WO2004085625A2 - 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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WO2004085625A2
WO2004085625A2 PCT/US2004/009388 US2004009388W WO2004085625A2 WO 2004085625 A2 WO2004085625 A2 WO 2004085625A2 US 2004009388 W US2004009388 W US 2004009388W WO 2004085625 A2 WO2004085625 A2 WO 2004085625A2
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polynucleotide
seq
polypeptide
amino acid
sequence
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PCT/US2004/009388
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WO2004085625A3 (fr
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Joseph P. Marquis
Uyen K. Tran
Yonghong G. Yang
Tom Y. Tang
Narinder K. Chawla
Jagi Murage
Jonathan T. Wang
Vicki S. Elliott
David Chien
Henry Yue
Yalda Azimzai
Craig H. Ison
Reena Khare
Pei Jin
Jayalaxmi Ramkumar
Kristin D. Favero
Thomas W. Richardson
April J.A. Hafalia
Mariah R. Baughn
Shanya D. Becha
Amy D. Wilson
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Incyte Corporation
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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

Definitions

  • the invention relates to novel nucleic acids, proteins associated with cell growth, differentiation, and death encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell proliferative disorders including cancer, developmental disorders, neurological disorders, autoimmune/inflammatory disorders, reproductive disorders, and disorders of the placenta.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and 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. 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.
  • 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.
  • 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.
  • Mitosis marks the end of interphase and concludes with the onset of cytokinesis.
  • Prophase includes the formation of bi-polar mitotic spindles, composed of microtubules and associated proteins such as dynein, which originate from polar mitotic centers.
  • metaphase the nuclear material condenses and develops kinetochore fibers which aid in its physical attachment to the mitotic spindles.
  • Telophase includes the disappearance of the mitotic spindles and kinetochore fibers from the nuclear material.
  • Mitosis depends on the interaction of numerous proteins.
  • centromere-associated proteins such as CENP-A, -B, and -C, play structural roles in kinetochore formation and assembly (Saffery, R. et al. (2000) Human Mol. Gen. 9:175-185).
  • M phase phosphorylation a component of U3 small nucleolar ribonucleoprotein (snoRNP), and relocalize to the nucleolus once mitosis is complete (Westendorf, J.M. et al. (1998) J. Biol. Chem.
  • U3 snoRNPs are essential mediators of RNA processing events. Proteins involved in the regulation of cellular processes such as mitosis include the Ser/Thr- protein phosphatases type 1 (PP-1). PP-ls act by dephosphorylation of key proteins involved in the metaphase-anaphase transition.
  • the gene PP1R7 encodes the regulatory polypeptide sds22, having at least six splice variants (Ceulemans, H. et al. (1999) Eur. J. Biochem. 262:36-42). Sds22 modulates the activity of the catalytic subunit of PP-ls, and enhances the PP-1 -dependent dephosphorylation of mitotic substrates.
  • mMOB 1 A recently identified protein, is the mammalian homolog of yeast MOB 1 , an essential yeast gene required for completion of mitosis and maintenance of ploidy.
  • the mammalian mMOB 1 is a member of protein complexes including protein phosphatase 2A (PP2A), and its phosphorylation appears to be regulated by PP2A (Moreno, C.S. et al. (2001) J. Biol. Chem. 276:24253-24260).
  • PP2A has been implicated in the development of human cancers, including lung and colon cancers and leukemias.
  • Cell cycle regulation involves numerous proteins interacting in a sequential manner.
  • the eukaryotic cell cycle consists of several highly controlled events whose precise order ensures successful DNA replication and cell division. Cells maintain the order of these events by making later events dependent on the successful completion of earlier events. This dependency is enforced by cellular mechanisms called checkpoints.
  • HDACs histone deacetylases
  • HDACs are involved in cell cycle regulation, and modulate chromatin structure.
  • Human HDAC1 has been found to interact in vitro with the human Husl gene product, whose Schizosaccharomyces pombehomolog has been implicated in G 2 /M checkpoint control (Cai, R.L. et al. (2000) J. Biol. Chem. 275:27909-27916).
  • DNA damage (G ⁇ and DNA replication (S -phase) checkpoints arrest eukaryotic cells at the G 2 /M transition. This arrest provides time for DNA repair or DNA replication to occur before entry into mitosis. Thus, the G 2 M checkpoint ensures that mitosis only occurs upon completion of DNA replication and in the absence of chromosomal damage.
  • the Husl gene of Schizosaccharomyces pombe is a cell cycle checkpoint gene, as are the rad family of genes (e.g., radl and rad9) (Volkmer, E. and L.M. Kamitz (1999) J. Biol. Chem. 274:567-570; Kostrub C.F. et al. (1998) EMBO J. 17:2055-2066).
  • Cyclins act by binding to and activating a group of cyclin-dependent protein kinases (Cdks) which then phosphorylate 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 "cyclinbox" (Chapman, D.L. and D.J. Wolgemuth (1993) Development 118:229-240).
  • cyclins contain a conserved 9 amino acid sequence in the N-terminal region of the molecule called the "destruction box.” This sequence is believed to be a recognition code that triggers ubiquitin-mediated degradation of cyclin B (Hunt, T. (1991) Nature 349:100-101).
  • Several types of cyclins exist (Ciechanover, A. (1994) Cell 79:13-21). Progression through Gl and S phase is driven by the Gl cyclins and their catalytic subunits, including Cdk2-cyclin A, Cdk2-cyclin E, Cdk4-cyclin D and Cdk6-cyclin D.
  • Progression through the G2-M transition is driven by the activation of mitotic CDK-cyclin complexes such as Cdc2-cyclin A, Cdc2-cyclh Bl and Cdc2-cyclin B2 complexes (reviewed in Yang, J. and S. Kornbluth (1999) Trends Cell Biol. 9:207-210).
  • mitotic CDK-cyclin complexes such as Cdc2-cyclin A, Cdc2-cyclh Bl and Cdc2-cyclin B2 complexes
  • Human dual-specificity phosphatases CDC25 (A, B and C) play an important role in the control of cell cycle progression by activating the cyclin-dependent kinases (CDKs). Regulation of these phosphatases during the cell cycle involves post-translational modifications such as phosphorylation and protein-protein interactions (Theis-Febvre, N. et al. (2003) Oncogene
  • rhodanese homology domain found in human dual-specificity phosphatases CDC25 proteins.
  • the rhodanese homology domain forms the catalytic domain in phosphatases such as the human dual-specificity phosphatases CDC25 (Hofmann, K., Bucher, P., and Kajava, A.V. (1998) J. Mol. Biol. 282:195-208).
  • the enzyme rhodanese thiosulfate/cyanide sulfurtransferase, EC 2.8.1.1
  • EC 2.8.1.1 is an ubiquitous enzyme and its activity is present in all living organisms from bacteria to man.
