogenic fragment also includes any polypeptide or oMgopeptide fragment of CGDD which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a pluraMty of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • array element refers to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of CGDD.
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CGDD.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oMgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-Mke or RNA-Mke material.
  • PNA peptide nucleic acid
  • operably Mnked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably Mnked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably Mnked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oMgonucleotide of at least about 5 nucleotides in length Mnked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubiMty to the composition.
  • PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Mfespan in the ceU.
  • Post-translational modification of an CGDD may involve Mpidation, glycosylation, phosphorylation, acetylation,.racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic miUeu of CGDD.
  • Probe refers to nucleic acid sequences encoding CGDD, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acid sequences. Probes are isolated oMgonucleotides or polynucleotides attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, Mgands, chemiluminescent agents, and enzymes.
  • "Primers" are short nucleic acids, usuaUy DNA oMgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for ampMfication (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR). Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence.
  • probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • OMgonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oMgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabiMties. For example, the PrimOU primer selection program (available to the pubMc from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome- wide scope.
  • Primer3 primer selection program (available to the pubMc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming Mbrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oMgonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the pubMc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aMgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aMgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oMgonucleotides and polynucleotide fragments.
  • oMgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oMgonucleotide selection are not Mmited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturaUy occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accompMshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably Mnked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stabiUty.
  • Reporter molecules are chemical or biochemical moieties used for labeMng a nucleic acid, amino acid, or antibody. Reporter molecules include radionucMdes; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the ait.
  • RNA equivalent in reference to a DNA sequence, is composed of the same Mnear sequence of nucleotides as the reference DNA sequence with the exception that aU occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing CGDD, nucleic acids encoding CGDD, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturaUy associated.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, sMdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capiUaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not Mmited to, bacteriophage or viral infection, electroporation, heat shock, Mpofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of repMcation either as an autonomously repMcating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for Mmited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not Mmited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art.
  • the nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of deUberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertiMzation, but rather is directed to the intioduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an
  • a spMce variant may have significant identity to a reference molecule, but will generaUy have a greater or lesser number of polynucleotides due to alternate spMcing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human proteins associated with ceU growth, differentiation, and death (CGDD), the polynucleotides encoding CGDD, and the use of these compositions for the diagnosis, treatment, or prevention of ceU proMferative, autoimmune, •5- , developmental, and reproductive disorders.
  • CGDD ceU growth, differentiation, and death
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ED). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte 0 polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ED) as shown.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. 5
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probabiUty scores for the matches 0 between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where appMcable, aU of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2
  • FIG. 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 0 shows analytical methods for protein stracture/function analysis and in some cases, searchable databases to which the analytical methods were appMed.
  • Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabMsh that the claimed polypeptides are proteins associated with ceU growth, differentiation, and death.
  • SEQ ID N0:1 is 48% identical to worm E6-AP ubiquitin-protein Mgase (GenBank ID g2340821) as determined by the Basic Local AMgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiMty score is 5.9e-143, which indicates the probabiMty of obtaining the observed polypeptide sequence aMgnment by chance.
  • SEQ ID NO:l also contains a HECT (ubiquitin transferase) domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HECT ubiquitin transferase
  • SEQ ID NO:l is an ubiquitin- protein Mgase.
  • SEQ ID NO:2 is 64% identical to human nucleotide-binding site protein 1
  • SEQ ID NO:4 is 48% identical to Human leucine-rich gMoma-inactivated protein precursor (GenBank ID g4091819) as determined by the Basic Local AMgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiMty score is 1.0e-136, which indicates the probabiMty of obtaining the observed polypeptide sequence aMgnment by chance. SEQ ED NO:4 also contains five leucine rich repeats as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS analysis provides fiirther corroborative evidence that SEQ ID NO:4 is a tumor suppressor protein.
  • HMM hidden Markov model
  • SEQ ED NO:3 and SEQ ED NO:5 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO: 1 -5 are described in Table 7.
  • the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 Msts the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU lengthpolynucleoti.de sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or ampMfication technologies that identify SEQ ED NO:6-10 or that distinguish between SEQ ID NO: 6- 10 and related polynucleotide sequences.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Mbraries or from pooled cDNA Mbraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotide sequences.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i. e. , those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as ⁇ L_XXXXX_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was appMed, and -- ⁇ Y-r ⁇ is the number of the prediction generated by the algorithm, and N 1 ⁇ 3 _ Struktur , if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • FLXXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was appMed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by " ⁇ M,” “ ⁇ P,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the foUowing Table Mst s examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Table 5 shows the representative cDNA Mbraries for those fuU length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA Mbrary is the Incyte cDNA Mbrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA Mbraries shown in Table 5 are described in Table 6.
  • the invention also encompasses CGDD variants.
  • a preferred CGDD variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the CGDD amino acid sequence, and which contains at least one functional or structural characteristic of CGDD.
  • the invention also encompasses polynucleotides which encode CGDD.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 6- 10, which encodes CGDD.
  • the polynucleotide sequences of SEQ ID NO:6-10 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses a variant of a polynucleotide sequence encoding CGDD.
  • such a variant polynucleotide sequence wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding CGDD.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO: 6- 10 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:6-10. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CGDD.
  • a polynucleotide variant of the invention is a spMce variant of a polynucleotide sequence encoding CGDD.
  • a spMce variant may have portions which have significant sequence identity to the polynucleotide sequence encoding CGDD, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spMcing of exons during mRNA processing.
  • a spMce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding CGDD over its entire length; however, portions of the spMce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide sequence encoding CGDD.
  • a polynucleotide comprising a sequence of SEQ ID NO:5 is a spMce variant of a polynucleotide comprising a sequence of SEQ ID NO:4. Any one of the spMce variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CGDD.
  • nucleotide sequences which encode CGDD and its variants are generaUy capable of hybridizing to the nucleotide sequence of the naturaUy occurring CGDD under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CGDD or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utiMzed by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of DNA sequences which encode CGDD and CGDD derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding CGDD or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ED NO:6-10 and fragments thereof under various conditions of stringency.
  • Hybridization conditions including anneaMng and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppMed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampMfication system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 Mquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppMed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppMed Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
  • the nucleic acid sequences encoding CGDD may be extended utiMzing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampMfy unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods AppMc. 2:318-322.)
  • Another method uses primers that extend in divergent directions to ampMfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR ampMfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstiom, M. et al. (1991) PCR Methods AppMc.
  • Biosciences, Beverly MN) or another appropriate program to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • Mbraries When screening for fuil length cDNAs, it is preferable to use Mbraries that have been size-selected to include larger cDNAs. In addition, random-primed Mbraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oMgo d(T) Mbrary does not yield a full-length cDNA. Genomic Mbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • CapiUary electrophoresis systems which are commerciaUy available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/Mght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppMed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • Capillary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in Mmited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode CGDD may be cloned in recombinant DNA molecules that direct expression of CGDD, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantiaUy the same or a functionaUy equivalent amino acid sequence may be produced and used to express CGDD.
  • nucleotide sequences of the present invention can be engineered using methods generaUy known in the art in order to alter CGDD-encoding sequences for a variety of purposes including, but not Mmited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oMgonucleotides may be used to engineer the nucleotide sequences.
  • oMgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spMce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of CGDD, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al.
  • DNA shuffling is a process by which a Mbrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Mbrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • sequences encoding CGDD may be synthesized, in whole or in part, using chemical methods weU known in the art.
  • chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • CGDD itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or soMd-phase techniques.
  • the peptide may be substantiaUy purified by preparative high performance Mquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences encoding CGDD or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 ' untranslated regions in the vector and in polynucleotide sequences encoding CGDD.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CGDD. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding CGDD. These include, but are not Mmited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauMflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g.,
  • Expression vectors derived from retroviruses, adenovirases, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for deUvery of nucleotide sequences to the targeted organ, tissue, or ceU population.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CGDD.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding CGDD can be achieved using a multifunctional E. coM vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Life Technologies).
  • PBLUESCRIPT Stratagene, La JoUa CA
  • PSPORT1 plasmid Life Technologies
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of CGDD may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of CGDD.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of CGDD. Transcription of sequences encoding CGDD may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al.
  • a number of viral-based expression systems may be utiUzed.
  • sequences encoding CGDD may be Mgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses CGDD in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host cells.
  • SV40 or EB V- based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deUver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and deUvered via conventional deMvery methods (Mposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • Miposomes polycationic amino polymers, or vesicles
  • sequences encoding CGDD can be transformed into ceU Mnes using expression vectors which may contain viral origins of repMcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • ceUs may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU Mnes. These include, but are not Mmited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltiansferase genes, for use in tk and apr ceUs, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetaboMte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • d fr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboMtes.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol.
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding CGDD is inserted within a marker gene sequence
  • transformed ceUs containing sequences encoding CGDD can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding CGDD under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
  • host cells that contain the nucleic acid sequence encoding CGDD and that express CGDD may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not Mmited to, DNA-DNA or DNA-RNA hybridizations, PCR ampMfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences, --mmunological methods for detecting and measuring the expression of CGDD using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-Mnked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CGDD include oMgolabeMng, nick translation, end-labeMng, or PCR ampMfication using a labeled nucleotide.
  • the sequences encoding CGDD, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionucMdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the Mke.
  • Host ceUs transformed with nucleotide sequences encoding CGDD may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed ceU may be secreted or retained intiaceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode CGDD may be designed to contain signal sequences which direct secretion of CGDD through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its abiUty to modulate expression of the inserted sequences or to process tlie expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not Mmited to, acetylation, carboxylation, glycosylation, phosphorylation, Mpidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host ceUs which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant nucleic acid sequences encoding CGDD may be Mgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric CGDD protein containing a heterologous moiety that can be recognized by a commercially available antibody may faciMtate the screening of peptide Mbraries for inhibitors of CGDD activity.
  • Heterologous protein and peptide moieties may also faciMtate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not Mmited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmoduMn binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobiMzed glutathione, maltose, phenylarsine oxide, calmoduMn, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the CGDD encoding sequence and the heterologous protein sequence, so that CGDD may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commerciaUy available kits may also be used to faciMtate expression and purification of fusion proteins.
  • synthesis of radiolabeled CGDD may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • CGDD of the present invention or fragments thereof may be used to screen for compounds that specificaUy bind to CGDD. At least one and up to a pluraUty of test compounds may be screened for specific binding to CGDD. Examples of test compounds include antibodies, oMgonucleotides, proteins (e.g., receptors), or smaU molecules.
  • the compound thus identified is closely related to the natural Mgand of CGDD, e.g., a Mgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • the compound can be closely related to the natural receptor to which CGDD binds, or to at least a fragment of the receptor, e.g., the Mgand binding site. In either case, the compound can be rationaUy designed using known techniques.
  • screening for these compounds involves producing appropriate ceUs which express CGDD, either as a secreted protein or on the ceU membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coM.