  • mammalian rhodanese catalyzes the transfer of sulfane sulfur from donors such as S202-(3) and organic thiosulfonate anions (RS(0)2S-) to nucleophilic acceptors such as CN- and dithiols (Nandi, D.L. and Westley, J. (1998) Int. J. Biochem. Cell Biol. 30:973-977).
  • Cyclins are degraded through the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaroytic cells 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 cellular processes such as gene transcription and cell cycle progression.
  • the UCS is implicated in the degradation of mitotic cyclin kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell 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 small, 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.
  • Second, activated Ub is transferred to one of several Ub-conjugating enzymes (E2).
  • E2 Ub-conjugating enzymes
  • Different ubiquitin-dependent proteolytic pathways employ structurally 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 the proteasome, a large, multisubunit proteolytic enzyme complex, and Ub is released for re-utilization.
  • Ub Prior to activation, Ub is usually expressed as a fusion protein composed of an N-terminal ubiquitin and a C-terminal extension protein (CEP) or as a polyubiquitin protein with Ub monomers attached head to tail.
  • CEPs have characteristics of a variety of regulatory proteins; most are highly basic, contain up to 30% lysine and arginine residues, and have nucleic acid-binding domains (Monia, B.P. et al. (1989) J. Biol. Chem. 264:4093-4103).
  • the fusion protein is an important intermediate which appears to mediate co-regulation of the cell's translational and protein degradation activities, as well as localization of the inactive enzyme to specific cellular sites.
  • Ub-conjugating enzymes are important for substrate specificity in different UCS pathways. All E2s have a conserved domain of approximately 16 kDa called the UBC domain that is at least 35% identical in all E2s and contains a centrally located cysteine residue required for ubiquitin-enzyme thiolester formation (Jentsch, supra). A well 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 cellular localization. Class III 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.
  • E3 ligases is the HECT (homologous to E6-AP C-terminus) domain protein family.
  • E6-AP E6-associated protein
  • HPV human papillomavirus
  • the C-terminal domain of HECT proteins contains the highly conserved ubiquitin-binding cysteine residue.
  • the N-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. Natl Acad. Sci.
  • the SCF (Skpl-Cdc53/Cullin-Fbox receptor) family of proteins comprise another group of ubiquitin ligases (Deshaies, R. (1999) Annu. Rev. Dev. Biol. 15:435-467). Multiple proteins are recruited into the SCF complex, including Skpl, cullin, and an F box domain containing protein.
  • the F box protein binds the substrate for the ubiquitination reaction and may play roles in determining substrate specificity and orienting the substrate for reaction.
  • Skpl interacts with both the F box protein and cullin and may be involved in positioning the F box protein and cullin in the complex for transfer of ubiquitin from the E2 enzyme to the protein substrate.
  • Substrates of SCF ligases include proteins involved in regulation of CDK activity, activation of transcription, signal transduction, assembly of kinetochores, and DNA replication.
  • Sgtl was identified in a screen for genes in yeast that suppress defects in kinetochore function caused by mutations in Skpl (Kitagawa, K. et al. (1999) Mol. Cell 4:21-33). Sgtl interacts with Sk l and associates with SCF ubiquitin ligase. Defects in Sgtl cause arrest of cells at either Gl or G2 stages of the cell cycle. A yeast Sgtl null mutant can be rescued by human Sgtl , an indication of the conservation of Sgtl function across species. Sgtl is required for assembly of kinetochore complexes in yeast.
  • Abnormal activities of the UCS are implicated in a number of diseases and disorders. These include, e.g., cachexia (Llovera, M. et al. (1995) Int. J. Cancer 61:138-141), degradation of the tumor-suppressor protein, p53 (Ciechanover, supra), and neurodegeneration such as observed in Alzheimer's disease (Gregori, L. et al. (1994) Biochem. Biophys. Res. Commun. 203:1731-1738). Since ubiquitin conjugation is a rate-limiting step in antigen presentation, the ubiquitin degradation pathway may also have a critical role in the immune response (Grant, E.P. et al. (1995) J. Immunol. 155:3750-3758).
  • Certain cell proliferation disorders can be identified by changes in the protein complexes that normally control progression through the cell cycle.
  • a primary treatment strategy involves reestablishing control over cell cycle progression by manipulation of the proteins involved in cell cycle regulation (Nigg, E.A. (1995) BioEssays 17:471-480).
  • Mammalian embryogenesis is a process which encompasses the first few weeks of development following conception. During this period, embryogenesis proceeds from a single fertilized egg to the formation of the three embryonic tissues, then to an embryo which has most of its internal organs and all of its external features.
  • the normal course of mammalian embryogenesis depends on the correct temporal and spatial regulation of a large number of genes and tissues. These regulation processes have been intensely studied in mouse. An essential process that is still poorly understood is the activation of the embryonic genome after fertilization. As mouse oocytes grow, they accumulate transcripts that are either translated directly into proteins or stored for later activation by regulated polyadenylation. During subsequent meiotic maturation and ovulation, the maternal genome is transcriptionally inert, and most maternal transcripts are deadenylated and/or degraded prior to, or together with, the activation of the zygotic genes at the two-cell stage (Stutz, A. et al. (1998) Genes Dev. 12:2535- 2548). The maternal to embryonic transition involves the degradation of oocyte, but not zygotic transcripts, the activation of the embryonic genome, and the induction of cell cycle progression to accommodate early development.
  • MATER Major Antigen That Embryos Require
  • ovarian immunity Tong, Z-B. and L.M. Nelson (1999) Endocrinology 140:3720-3726.
  • Expression of the gene encoding MATER is restricted to the oocyte, making it one of a limited number of known maternal-effect genes in mammals (Tong, Z-B. et al. (2000) Mamm. Genome 11 :281-287).
  • the MATER protein is required for embryonic development beyond two cells, based upon preliminary results from mice in which this gene has been inactivated.
  • the 1111 -amino acid MATER protein contains a hydrophilic repeat region in the amino terminus, and a region containing 14 leucine-rich repeats in the carboxyl terminus. These repeats resemble the sequence found in porcine ribonuclease inhibitor that is critical for protein-protein interactions.
  • the degradation of maternal transcripts during meiotic maturation and ovulation may involve the activation of a ribonuclease just prior to ovulation.
  • the function of MATER may be to bind to the maternal ribonuclease and prevent degradation of zygotic transcripts (Tong et al., supra).
  • MATER may also be relevant to the pathogenesis of ovarian immunity, as it is a target of autoantibodies in mice with autoimmune oophoritis (Tong and Nelson, supra).
  • the maternal mRNA D7 is a moderately abundant transcript ⁇ nXenopus laevis whose expression is highest in, and perhaps restricted to, oogenesis and early embryogenesis.
  • D7 protein is absent from oocytes and first begins to accumulate during oocyte maturation. Its levels are highest during the first day of embryonic development and then they decrease. The loss of D7 protein affects the maturation process itself, significantly delaying the time course of germinal vesicle breakdown. Thus, D7 is a newly described protein involved in oocyte maturation (Smith, R.C. et al. (1988) Genes Dev. 2(10):1296-306.)
  • Implantation results from the action of trophoblast cells that develop over the surface of the blastocyst. These cells secrete proteolytic enzymes that digest and liquefy the cells of the endometrium.
  • the invasive process is reviewed in Fisher, S.J. and CH. Damsky (1993; Semin Cell Biol 4:183-188) and Graham, CH. and P.K. Lala (1992; Biochem Cell Biol 70:867-874).
  • the trophoblast and other sublying cells proliferate rapidly, forming the placenta and the various membranes of pregnancy. (See Guyton, A.C. (1991) Textbook of Medical Physiology, 8 th ed. W.B. Saunders Company, Philadelphia PA, pp.
  • the placenta has an essential role in protecting and nourishing the developing fetus.
  • the syncvtiotrophoblast layer is present on the outside of the placenta at the fetal-maternal interface. This is a continuous structure, one cell deep, formed by the fusion of the constituent trophoblast cells.
  • the syncvtiotrophoblast cells play important roles in maternal-fetal exchange, in tissue remodeling during fetal development, and in protecting the developing fetus from the maternal immune response (Stoye, J.P. and J.M. Coffin (2000) Nature 403:715-717).
  • syncytin is the envelope gene of a human endogenous defective provirus. Syncytin is expressed in high levels in placenta, and more weakly in testis, but is not detected in any other tissues (Mi, S. et al. (2000) Nature 403:785-789). Syncytin expression in the placenta is restricted to the syncytiotrophoblasts. Since retroviral env proteins are often involved in promoting cell fusion events, it was thought that syncytin might be involved in regulating the fusion of trophoblast cells into the syncytiotrophoblast layer.
  • syncytin can mediate cell fusion in vitro, and that anti-syncytin antibodies can inhibit the fusion of placental cytotrophoblasts (Mi et al., supra).
  • a conserved immunosuppressive domain present in retroviral envelope proteins, and found in syncytin at amino acid residues 373-397, might be involved in preventing maternal immune responses against the developing embryo.
  • Syncytin may also be involved in regulating trophoblast invasiveness by inducing trophoblast fusion and terminal differentiation (Mi et al., supra). Insufficient trophoblast infiltration of the uterine wall is associated with placental disorders such as preeclampsia, or pregnancy induced hypertension, while uncontrolled trophoblast invasion is observed in choriocarcinoma and other gestational trophoblastic diseases. Thus syncytin function may be involved in these diseases.
  • Multicellular organisms are comprised of diverse cell types that differ dramatically both in structure and function, despite the fact that each cell is like the others in its hereditary endowment.
  • Cell differentiation is the process by which cells come to differ in their structure and physiological function.
  • the cells of a multicellular organism all arise from mitotic divisions of a single-celled zygote.
  • the zygote is totipotent, meaning that it has the ability to give rise to every type of cell in the adult body.
  • the cellular descendants of the zygote lose their totipotency and become determined. Once its prospective fate is achieved, a cell is said to have differentiated. All descendants of this cell will be of the same type.
  • Human growth and development requires the spatial and temporal regulation of cell differentiation, along with cell proliferation and regulated cell death.
  • the processes involved in cell differentiation are also relevant to disease states such as cancer, in which case the factors regulating normal cell differentiation have been altered, allowing the cancerous cells to proliferate in an anaplastic, or undifferentiated, state.
  • the mechanisms of differentiation involve cell-specific regulation of transcription and translation, so that different genes are selectively expressed at different times in different cells.
  • Genetic experiments using the fruit fly Drosophila melanogaster have identified regulated cascades of transcription factors which control pattern formation during development and differentiation. These include the homeotic genes, which encode transcription factors containing homeobox motifs.
  • the products of homeotic genes determine how the insect's imaginal discs develop from masses of undifferentiated cells to specific segments containing complex organs.
  • Many genes found to be involved in cell differentiation and development in Drosophila have homologs in mammals. Some human genes have equivalent developmental roles to their Drosophila homologs.
  • the human homolog of the Drosophila eyes absent gene underlies branchio-oto-renal syndrome, a developmental disorder affecting the ears and kidneys (Abdelhak, S. et al. (1997) Nat. Genet. 15:157- 164).
  • the Drosophila slit gene encodes a secreted leucine-rich repeat containing protein expressed by the midline glial cells and required for normal neural development.
  • growth and development are governed by the cell's decision to enter into or exit from the cell cycle and by the cell's commitment to a terminally differentiated state.
  • Differential gene expression within cells is triggered in response to extracellular signals and other environmental cues.
  • signals include growth factors and other mitogens such as retinoic acid; cell-cell and cell-matrix contacts; and environmental factors such as nutritional signals, toxic substances, and heat shock.
  • Candidate genes that may play a role in differentiation can be identified by altered expression patterns upon induction of cell differentiation in vitro.
  • the final step in cell differentiation results in a specialization that is characterized by the production of particular proteins, such as contractile proteins in muscle cells, serum proteins in liver cells and globins in red blood cell precursors.
  • the expression of these specialized proteins depends at least in part on cell-specific transcription factors.
  • the homeobox-containing transcription factor PAX-6 is essential for early eye determination, specification of ocular tissues, and normal eye development in vertebrates.
  • differentiation-specific genes In the case of epidermal differentiation, the induction of differentiation-specific genes occurs either together with or following growth arrest and is believed to be linked to the molecular events that control irreversible growth arrest.
  • Irreversible growth arrest is an early event which occurs when cells transit from the basal to the innermost suprabasal layer of the skin and begin expressing squamous-specific genes.
  • These genes include those involved in the formation of the cross-linked envelope, such as tiansglutaminase I and III, involucrin, loricin, and small proline-rich repeat (SPRR) proteins.
  • SPRR proteins are 8-10 kDa in molecular mass, rich in proline, glutamine, and cysteine, and contain similar repeating sequence elements.
  • the SPRR proteins may be structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins.
  • the Wnt gene family of secreted signaling molecules is highly conserved throughout eukaryotic cells. Members of the Wnt family are involved in regulating chondrocyte differentiation within the cartilage template. Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb, Wnt-5a being expressed in the perichondrium (mesenchymal cells immediately surrounding the early cartilage template). Wnt-5a misexpression delays the maturation of chondrocytes and the onset of bone collar formation in chicken limb (Hartmann, C. and C.J. Tabin (2000) Development 127:3141-3159).
  • Glypicans are a family of cell surface heparan sulfate proteoglycans that play an important role in cellular growth control and differentiation. Cerebroglycan, a heparan sulfate proteoglycan expressed in the nervous system, is involved with the motile behavior of developing neurons (Stipp, C.S. et al. (1994) J. CeU Biol. 124:149-160).
  • Notch plays an active role in the differentiation of glial cells, and influences the length and organization of neuronal processes (for a review, see Frisen, J. and U. Lendahl (2001) Bioessays 23:3-7).