  • CeUs expressing CGDD or ceU membrane fractions which contain CGDD are then contacted with a test compound and binding, stimulation, or inhibition of activity of either CGDD or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with CGDD, either in solution or affixed to a soMd support, and detecting the binding of CGDD to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Mbraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soMd support.
  • CGDD of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of CGDD.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for CGDD activity, wherein CGDD is combined with at least one test compound, and the activity of CGDD in the presence of a test compound is compared with the activity of CGDD in the absence of the test compound. A change in the activity of CGDD in the presence of the test compound is indicative of a compound that modulates the activity of CGDD.
  • a test compound is combined with an in vitro or ceU-free system comprising CGDD under conditions suitable for CGDD activity, and the assay is performed.
  • a test compound which modulates the activity of CGDD may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraMty of test compounds may be screened.
  • polynucleotides encoding CGDD or their mammaMan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
  • mouse ES ceUs such as uie mouse 129/SvJ ceU Mne
  • the ES ceUs are tiansformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) CMn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding CGDD may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceUs have the potential to differentiate into at least eight separate ceU Mneages including endoderm, mesoderm, and ectodermal ceU types. These ceU Mneages differentiate into, for example, neural ceUs, hematopoietic Mneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding CGDD can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding CGDD is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred Mnes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • CGDD neurotrophic factor
  • CGDD Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CGDD and proteins associated with ceU growth, differentiation, and death.
  • the expression of CGDD is closely associated with diseased lung tissue, tumors of the testicle, pulmonary tumors and tumors involving connective tissues. Tissues expressing CGDD can also be found in Table 6. Therefore, CGDD appears to play a role in ceU proMferative, autoimmune, developmental, and reproductive disorders. In the treatment of disorders associated with increased CGDD expression or activity, it is desirable to decrease the expression or activity of CGDD. In the treatment of disorders associated with decreased CGDD expression or activity, it is desirable to increase the expression or activity of CGDD.
  • CGDD or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CGDD.
  • disorders include, but are not Mmited to, a ceU proMferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangMa, gastrointestinal tract, heart,
  • a vector capable of expressing CGDD or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CGDD including, but not Mmited to, those described above.
  • compositions comprising a substantiaUy purified CGDD in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CGDD including, but not Mmited to, those provided above.
  • an agonist which modulates the activity of CGDD may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CGDD including, but not Mmited to, those Msted above.
  • an antagonist of CGDD may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CGDD. Examples of such disorders include, but are not Mmited to, those ceU proMferative, autoimmune, developmental, and reproductive disorders described above.
  • an antibody which specifically binds CGDD may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express CGDD.
  • a vector expressing the complement of the polynucleotide encoding CGDD may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CGDD including, but not Mmited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of CGDD may be produced using methods which are generaUy known in the art.
  • purified CGDD may be used to produce antibodies or to screen Mbraries of pharmaceutical agents to identify those which specificaUy bind CGDD.
  • Antibodies to CGDD may also be generated using methods that are weU known in the art. Such antibodies may include, but are not Mmited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Mbrary.
  • NeutraMzing antibodies i.e., those which inhibit di er formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with CGDD or with any fragment or oMgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Mmited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • the oMgopeptides, peptides, or fragments used to induce antibodies to CGDD have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oMgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of CGDD amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to CGDD may be prepared using any technique which provides for the production of antibody molecules by continuous ceU Mnes in culture. These include, but are not Mmited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique.
  • Mmited to the hybridoma technique
  • human B-ceU hybridoma technique the human B-ceU hybridoma technique
  • EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. hnmunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuMn Mbraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuMn Mbraries or panels of highly specific binding reagents as disclosed in the Mterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for CGDD may also be generated.
  • such fragments include, but are not Mmited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression Mbraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with estabMshed specificities are weU known in the art.
  • Such immunoassays typicaUy involve the measurement of complex formation between CGDD and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CGDD epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentiation of CGDD-antibody complex divided by the molar concentrations of free antigen and free antibody under equiMbrium conditions.
  • K a association constant
  • the K- determined for a preparation of monoclonal antibodies, which are monospecific for a particular CGDD epitope, represents a true measure of affinity.
  • High-affinity antibody preparations with K- ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the CGDD- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 s to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of CGDD, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • the titer and avidity of polyclonal antibody preparations may be -further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstream appMcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of CGDD-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guideUnes for antibody quaMty and usage in various appMcations, are generaUy available. (See, e.g., Catty, supra, and CoMgan et al.
  • the polynucleotides encoding CGDD may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oMgonucleotides) to the coding or regulatory regions of the gene encoding CGDD.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oMgonucleotides
  • antisense oMgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CGDD. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press Inc., Totawa NJ.)
  • Antisense sequences can be deUvered intiaceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein.
  • Slater J.E. et al. (1998) J. AUergy CMn. Immunol. 102(3):469-475; and Scanlon, KJ. et al.
  • Antisense sequences can also be intioduced intiaceUularly through the use of viral vectors, such as retiovirus and adeno-associated virus vectors.
  • viral vectors such as retiovirus and adeno-associated virus vectors.
  • Other gene deUvery mechanisms include Mposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • polynucleotides encoding CGDD may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCED)-Xl disease characterized byX- Mnked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
  • SCED severe combined immunodeficiency
  • ADA adenosine deaminase
  • hepatitis B or C vims HBV, HCV
  • fungal parasites such as Candida albicans and Paracoccidioides brasiMensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi.
  • the expression of CGDD from an appropriate population of transduced ceUs may aUeviate the cMnical manifestations caused by the genetic deficiency.