  • the Notch receptor signaling pathway is important for morphogenesis and development of many organs and tissues in multicellular species. Drosophila fringe proteins modulate the activation of the Notch signal transduction pathway at the dorsal- ventral boundary of the wing imaginal disc. Mammalian fringe-related family members participate in boundary determination during segmentation (Johnston, S.H. et al. (1997) Development 124:2245-2254).
  • LIM domain conserved cysteine-rich domain of about 60 amino-acid residues called the LIM domain (for Lin-11 Isl-1 Mec-3) (Freyd, G. et al. (1990) Nature 344:876-879; Baltz, R. et al. (1992) Plant CeU 4:1465-1466).
  • LIM domain there are seven conserved cysteine residues and a histidine.
  • the LIM domain binds two zinc ions (Michelsen, J.W. et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:4404-4408). LIM does not bind DNA; rather, it seems to act as an interface for protein-protein interaction. Apoptosis
  • Apoptosis is the geneticaUy controlled process by which unneeded or defective ceUs undergo programmed ceU death. Selective elimination of ceUs is as important for morphogenesis and tissue remodeling as is ceU prohferation and differentiation. Lack of apoptosis may result inhyperplasia 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 killer 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.
  • Apoptotic ceUs 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. The molecular mechanisms of apoptosis are highly conserved, and many of the key protein regulators and effectors of apoptosis have been identified. 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. Transcription factors also play an important role in the onset of apoptosis.
  • Bcl-2 family of proteins are key regulators of apoptosis.
  • Bcl-2 famUy proteins contain the BHl and BH2 domains, which are found in members of the pro-survival subfamily, while those proteins which are most similar to Bcl-2 have all four conserved domains, enabling inhibition of apoptosis following encounters with a variety of cytotoxic chaUenges.
  • Members of the pro-survival subfamily include Bcl-2, Bcl-x L , Bcl-w, Mcl-1, and Al in mammals; NF-13 (chicken); CED-9
  • 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 caUed Mtd); and Bik, Blk, Hrk, BNIP3, Bim jL , Bad, Bid, and Egl-1 (C. elegans); respectively.
  • Members of the Bax subfamily contain the BHl, BH2, and BH3 domains, and resemble Bcl-2 rather closely.
  • members of the BH3 subfamily have only the 9-16 residue BH3 domain, being otherwise unrelated to any known protein, and only Bik and Blk share sequence similarity.
  • the proteins of the two pro-apoptosis subfamilies may be the antagonists of pro-survival subfarruly proteins. This is illustrated 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 subfamilies, 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 BHl 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 immunoglobulin 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-lived post-mitotic cells.
  • the BHl, BH2, and BH4 domains are involved in pro-survival activity.
  • the Bcl-w protein is found within the cytoplasm of almost all myeloid ceU Unes and in numerous tissues, with the highest levels of expression in brain, colon, and salivary gland. This protein is expressed in low levels in testis, hver, heart, stomach, skeletal muscle, and placenta, and a few lymphoid ceU lines. Bcl-w contains the BHl, BH2, and BH4 domains, aU of which are needed for its ceU survival promotion activity. Although 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.
  • 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.
  • 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 NH2-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 EBV- BHRF1 (D'Sa-Eipper, C. et al. (1996) Cancer Res. 56:3879-3882).
  • Bfl-1 is found intracellularly, 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 uncontrolled cell proliferation. Some cancers are associated with suppression of normal apoptotic cell death. Strategies for treatment may involve either reestabhsbing control over ceU cycle progression, or selectively stimulating apoptosis in cancerous ceUs (Nigg, E.A. (1995) BioEssays 17:471-480). Imrnunological 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.
  • the Rel/NF-kappa B family of vertebrate transcription factors plays a pivotal role in inflammatory and immune responses to radiation.
  • the NF-kappa B family includes p50, p52, RelA, RelB, cRel, and other DNA-binding proteins.
  • the p52 protein induces apoptosis, upregulates the transcription factor c-Jun, and activates c-Jun N-terminal kinase 1 (JNK1) (Sun, L. et al. (1998) Gene 208:157-166).
  • Most NF-kappa B proteins form DNA-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 (TNF) receptor family. Upon binding its ligand (Fas Ugand), the membrane-spanning FAS induces apoptosis by recruiting several cytoplasmic proteins that transmit the death signal.
  • FAFl FAS-associated protein factor 1
  • FAS -associated factors have been isolated fromnumerous other species, including fruit fly and quaU (Frohlich, T. et al. (1998) J. CeU Sci. 111:2353-2363).
  • Another cytoplasmic protein that functions in the transmittal of the death signal from Fas is the Fas- associated death domain protein, also known as FADD.
  • FADD transmits the death signal in both
  • DFF DNA fragmentation factor
  • CAD DNA fragmentation factor
  • ICAD Idenose-like nuclease
  • CIDE-A and CIDE-B Two mouse homologs of DFF45/ICAD, termed CIDE-A and CIDE-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 CIDE-B, further implicating these proteins as effectors that mediate apoptosis.
  • a number of downstream effector molecules 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, C.G. 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 among the most specific endopeptidases, cleaving after aspartate residues. Caspases are synthesized as inactive zymogens consisting of one large (p20) and one smaU ( lO) 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.
  • the long prodomains of some caspase famUy 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.
  • TNF Tumor necrosis factor
  • ICE Interleukin-l ⁇ converting enzyme
  • ICE is expressed primarily in monocytes.
  • ICE processes the cytokine precursor, interleukin-l ⁇ , into its active form, which plays a central role in acute and chronic inflammation, bone resorption, myelogenous leukemia, and other pathological processes.
  • ICE and related caspases cause apoptosis when overexpressed in transfected ceU lines.
  • 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, RAIDD, and ceUular inhibitors of apoptosis proteins (lAPs) (Hofmann, K. et al. (1997) Trends Biochem. Sci.
  • CARD apoptosis repressor with a CARD (ARC) which is expressed in both skeletal and cardiac muscle has been identified and characterized. ARC functions as an inhibitor of apoptosis and interacts selectively with 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.
  • ESI 8 was identified as a potential regulator of apoptosis in mouse T-ceUs (Park, E.J. et al. (1999) Nuc. Acid. Res. 27:1524-1530).
  • ES18 is 428 amino acids in length, contains an N-terminal proline-rich region, an acidic glutamic acid-rich domain, and a putative LXXLL nuclear receptor binding motif. The protein is preferentiaUy expressed in lymph nodes and thymus. The level of ES 18 expression increases in T-ceU thymoma S49.1 in response to treatment with dexamethasone, staurosporine, or C2-ceramide, which induce apoptosis. ES 18 may play a role in stimulating apoptotic ceU death in T-ceUs.
  • the rat ventral prostate is a model system for the study of hormone-regulated apoptosis.
  • RVP epitheUal ceUs undergo apoptosis in response to androgen deprivation.
  • Messenger RNA (mRNA) transcripts that are up-regulated in the apoptotic RVP have been identified (Briehl, M. M. and R.L. Miesfeld (1991) Mol. Endocrinol. 5:1381-1388).
  • mRNA Messenger RNA transcripts that are up-regulated in the apoptotic RVP have been identified (Briehl, M. M. and R.L. Miesfeld (1991) Mol. Endocrinol. 5:1381-1388).
  • One such transcript encodes RVP.l, the precise role of which in apoptosis has not been determined.
  • the human homolog of RVP.1 , hRVPl is 89% identical to the rat protein (Katahira, J. et al. (
  • hRVPl is 220 amino acids in length and contains four transmembrane domains. hRVPl is highly expressed in the lung, intestine, and hver. Interestingly, hRVPl functions as a low affinity receptor for the Clostridium perfringens enterotoxin, a causative agent of diarrhea in humans and other animals.
  • Cytokine-mediated apoptosis plays an important role in hematopoiesis and the immune response.
  • Myeloid cells which are the stem ceU progenitors of macrophages, neutrophils, erythrocytes, and other blood ceUs, prohferate in response to specific cytokines such as granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3).
  • GM-CSF granulocyte/macrophage-colony stimulating factor
  • IL-3 interleukin-3
  • myeloid ceUs undergo apoptosis.
  • the murine requiem (req) gene encodes a putative transcription factor required for this apoptotic response in the myeloid ceU line FDCP-1 (Gabig, T.
  • the Req protein is 371 amino acids in length and contains a nuclear locahzation signal, a single K ⁇ -uppel-t pe zinc finger, an acidic domain, and a cluster of four unique zinc-finger motifs enriched in cysteine and histidine residues involved in metal binding. Expression of req is not myeloid- or apoptosis-specific, suggesting that additional factors regulate Req activity in myeloid ceU apoptosis.
  • Dysregulation of apoptosis has recently been recognized as a significant factor in the pathogenesis of many human diseases.
  • excessive ceU survival caused by decreased apoptosis can contribute to disorders related to ceU prohferation and the immune response.
  • disorders include cancer, autoimmune diseases, viral infections, and inflammation.
  • excessive ceU death caused by increased apoptosis can lead to degenerative and immunodeficiency disorders such as AIDS, neurodegenerative diseases, and myelodysplastic syndromes. (Thompson, C.B. (1995) Science 267:1456-1462.)
  • Alzheimer's disease is a progressive neurodegenerative disorder that is characterized by the formation of senUe plaques and neurofibriUary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. B-amyloid peptide participates in signaling pathways that induce apoptosis and lead to the death of neurons (Kajkowski, C et al. (2001) J. Biol. Chem 276:18748-18756).
  • Cancers are characterized by continuous or uncontrolled cell prohferation. Some cancers are associated with suppression of normal apoptotic cell death. Understanding of the neoplastic process can be aided by the identification of molecular markers of prognostic and diagnostic importance. Cancers are associated with oncoproteins which are capable of transforming normal cells into malignant cells. Some oncoproteins are mutant isoforms of the normal protein while others are abnormaUy expressed with respect to location or level of expression. Normal cell prohferation begins with binding of a growth factor to its receptor on the cell membrane, resulting in activation of a signal system that induces and activates nuclear regulatory factors to initiate DNA transcription, subsequently leading to cell division.
  • oncoproteins known to affect the cell cycle controls include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and ceU-cycle control proteins.
  • growth factors growth factor receptors
  • intracellular signal transducers such as tumor antigens and tumor suppressors
  • ceU-cycle control proteins such as tumor antigens and tumor suppressors.
  • Oncogenes are encoded by genes, caUed oncogenes, that are derived from genes that normaUy control ceU growth and development. Many oncogenes have been identified and characterized.
  • growth factors such as sis, receptors such as erbA, erbB, neu, and ros, intracellular receptors such as src, yes, fps, abl, and met, protein-serine/threonine kinases such as mos and raf, nuclear transcription factors such asjun, fos, myc, N-myc, myb, ski, and rel, cell cycle control proteins such as RB mdp53, mutated tumor-suppressor genes such as mdm2, dpi, pl6, and cyclin D, ras, set, can, sec, and gag R10.
  • growth factors such as sis, receptors such as erbA, erbB, neu, and ros
  • intracellular receptors such as src, yes, fps, abl, and met
  • protein-serine/threonine kinases such as mos and raf
  • nuclear transcription factors such asjun, fos, myc, N
  • Viral oncogenes are integrated into the human genome after infection of human ceUs by certain viruses.
  • Examples of viral oncogenes include v-src, v-abl, and v-fps. Transformation of normal genes to oncogenes may also occur by chromosomal translocation.
  • Philadelphia chromosome characteristic of chronic myeloid leukemia and a subset of acute lymphoblastic leukemias, results from a reciprocal translocation between chromosomes 9 and 22 that moves a truncated portion of the proto-oncogene c-abl to the breakpoint cluster region (bcr) on chromosome 22.
  • the hybrid c-abl-bcr gene encodes a chimeric protein that has tyrosine kinase activity.
  • the chimeric protein In chronic myeloid leukemia, the chimeric protein has a molecular weight of 210 kd, whereas in acute leukemias a more active 180 kd tyrosine kinase is formed (Robbins, S.L. et al. (1994) Pathologic Basis of Disease, W.B. Saunders Co., Philadelphia PA).
  • Ras superfamily of smaU GTPases is involved in the regulation of a wide range of ceUular signaling pathways.
  • Ras family proteins are membrane-associated proteins acting as molecular switches that bind GTP and GDP, hydrolyzing GTP to GDP.
  • Ras family proteins interact with a variety of ceUular targets to activate downstream signaling pathways.
  • members of the Ras subfamily are essential in transducing signals from receptor tyrosine kinases (RTKs) to a series of serine/threonine kinases which control ceU growth and differentiation.
  • Ras proteins which bind but can not hydrolyze GTP, are permanently activated, and cause continuous ceU prohferation or cancer.
  • Ras famUy proteins Activation of Ras famUy proteins is catalyzed by guanine nucleotide exchange factors (GEFs) which catalyze the dissociation of bound GDP and subsequent binding of GTP.
  • GEFs guanine nucleotide exchange factors
  • RGL3 RalGEF-like protein
  • Tumor antigens are ceU surface molecules that are differentiaUy expressed in tumor cells relative to non-tumor tissues. Tumor antigens make tumor ceUs immunologically distinct from normal ceUs and are potential diagnostics for human cancers.
  • monoclonal antibodies have been identified which react specificaUy with cancerous ceUs such as T-ceU acute lymphoblastic leukemia and neuroblastoma (Minegishi, M. et al. (1989) Leukemia Res. 13:43-51; Takagi, S. et al. (1995) Int. J. Cancer 61:706-715).