  • CGDD are treated by constructing mammaMan expression vectors encoding CGDD and introducing these vectors by mechanical means into CGDD-deficient cells.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) ballistic gold particle deUvery, (iii) Mposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA tiansposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
  • Expression vectors that may be effective for the expression of CGDD include, but are not
  • PCDNA 3.1 EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCREPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • CGDD may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 viras, thymidine kinase (TK), or ⁇ -actin genes), (n) an inducible promoter (e.g., the tetracycMne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.N. and H.M. Blau (1998) Curr. Opin.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 viras, thymidine kinase (TK), or ⁇ -act
  • CommerciaUy available Mposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
  • aUow one with ordinary skill in the art to deUver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammaUan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to CGDD expression are treated by constructing a retiovirus vector consisting of (i) the polynucleotide encoding CGDD under the control of an independent promoter or the retiovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (Mi) a Rev-responsive element (RRE) along with additional retiovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retiovirus vectors e.g., PFB and PFBNEO
  • PFB and PFBNEO are commerciaUy available (Stratagene) and are based onpubMshed data (Riviere, I. et al. (1995) Proc.
  • the vector is propagated in an appropriate vector producing ceU Mne (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. MiUer (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol.
  • VPCL ceU Mne
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retiovirus packaging ceU Mnes and is hereby incorporated by reference. Propagation of retiovirus vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been weU documented (Ranga, U.
  • an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding CGDD to ceUs which have one or more genetic abnormaUties with respect to the expression of CGDD.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art.
  • RepMcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No.
  • Adadenovirus vectors for gene therapy hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P. A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
  • a herpes-based, gene therapy deUvery system is used to deMver polynucleotides encoding CGDD to target ceUs which have one or more genetic abnormaUties with respect to the expression of CGDD.
  • the use of herpes simplex virus (HSV)-based vectors may be especiaUy valuable for intioducing CGDD to ceUs of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are weU known to those with ordinary skiU in the art.
  • HSV herpes simplex viras
  • 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163 : 152-161 , hereby incorporated by reference.
  • herpesviras sequences The manipulation of cloned herpesviras sequences, the generation of recombinant viras foUowing the transfection of multiple plasmids contair ⁇ ng different segments of the large herpesviras genomes, the growth and propagation of herpesviras, and the infection of ceUs with herpesviras are techniques weU known to those of ordinary skiU in the art.
  • an alphaviras (positive, single-stranded RNA virus) vector is used to deMver polynucleotides encoding CGDD to target ceUs.
  • SFV SemMki Forest Virus
  • SFV SemMki Forest Virus
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for CGDD into the alphaviras genome in place of the capsid-coding region results in the production of a large number of CGDD-coding RNAs and the synthesis of high levels of CGDD in vector transduced ceUs.
  • alphaviras infection is typically associated with ceU lysis within a few days
  • the abiUty to estabMsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis viras (SIN) indicates that the lytic repMcation of alphavirases can be altered to suit the needs of the gene therapy appMcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphavirases will aUow the introduction of CGDD into a variety of ceU types.
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphavirases, performing alphaviras cDNA and RNA tiansfections, and performing alphaviras infections, are weU known to those with ordinary skiU in the art.
  • OMgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heMx base-pairing methodology.
  • Triple heMx pairing is useful because it causes inhibition of the abiMty of the double heMx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the Mterature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and hnmunologic Approaches. Futura PubMshing, Mt. Kisco NY, pp. 163-177.)
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of sequences encoding CGDD.
  • RNA sequences of between 15 and 20 ribonucleotides may be evaluated for secondary structural features which may render the oMgonucleotide inoperable.
  • the suitabiMty of candidate targets may also be evaluated by testing accessibiMty to hybridization with complementary oMgonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oUgonucleotides such as soMd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding CGDD. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU Unes, ceUs, or tissues.
  • RNA molecules may be modified to increase intracellular stabiUty and half-Mfe. Possible modifications include, but are not Mmited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase Mnkages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding CGDD.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not Mmited to, oMgonucleotides, antisense oMgonucleotides, triple heMx-forming oMgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encoding CGDD may be therapeuticaUy useful, and in the treatment of disorders associated with decreased CGDD expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding CGDD may be therapeuticaUy useful.
  • At least one, and up to a pluraUty, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary Mbrary of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a Ubrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding CGDD is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding CGDD are assayed by any method commonly known in the art.
  • TypicaUy the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding CGDD.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, GM. et al. (2000) Nucleic Acids Res. 28.E15) or a human ceU Mne such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
  • a particular embodiment of the present invention involves screening a combinatorial Mbrary of oMgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oMgonucleotides) for antisense activity against a specific polynucleotide sequence (Braice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Braice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
  • oMgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oMgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeUvery by transfection, by Mposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • Any of the therapeutic methods described above may be appMed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the administration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubMshing, Easton PA).
  • Such compositions may consist of CGDD, antibodies to CGDD, and mimetics, agonists, antagonists, or inhibitors of CGDD.
  • compositions utiMzed in this invention may be administered by any number of routes including, but not Mmited to, oral, intravenous, intramuscular, intia-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subUngual, or rectal means.
  • compositions for pulmonary administiation may be prepared in Mquid or dry powder form. These compositions are generaUy aerosoMzed immediately prior to inhalation by the patient.
  • aerosol deUvery of fast- acting formulations is weU-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary deMvery has the advantage of administration without needle injection, and obviates the need for potentiaUy toxic penetration enhancers.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is weU within the capabiUty of those skiUed in the art.