  • T-ceU acute lymphoblastic leukemia and neuroblastoma Minegishi, M. et al. (1989) Leukemia Res. 13:43-51; Takagi, S. et al. (1995) Int. J. Cancer 61:706-715.
  • the discovery of high level expression of the HER2 gene in breast tumors has led to the development of therapeutic treatments (Liu
  • MAGE genes encode a famUy of tumor antigens recognized on melanoma ceU surfaces by autologous cytolytic T lymphocytes.
  • melanoma ceU surfaces by autologous cytolytic T lymphocytes.
  • 12 human MAGE genes isolated half are differentiaUy expressed in tumors of various histological types (De Plaen, E. et al. (1994) Immunogenetics 40:360-369). None of the 12 MAGE genes, however, is expressed in healthy tissues except testis and placenta.
  • Tumor suppressor genes are generaUy defined as genetic elements whose loss or inactivation contributes to the deregulation of ceU prohferation 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.
  • Several tumor suppressor genes have been identified including the genes encoding the retinoblastoma (Rb) protein, p53, and the breast cancer 1 and 2 proteins (BRCA1 and BRCA2). Mutations in these genes are associated with acquired and inherited genetic predisposition to the development of certain cancers.
  • 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 DNA binding. Most cancer- associated mutations in p53 locahze 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.
  • KAIl The metastasis-suppressor gene KAIl (CD82) has been reported to be related to the tumor suppressor gene p53.
  • KAIl is involved in the progression of human prostatic cancer and possibly lung and breast cancers when expression is decreased.
  • KAIl encodes a member of a structurally distinct family of leukocyte surface glycoproteins.
  • the family is known as either the tetraspan transmembrane protein family or transmembrane 4 superfamily (TM4SF) as the members of this family span the plasma membrane four times.
  • 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.
  • TM4SF proteins have three additional transmembrane regions, seven or more conserved cysteine residues, are similar in size (218 to 284 residues), and all have a large extraceUular 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 KAIl and seven other members of the TM4SF indicate that the splicing 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.
  • LGI1 Leucine-rich gene-Glioma Inactivated
  • LGI1 expression is seen predominantly in neural tissues, especially brain.
  • the loss of tumor suppressor activity is seen in the inactivation of the LGI1 protein which occurs during the transition from low to high-grade tumors in malignant gliomas.
  • the reduction of LGI1 expression in low grade brain tumors and its significant reduction or absence of expression in malignant gliomas suggests that it could be used for diagnosis of glial tumor progression (Chernova, O.B. et al. (1998) Oncogene 17:2873-2881).
  • the ST13 tumor suppressor was identified in a screen for factors related to colorectal carcinomas by subtractive hybridization between cDNA of normal mucosal tissues and mRNA of colorectal carcinoma tissues (Cao, J. et al. (1997) J. Cancer Res. Clin. Oncol. 123:447-451). ST13 is down-regulated in human colorectal carcinomas. Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are associated with retinal and central nervous system hemangioblastomas, clear cell renal carcinomas, and pheochromocytomas (Hoffman, M. et al. (2001) Hum. Mol. Genet. 10:1019-1027; Kamada, M. (2001) Cancer Res.
  • VHL von Hippel-Lindau
  • VHL regulates the expression of transforming growth factor- , the GLUT-1 glucose transporter and vascular endothelial growth factor.
  • the VHL protein associates with elongin B, elongin C, Cul2 and Rbxl to form a complex that regulates the transcriptional activator hypoxia-inducible factor (HIF).
  • HIF induces genes involved in angiogenesis such as vascular endothelial growth factor and platelet- derived growth factor B. Loss of control of HIF caused by defects in VHL results in the excessive production of angiogenic peptides.
  • VHL may play roles in inhibition of angiogenesis, cell cycle control, fibronectin matrix assembly, cell adhesion, and proteolysis.
  • PR-domain genes were recently recognized as playing a role inhuman tumorigenesis.
  • PR-domain genes normaUy produce two protein products: the PR-plus product, which contains the PR domain, and the PR-minus product which lacks this domain.
  • PR-plus is disrupted or overexpressed
  • wMle PR-minus is present or overexpressed.
  • the imbalance in the amount of these two proteins appears to be an important cause of malignancy (Jiang, G.L. and S. Huang (2000) Histol. Histopathol. 15:109-117).
  • neoplastic disorders in humans can be attributed to inappropriate gene transcription. Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer Surv. 15:89-104). Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene.
  • An important class of transcriptional regulators are the zinc finger proteins.
  • the zinc finger motif which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodicaUy spaced cysteine, and histidine residues.
  • Examples of this sequence pattern include the C2H2-type, C4-type, and C3HC4- type zinc fingers, and the PHD domain (Lewin, B. (1990) Genes IV, Oxford University Press, New York, NY, and Cell Press, Cambridge, MA, pp. 554-570; Aasland, R., et al. (1995) Trends Biochem. Sci. 20:56-59).
  • WTl a tumor-suppressor protein that is inactivated in children with Wilm's tumor.
  • the oncogene bcl-6 which plays an important role in large-cell lymphoma, is also a zinc-finger protein (Papavassiliou, A.G. (1995) N. Engl. J. Med. 332:45-47).
  • Cancers also called neoplasias, are characterized by continuous and uncontrolled cell proliferation. They can be divided into three categories: carcinomas, sarcomas, and leukemias.
  • Carcinomas are malignant growths of soft epithelial ceUs that may infiltrate surrounding tissues and give rise to metastatic tumors.
  • Sarcomas may be of epithelial origin or arise from connective tissue.
  • Leukemias are progressive malignancies of blood-forming tissue characterized by proliferation of leukocytes and their precursors, and may be classified as myelogenous (granulocyte- or monocyte- derived) or lymphocytic (lymphocyte-derived).
  • Tumorigenesis refers to the progression of a tumor's growth from its inception.
  • Malignant ceUs may be quite similar to normal cells within the tissue of origin or may be undifferentiated (anaplastic). Tumor cells may possess few nuclei or one large polymorphic nucleus.
  • Anaplastic cells may grow in a disorganized mass that is poorly vascularized and as a result contains large areas of ischemic necrosis.
  • Differentiated neoplastic ceUs may secrete the same proteins as the tissue of origin. Cancers grow, infiltrate, invade, and destroy the sunounding tissue through direct seeding of body cavities or surfaces, through lymphatic spread, or through hematogenous spread. Cancer remains a major pubhc health concern and current preventative measures and treatments do not match the needs of most patients. Understanding of the neoplastic process of tumorigenesis can be aided by the identification of molecular markers of prognostic and diagnostic importance. Current forms of cancer treatment include the use of itnmunosuppressive drugs (Morisaki, T. et al.
  • immunophihns are a family of conserved proteins found in both prokaryotes and eukaryotes that bind to i munosuppressive drugs with varying degrees of specificity.
  • immunophihc proteins is the peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) family (PPIase, rotamase).