  • SpeciaMzed forms of compositions may be prepared for direct intiaceUular deMvery of macromolecules comprising CGDD or fragments thereof.
  • Mposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intiaceUular deMvery of the macromolecule.
  • CGDD or a fragment thereof may be joined to a short cationic N- terminal portion from the H_V Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • ceU culture assays e.g., of neoplastic ceUs
  • animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administiation. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example CGDD or fragments thereof, antibodies of CGDD, and agonists, antagonists or inhibitors of CGDD, which ameUorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Mttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route
  • Dosage and administiation are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
  • Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drag combination(s), reaction sensitivities, and response to therapy.
  • Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administiation.
  • Guidance as to particular dosages and methods of deUvery is provided in the Uterature and generaUy available to practitioners in the art. Those skiUed in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deMvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specificaUy bind CGDD may be used for the diagnosis of disorders characterized by expression of CGDD, or in assays to monitor patients being treated with CGDD or agonists, antagonists, or inhibitors of CGDD.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CGDD include methods which utiUze the antibody and a label to detect CGDD in human body fluids or in extracts of ceUs or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • CGDD neurotrophic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor-dependent cytoplasmic factor, for example, antibodies to CGDD under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of CGDD expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • the polynucleotides encoding CGDD may be used for diagnostic purposes.
  • the polynucleotides which may be used include oUgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of CGDD may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of CGDD, and to monitor regulation of CGDD levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CGDD or closely related molecules may be used to identify nucleic acid sequences which encode CGDD.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampMfication will determine whether the probe identifies only naturaUy occurring sequences encoding CGDD, aUeMc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the CGDD encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO:6-10 or from genomic sequences including promoters, enhancers, and introns of the CGDD gene.
  • Means for producing specific hybridization probes for DNAs encoding CGDD include the cloning of polynucleotide sequences encoding CGDD or CGDD derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the ait, are commerciaUy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucMdes such as 32 P or 35 S, or by enzymatic labels, such as alkaMne phosphatase coupled to the probe via avidin/biotin coupMng systems, and the Mke.
  • Polynucleotide sequences encoding CGDD may be used for the diagnosis of disorders associated with expression of CGDD.
  • a ceU proMferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangMa, gastrointestinal tract, heart, kidney, Mver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saMvary glands, skin, sple
  • the polynucleotide sequences encoding CGDD may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Mke assays; and in microarrays utiMzing fluids or tissues from patients to detect altered CGDD expression.
  • Such quaMtative or quantitative methods are weU known in the art.
  • the nucleotide sequences encoding CGDD may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding CGDD may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding CGDD in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in cUnical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabMshed. This may be accompMshed by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CGDD, under conditions suitable for hybridization or ampUfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual cMnical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earUer thereby preventing the development or further progression of the cancer.
  • oUgonucleotides designed from the sequences encoding CGDD may involve the use of PCR. These oMgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OUgomers will preferably contain a fragment of a polynucleotide encoding CGDD, or a fragment of a polynucleotide complementary to the polynucleotide encoding CGDD, and wiU be employed under optimized conditions for identification of a specific gene or condition. OMgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oMgonucleotide primers derived from the polynucleotide sequences encoding CGDD may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not Mmited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oMgonucleotide primers derived from the polynucleotide sequences encoding CGDD are used to ampMfy DNA using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the Mke.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oMgonucleotide primers are fluorescently labeled, which aUows detection of the ampUmers in high-throughput equipment such as DNA sequencing machines.
  • sequence database analysis methods termed in siUco SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • Methods which may also be used to quantify the expression of CGDD include radiolabeUng or biotinylating nucleotides, coampMfication of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oMgomer or polynucleotide of interest is presented in various dilutions and a spectiOphotometiic or colorimetric response gives rapid quantitation.
  • oMgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • CGDD CGDD
  • the microarray may be used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, expressly incorporated by reference herein.)
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totaUty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraMty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU Unes, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU Mne.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and precMnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental compounds.
  • AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein).
  • a test compound has a signature similar to that of a compound with known toxicity, it is Mkely to share those toxic properties.
  • These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene famiMes.
  • IdeaUy a genome- wide measurement of expression provides tlie highest quaMty signature. Even genes whose expression is not altered by any tested compounds are important as weU, as the levels of expression of these genes are used to normaMze the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity.
  • the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or cell type.
  • proteome can be subjected individuaUy to further analysis.
  • Proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
  • a profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectiic focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuaUzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for CGDD to quantify the levels of CGDD expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reMable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the tieated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the tieated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
  • nucleic acid sequences encoding CGDD may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
  • Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (B ACs), bacterial PI constructions, or single chromosome cDNA Ubraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • B ACs bacterial artificial chromosomes
  • bacterial PI constructions or single chromosome cDNA Ubraries.
  • nucleic acid sequences of the invention may be used to develop genetic Mnkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymo ⁇ hism (RFLP).
  • RFLP restriction fragment length polymo ⁇ hism
  • Fluorescent in situ hybridization may be correlated with other physical and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online MendeMan Inheritance in Man (OMEvI) World Wide Web site. Correlation between the location of the gene encoding CGDD on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
  • placement of a gene on the chromosome of another mammaMan species, such as mouse may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • Once the gene or genes responsible for a disease or syndrome have been crudely locaMzed by genetic Mnkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to tianslocation, inversion, etc., among normal, carrier, or affected individuals.
  • CGDD its catalytic or immunogenic fragments, or oMgopeptides thereof can be used for screening Mbraries of compounds in any of a variety of drag screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soMd support, borne on a ceU surface, or located intiaceUularly. The formation of binding complexes between CGDD and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different smaU test compounds are synthesized on a soMd substrate.
  • the test compounds are reacted with CGDD, or fragments thereof, and washed.
  • Bound CGDD is then detected by methods weU known in the art.
  • Purified CGDD can also be coated directly onto plates for use in the aforementioned drag screening techniques.
  • non-neutiaUzing antibodies can be used to capture the peptide and immobiMze it on a soMd support.
  • nucleotide sequences which encode CGDD may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Mmited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Mbraries described in the LE ⁇ SEQ GOLD database (hicyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A)+ RNA was isolated using oMgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stiatagene was provided with RNA and constructed the corresponding cDNA Mbraries. Otherwise, cDNA was synthesized and cDNA Mbraries were constructed with the UNIZAP vector system (Stiatagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the ait. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oMgo d(T) or random primers. Synthetic oMgonucleotide adapters were Mgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were Mgated into compatible restriction enzyme sites of the polyMnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stiatagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.
  • a suitable plasmid e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-
  • coM ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stiatagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies.
  • Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stiatagene) or by ceU lysis.
  • Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophiUzation, at 4°C
  • plasmid DNA was ampUfied from host ceU lysates using direct Mnk PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycMng steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampUfied plasmid DNA was quantified fiuorometiicaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Incyte cDNA recovered in plasmids as described in Example ⁇ were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppMed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or suppMed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppMed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGAB ACE 1000 DNA sequencing system (Molecular Dynamics) ; the ABI PRISM 373 or 377 sequencing system (AppMed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.1). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VHI.
  • the polynucleotide sequences derived from Incyte cDNAs were vaMdated by removing vector, Mnker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubMc databases such as the GenBank primate, rodent, mammaMan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • pubMc databases such as the GenBank primate, rodent, mammaMan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM
  • PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus
  • HMM is a probabiMstic approach which analyzes consensus primary stractures of gene famiMes. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce fuU lengthpolynucleoti.de sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to full length.
  • FuU length polypeptide sequences were translated to derive the corresponding fuU length polypeptide sequences.
  • a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aMgnments are generated using default parameters specified by the CLUSTAL algorithm as inco ⁇ orated into the MEGALIGN multisequence aMgnment program (DNASTAR), which also calculates the percent identity between aMgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides appMcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where appUcable, the scores, probabiMty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiMty value, the greater the identity between two sequences).
  • Genscan is a general-pvupose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. KarUn (1997) J. Mol. Biol. 268:78-94, and Burge, C and S. KarUn (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • Genscan is a FASTA database ofpolynucleoti.de and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
  • the encoded polypeptides were analyzed by querying against PFAM models for proteins associated with ceU growth, differentiation, and death. Potential proteins associated with ceU growth, differentiation, and death were also identified by homology to Incyte cDNA sequences that had been annotated as proteins associated with ceU growth, differentiation, and death.
  • Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubMc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or pubMc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • FuU length polynucleotide sequences were obtained by assembUng Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubMc cDNA sequences using the assembly process described in Example m. Alternatively, fuU length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a f ⁇ U length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by tiansitivity.
  • GenBank protein homolog the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubMc human genome databases. Partial DNA sequences were therefore "stietched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of CGDD Encoding Polynucleotides
  • sequences which were used to assemble SEQ ID NO:6-10 were compared with sequences from the Incyte LIFESEQ database and pubMc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ED NO:6-10 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubMc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of aU sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • pubMc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound.
  • a membrane on which RNAs from a particular ceU type or tissue have been bound See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.
  • Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
  • the basis of the search is the product score, which is defined as:
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quaUty in a BLAST aMgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding CGDD are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI). Each cDNA sequence is derived from a cDNA Mbrary constructed from a human tissue.
  • Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic stractures; endocrine system; exocrine glands; genitaMa, female; genitaMa, male; germ ceUs; hemic and immune system; Mver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Mbraries in each category is counted and divided by the total number of Mbraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU Une, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Mbraries across aU categories. The resulting percentages reflect the tissue-* and disease-specific expression of cDNA encoding CGDD.
  • cDNA sequences and cDNA Ubrary/tissue information are found in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hai ⁇ in stractures and primer-primer dimerizations was avoided.
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ were as foUows: Step 1 : 94 °C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 1
  • the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentiation of DNA.
  • Step 1 94°C, 3 min
  • Step 2 94°C, 15 sec
  • Step 3 60°C, 1 min
  • Step 4 72°C, 2 min
  • Step 5 steps 2, 3, and 4 repeated 29 times
  • Step 6 72°C, 5 min
  • Step 7 storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampMfied using the same conditions as described above.
  • fuU length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oMgonucleotides designed for such extension, and an appropriate genomic Mbrary.
  • Hybridization probes derived from SEQ ID NO:6-10 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeMng of oMgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oMgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine tiiphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oMgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
  • An aMquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl H, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saUne sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaMzed using autoradiography or an alternative imaging means and compared. X. Microarrays
  • the Mnkage or synthesis of array elements upon a microarray can be achieved utilizing photoMthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and soMd with a non-porous surface (Schena (1999), supra). Suggested substiates include siMcon, siMca, glass sMdes, glass chips, and siMcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and Mnk elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines weU known to those of ordinary skiU in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oMgomers thereof may comprise the elements of the microarray. Fragments or oMgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oMgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse tianscribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oUgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMB RIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeUng) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampMfied from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR ampMfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampMfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • AmpMfied array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified array elements are immobiMzed on polymer-coated glass sMdes.