  • cyclophihns e.g., peptidyl-prolyl isomerase A or PPIA
  • FKBP FK-binding protein
  • Cyclophihns are multifunctional receptor proteins which participate in signal transduction activities, including those mediated by cyclosporin (or cyclosporine).
  • the PPIase domain of each famUy is highly conserved between species. Although structuraUy distinct, these multifunctional receptor proteins are involved in numerous signal transduction pathways, and have been implicated in folding and trafficking events.
  • the immunophilin protein cyclophilin binds to the irnmunosuppressant drug cyclosporin A.
  • FKBP another immunophilin, binds to FK506 (or rapamycin). Rapamycin is an irnmunosuppressant agent that arrests ceUs in the G t phase of growth, inducing apoptosis.
  • this macrolide antibiotic produced by Streptomyces tsukubaensis acts by binding to ubiquitous, predominantly cytosolic immunophilin receptors.
  • immunophihn/immunosuppressant complexes e.g., cyclophilin A cyclosporin A (CypA CsA) and FKBP12 FK506
  • cyclophilin A cyclosporin A CypA CsA
  • FKBP12 FK506 FKBP12 FK506
  • phosphatase calcineurin a calcium calmodulin-dependent protein kinase that participates in T-ceU activation
  • the murine fkbp51 gene is abundantly expressed in imrnunological tissues, including the thymus and T lymphocytes (Baughman, G. et al. (1995) Molec. CeU. Biol.
  • FKBP12/ra ⁇ amycin-directed immunosuppression occurs through binding to TOR (yeast) or FRAP (FKBP12-rapamycin-associated protein, in mammalian cells), the kinase target of rapamycin essential for mamtaining normal cellular growth patterns.
  • Dysfunctional TOR signaling has been linked to various human disorders including cancer (Metcalfe, S.M. et al. (1997) Oncogene 15:1635-1642; Emami, S. et al. (2001) FASEB J. 15:351-361), and autoimmunity (Damilor, J.G. et al. (1996) Transplantation 62:994-1001).
  • cyclophilin isozymes include cyclophilin B, cyclophilin C, mitochondrial matrix cyclophilin, bacterial cytosolic and periplasmic PPIases, and natural-killer ceU cyclophilin-related protein possessing a cyclophilin-type PPIase domain, a putative tumor-recognition complex involved in the function of natural killer (NK) ceUs.
  • NK natural killer
  • NK ceUs specificaUy target ceUs that have lost their expression of major histocompatibihty complex (MHC) class I genes (common during tumorigenesis), endowing them with the potential for attenuating tumor growth.
  • MHC major histocompatibihty complex
  • a 150-kDa molecule has been identified on the surface of human NK ceUs that possesses a domain which is highly homologous to cyclophilin/peptidyl-prolyl cis-trans isomerase.
  • This cyclophilin-type protein may be a component of a putative tumor-recognition complex, a NK tumor recognition sequence (NK-TR) (Anderson, S.K. et al. (1993) Proc. Natl. Acad. Sci. USA 90:542-546).
  • the NKTR tumor recognition sequence mediates recognition between tumor ceUs and large granular lymphocytes (LGLs), a subpopulation of white blood ceUs (comprised of activated cytotoxic T ceUs and natural kiUer ceUs) capable of destroying tumor targets.
  • LGLs large granular lymphocytes
  • the protein product of the NKTR gene presents on the surface of LGLs and facilitates binding to tumor targets.
  • a mouse Nktr gene and promoter region have been located on chromosome 9.
  • the gene encodes a NK-ceU-specific 150-kDa protein (NK-TR) that is homologous to cyclophilin and other tumor-responsive proteins (Simons-Evelyn, M. et al. (1997) Genomics 40:94-100).
  • TNF tumor necrosis factor
  • TNF farr ⁇ fy of cytokines are produced by lymphocytes and macrophages, and can cause the lysis of transformed (tumor) endothelial ceUs.
  • Endothelial protein 1 (Edpl) has been identified as a human gene activated transcriptionaUy by TNF-alpha in endothelial cells, and a TNF- alpha inducible Edpl gene has been identified in the mouse (Swift, S. et al. (1998) Biochim. Biophys. Acta 1442:394-398).
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specificaUy related to a particular genetic predisposition, condition, disease, or disorder. Inflammation
  • LPS hpopolysacchari.de
  • LPS ehcits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections.
  • Gram-negative bacteria probably release minute amounts of endotoxin while growing.
  • endotoxins may be released in soluble form, especiaUy by young cultures.
  • endotoxins remain associated with the ceU waU until the bacteria disintegrate. In vivo, disintegration is the result of autolysis of the bacteria, external lysis mediated by complement and lysozyme, and phagocytic digestion of bacterial ceUs.
  • LPS -binding proteins LPS -binding proteins
  • the LPS -binding protein complex interacts with CD14 receptors on monocytes, macrophages, B ceUs, and other types of receptors on endofhehal ceUs.
  • Activation of human B ceUs with LPS results in mitogenesis as weU as immunoglobulin synthesis.
  • Jurkat is an acute T ceU leukemia cell line that grows actively in the absence of external stimuli.
  • Jurkat has been extensively used to study signaling in human T ceUs.
  • PMA phorbol myristate acetate
  • Ionomycin is a calcium ionophore that permits the entry of calcium into the cell, hence increasing the cytosolic calcium concentration.
  • the combination of PMA and ionomycin activates two of the major signaling pathways used by mammahan cells to interact with their environment. In T ceUs, the combination of PMA and ionomycin mimics the type of secondary signahng events elicited during optimal B ceU activation.
  • T ceUs can be subdivided into two classes according to their main function and the surface- antigens they express.
  • CD4 positive (+) T ceUs also known as T Helper cells, primarily regulate the immune response by producing soluble factors that, in turn, regulate the activity of effector ceUs such as B lymphocytes, NK ceUs, and macrophages.
  • CD8 positive (+) T ceUs also known as cytotoxic T ceUs, primarily kiU "abnormal" ceUs such as tumor ceUs or ceUs infected by viruses.
  • CD4+ T ceUs and CD8+ T ceUs represent 25% and 15% of the mononuclear ceUs, respectively.
  • PBMCs peripheral blood mononuclear ceUs
  • PHA phytohemagglutinin
  • IL-2 interleukin 2
  • both CD4+ and CD8+ T ceUs expand roughly 5 to 10 fold, yielding a ceU population composed of >90% T ceUs, also known as PHA blasts.
  • T ceU expansion occurs during the first 5 days of PHA stimulation; after 8 to 10 days in culture, most PHA blasts have returned to a resting state.
  • T ceUs require two distinct signals to achieve optimal activation. First, the "antigenic" signal dehvered through the binding of the TCR-CD3 complex. Second, the costimulatory signal dehvered through the binding of the CD28 molecules. Upon binding of the TCR-CD3 complex alone, T ceUs only achieve a partial state of activation. However, it is important to note that the signaling requirements of T ceUs depend greatly on the cycling state of those ceUs.