  • Glass microscope sMdes (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated sUdes are cured in a 110°C oven.
  • Array elements are appUed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, inco ⁇ orated herein by reference.
  • 1 ⁇ l of the array element DNA is loaded into the open capiUary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per sUde.
  • Microarrays are UV-crossUnked using a STRATALINKER UV-crossMnker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saUne (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distiUed water as before.
  • PBS phosphate buffered saUne
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aUquoted onto the microarray surface and covered with an 1.8 cm 2 coversMp.
  • the arrays are transferred to a wate ⁇ roof chamber having a cavity just sMghtly larger than a microscope sUde.
  • the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60° C
  • the arrays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1 % SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral Unes at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Ught is focused on the array using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the sMde containing the array is placed on a computer-contioUed X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipUer tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is typicaUy scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typicaUy caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentiation.
  • a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the caUbration is done by labeUng samples of the caMbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipUer tube is digitized using a 12-bit RTI-835H analog-to-digital
  • the digitized data are displayed as an image where the signal intensity is mapped using a Unear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
  • Sequences complementary to the CGDD-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring CGDD.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oMgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CGDD.
  • a complementary oMgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oMgonucleotide is designed to prevent ribosomal binding to the CGDD-encoding transcript.
  • CGDD CGDD expression and purification of CGDD is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not Mmited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are tiansformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express CGDD upon induction with isopropyl beta-D- thiogalactopyranoside (EPTG).
  • CGDD CGDD in eukaryotic ceUs
  • AcMNPV Autographica caMfornica nuclear polyhedrosis viras
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CGDD by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA tianscription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus.
  • Spodoptera frugiperda Sf9 insect ceUs
  • human hepatocytes in some cases. Infection of the latter requires additional genetic modifications to baculovirus.
  • CGDD is synthesized as a fusion protein with, e.g., glutathione S- tiansferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST a 26-kilodalton enzyme from Schistosoma iaponicum. enables the purification of fusion proteins on immobiUzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
  • the GST moiety can be proteolyticaUy cleaved from CGDD at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide
  • 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified CGDD obtained by these methods can be used directly in the assays shown in Examples XVI and XVII, where appUcable. XIII. Functional Assays
  • CGDD function is assessed by expressing the sequences encoding CGDD at physiologicaUy elevated levels in mammaUan ceU culture systems.
  • cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Mne, for example, an endotheUal or hematopoietic ceU Mne, using either Mposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs from nontiansfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Mght scatter and 90 degree side Mght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intiaceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
  • CGDD The influence of CGDD on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding CGDD and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected ceUs are efficiently separated from nontiansfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding CGDD and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • the CGDD amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions are weU described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • oMgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppMed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-m-dei---Mdobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MFS N-m-dei---Mdobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oMgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-CGDD activity by, for example, binding the peptide or CGDD to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • CGDD Naturally Occurring CGDD Using Specific Antibodies
  • CGDD is substantiaUy purified by irnmunoaffinity chromatography using antibodies specific for CGDD.
  • An irnmunoaffinity column is constructed by covalently coupMng anti-CGDD antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupMng, the resin is blocked and washed according to the manufacturer's instructions.
  • activated chromatographic resin such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
  • CGDD Media containing CGDD are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of CGDD (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/CGDD binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and CGDD is coUected.
  • Candidate molecules previously arrayed in the weUs of a multi- weU plate are incubated with the labeled CGDD, washed, and any weUs with labeled CGDD complex are assayed. Data obtained using different concentrations of CGDD are used to calculate values for the number, affinity, and association of CGDD with the candidate molecules.
  • molecules interacting with CGDD are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • CGDD may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • CGDD activity is demonstrated by measuring the induction of ceU cycle progression when CGDD is expressed at physiologicaUy elevated levels in mammaUan cell culture systems.
  • CGDD cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORTTM (Life Technologies, Gaithersburg, MD) and pCRTM 3.1 (Invitrogen, Carlsbad, CA), both of which contain the cytomegalo viras promoter.
  • recombinant vector 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, preferably of endotheUal or hematopoietic origin, using either Mposome formulations or electroporation. 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected ceUs from nontiansfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto, CA).
  • GFP Green Fluorescent Protein
  • Flow cytometry detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU cycle progression or terminal differentiation. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Mght scatter and 90 degree side Mght scatter; up or down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intiaceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface.
  • an assay for CGDD activity measures ceU proMferation as the amotmt of newly initiated DNA synthesis in Swiss mouse 3T3 ceUs.
  • a plasmid containing polynucleotides encoding CGDD is transfected into quiescent 3T3 cultured ceUs using methods weU known in the art. The transiently transfected ceUs are then incubated in the presence of [ 3 H]thymidine or a radioactive DNA precursor such as [ 32 P]ATP.
  • varying amounts of CGDD Ugand are added to the transfected ceUs. Inco ⁇ oration of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval, and the amotmt inco ⁇ orated is directly proportional to the amount of newly synthesized DNA and CGDD activity.
  • CGDD activity is measured by the cycMn-ubiquitin Mgation assay (Townsley, F. M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2362-2367).
  • the reaction contains in a volume of 10 ⁇ l, 40 mM Tris.HCl (pH 7.6), 5 mM Mg Cl 2 , 0.5 mM ATP, 10 mM phosphocreatine, 50 ⁇ g of creatine phosphokinase/ml, 1 mg reduced carboxymethylated bovine serum albumin ml, 50 ⁇ M ubiquitin, 1 ⁇ M ubiquitin aldehyde, 1-2 pmol 125 I-labeled cycMn B, 1 pmol El, 1 ⁇ M okadaic acid, 10 ⁇ g of protein of M-phase fraction 1 A (containing active E3-C and essentiaUy free of E2-C), and varying amounts of CGDD.
  • the reaction is incubated at 18 °C for 60 minutes. Samples are then separated by electrophoresis on an SDS polyacrylamide gel. The amount of 125 I- cycMn-ubiquitin formed is quantified by Phosphorhnager analysis. The amount of cycMn-ubiquitin formation is proportional to the activity of CGDD in the reaction.
  • an assay for CGDD activity measures the induction of apoptosis when CGDD is expressed at physiologicaUy elevated levels in mammaUan ceU culture systems.
  • cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORT (Life Technologies, Gaithersburg MD) and pCR 3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegaloviras promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, preferably of endotheUal or hematopoietic origin, using either Mposome formulations or electroporation. 1 -2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-tiansfected.
  • marker protein provides a means to distinguish transfected ceUs from nontiansfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto CA), CD64, or a CD64-GFP fusion protein.
  • GFP Green Fluorescent Protein
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death.
  • These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward Ught scatter and 90 degree side Ught scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intiaceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface.
  • an assay for CGDD activity uses radiolabeled nucleotides, such as [ ⁇ 32 P] ATP, to measure either the incorporation of radiolabel into DNA during DNA synthesis, or fragmentation of DNA that accompanies apoptosis.
  • radiolabeled nucleotides such as [ ⁇ 32 P] ATP
  • MammaUan cells are transfected with plasmid containing cDNA encoding CGDD by methods weU known in the art. Cells are then incubated with radiolabeled nucleotide for various lengths of time. Chromosomal DNA is coUected, and radioactivity is detected using a scintiUation counter. Inco ⁇ oration of radiolabel into chromosomal DNA is proportional to the degree of stimulation of the ceU cycle.
  • chromosomal DNA is collected as above, and analyzed using polyacrylamide gel electrophoresis, by methods well known in the art. Fragmentation of DNA is quantified by comparison to untransfected control ceUs, and is proportional to the apoptotic activity of CGDD.
  • an in vitro assay for CGDD activity measures the transformation of normal human fibroblast ceUs overexpressing antisense CGDD RNA.
  • cDNA encoding CGDD is subcloned into the PLNCX retioviral vector to enable expression of antisense CGDD RNA.
  • the resulting constract is tiansfected into the ecotiopic BOSC23 virus-packaging ceU Une.
  • Viras contained in the BOSC23 culture supernatant is used to infect the amphotropic CAK8 viras-packaging ceU Mne.
  • Virus contained in the CAK8 culture supernatant is used to infect normal human fibroblast (Hs68) ceUs.
  • Infected ceUs are assessed for the foUowing quantifiable properties characteristic of tiansformed ceUs: growth in culture to high density associated with loss of contact inhibition, growth in suspension or in soft agar, formation of colonies or foci, lowered serum requirements, and abiUty to induce tumors when injected into immunodeficient mice.
  • the activity of CGDD is proportional to the extent of transformation of Hs68 ceUs.
  • the abiUty of CGDD to suppress tumorigenesis can be demonstrated by designing an antisense sequence to the 5' end of the gene and transfecting NIH 3T3 ceUs with a vector transcribing this sequence.
  • the suppression of the endogenous gene wiU allow tiansformed fibroblasts to produce clumps of ceUs capable of forming metastatic tumors when introduced into nude mice.

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Abstract

L'invention concerne des protéines humaines associées à la croissance, à la différenciation et à la mort cellulaire (CGDD) et des polynucléotides identifiant et codant CGDD. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention concerne également des méthodes de diagnostic, de traitement ou de prévention de troubles associés à une expression anormale de CGDD.
PCT/US2001/047871 2000-12-14 2001-12-11 Proteines associees a la croissance, a la differenciation et a la mort cellulaire Ceased WO2002048368A2 (fr)

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CA002430906A CA2430906A1 (fr) 2000-12-14 2001-12-11 Proteines associees a la croissance, a la differenciation et a la mort cellulaire
AU2002227373A AU2002227373A1 (en) 2000-12-14 2001-12-11 Proteins associated with cell growth, differentiation, and death
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003027263A3 (fr) * 2001-09-28 2003-11-06 Incyte Genomics Inc Proteines associees a la croissance, a la differenciation et a la mort des cellules
EP1399474A2 (fr) * 2000-12-15 2004-03-24 Eli Lilly And Company Nouvelles proteines secretees et leurs utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUANG SUI ET AL: "Shape-dependent control of cell growth, differentiation, and apoptosis: Switching between attractors in cell regulatory networks" EXPERIMENTAL CELL RESEARCH, SAN DIEGO, CA, US, vol. 261, no. 1, 25 November 2000 (2000-11-25), pages 91-103, XP002205154 ISSN: 0014-4827 *
LOMAX MI ET AL: "Differential display and gene arrays to examine auditory plasticity" HEARING RESEARCH, vol. 147, - September 2000 (2000-09) pages 293-302, XP002220427 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1399474A2 (fr) * 2000-12-15 2004-03-24 Eli Lilly And Company Nouvelles proteines secretees et leurs utilisations
WO2003027263A3 (fr) * 2001-09-28 2003-11-06 Incyte Genomics Inc Proteines associees a la croissance, a la differenciation et a la mort des cellules

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