  • THP-1 is a promonocyte ceU line that was isolated from the peripheral blood of a 1 -year-old male with acute monocytic leukemia. Upon stimulation with PMA, the THP-1 ceU line acquires monocytic characteristics.
  • PBMCs Human peripheral blood mononuclear ceUs
  • PBMCs represent the major ceUular components of the immune system.
  • PBMCs contain about 12% B lymphocytes, 25% CD4+ and 15% CD8+ lymphocytes, 20% NK ceUs, 25% monocytes, and 3% various ceUs that include dendritic cells and progenitor ceUs.
  • the proportions, as well as the biology of these ceUular components tend to vary shghtly between healthy individuals, depending on factors such as age, gender, past medical history, and genetic background.
  • LPS lipopolysaccharide
  • Toxicity is associated with the hpid component (Lipid A) of LPS
  • immunogenicity is associated with the polysaccharide components of LPS.
  • LPS ehcits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections. For the most part, endotoxins remain associated with the cell waU until the bacteria disintegrate. LPS released into the bloodstream by lysing gram-negative bacteria is first bound by certain plasma proteins identified as LPS -binding proteins.
  • the LPS-binding protein complex interacts with CD14 receptors on monocytes, macrophages, B ceUs, and other types of receptors on endofhehal ceUs. Activation of human B ceUs with LPS results in mitogenesis as weU as immunoglobulin synthesis. In monocytes and macrophages three types of events are triggered during their interaction with LPS: 1) production of cytokines, including IL-1, IL-6, IL-8, TNF- ⁇ , and platelet-activating factor, which stimulate production of prostaglandins and leukotrienes that mediate inflammation and septic shock; 2) activation of the complement cascade; and 3) activation of the coagulation cascade.
  • Interleukin 10 initiaUy designated cytokine synthesis inhibitory factor (CSIF)
  • CCF cytokine synthesis inhibitory factor
  • Th2 murine T helper 2
  • APC antigen presenting ceUs
  • the human homolog of murine IL-10 was subsequently cloned by cross-hybridization.
  • Human IL-10 is produced by CD4 + T ceU clones as weU as by some CD8 + T ceU clones.
  • human B cells, EBV-transformed lymphoblastoid ceU lines, and monocytes can also produce IL-10 upon activation.
  • IL-10 is a pleiotrophic cytokine that can exert either i munostimulatory or immunosupressive effects on a variety of ceU types. It is a potent irnmunosuppressant of macrophage functions. In vitro, IL-10 can inhibit the accessory function and antigen-presenting capacity of monocytes by, among other effects, downregulating class II MHC expression. Thus, IL-10 can inhibit monocyte/ macrophage-dependent, antigen-specific prohferation of mouse Thl clones as weU as human ThO-, Thl-, and Th2-like T ceUs.
  • IL-10 can also inhibit the monocyte/macrophage-dependent, antigen stimulated cytokine synthesis (especiaUy IFN- ⁇ ) by human PBMC and NK ceUs.
  • especiaUy IFN- ⁇ monocyte/macrophage-dependent, antigen stimulated cytokine synthesis
  • IL-10 is a potent inhibitor of monocyte/macrophage activation and its resultant cytotoxic effects. It can suppress the production of numerous cytokines including TNF- ⁇ , IL-1, IL-6, and IL-10, as weU as the synthesis of superoxide anion, reactive oxygen intermediates, and reactive nitrogen intermediates by activated monocytes/macrophages.
  • IL-10 can act on B ceUs to enhance their viability, cell prohferation, Ig secretion, and class II MHC expression. Aside fromB lymphocytes, IL-10 is also a growth co- stimulator for fhymocytes and mast ceUs, as well as an enhancer of cytotoxic T cell development. Activation of Vascular Endothehum
  • HAECs Human aortic endothelial ceUs
  • HUVECs Human umbilical vein endofhehal cells
  • HAECs and HUVECs have both been used as experimental models for investigating the role of the endothehum in human vascular biology in vitro.
  • Activation of the vascular endothehum is considered to be a central event in a wide range of both physiological and pafhophysiological processes, such as vascular tone regulation coagulation and thrombosis, atherosclerosis, and inflammation.
  • Tumor necrosis factor-alpha is a pleiotropic cytokine that plays a central role in mediation of the inflammatory response through activation of multiple signal transduction pathways.
  • TNF ⁇ is produced by activated lymphocytes, macrophages, and other white blood ceUs, and activates endofhehal ceUs. Monitoring the endofhehal ceUs' response to TNF ⁇ at the level of the mRNA expression can provide information necessary for better understanding of both TNF ⁇ signaling pathways and endofhehal ceU biology.
  • Promonocvtes Leukocytes comprise lymphocytes, granulocytes, and monocytes. Lymphocytes include T- and B-ceUs, which specificaUy recognize and respond to foreign pathogens.
  • T-ceUs fight viral infections and activate other leukocytes, whUe B-ceUs secrete antibodies that neutrahze bacteria and other microbes.
  • Granulocytes and monocytes are primarily migratory, phagocytic ceUs that exit the bloodstream to fight infection in tissues.
  • Monocytes which are derived from immature promonocytes, further differentiate into macrophages that engulf and digest microorganisms and damaged or dead ceUs.
  • Monocytes and macrophages modulate the immune response by secreting signaling molecules such as growth factors and cytokines.
  • Tumor necrosis factor- ⁇ (TNF- ⁇ ), for example, is a macrophage-secreted protein with anti-tumor and anti-viral activity.
  • monocytes and macrophages are recruited to sites of infection and inflammation by signaling proteins secreted by other leukocytes.
  • the differentiation of the monocyte blood ceU hneage can be studied in vitro using cultured ceU lines.
  • THP-1 is a human promonocyte cell line that can be activated by treatment with both phorbol ester such as phorbol myristate acetate (PMA), and lipopolysaccharide (LPS).
  • PMA is a broad activator of the protein kinase C-dependent pathways.
  • Monocytes are involved in the initiation and maintenance of inflammatory immune responses.
  • the outer membrane of gram-negative bacteria expresses lipopolysaccharide (LPS) complexes caUed endotoxins.
  • LPS lipopolysaccharide
  • Toxicity is associated with the lipid component (Lipid A) of LPS, and immunogenicity is associated with the polysaccharide components of LPS.
  • LPS ehcits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections. For the most part, endotoxins remain associated with the ceU wall until the bacteria disintegrate.
  • LPS released into the bloodstream by lysing gram-negative bacteria is first bound by certain plasma proteins identified as LPS-binding proteins. The LPS-binding protein complex interacts with CD14 receptors on monocytes, macrophages, B ceUs, and other types of receptors on endothelial ceUs.
  • Activation of human B ceUs with LPS results in mitogenesis as well as immunoglobulin synthesis.
  • monocytes and macrophages three types of events are triggered during their interaction with LPS : 1) production of cytokines, including IL-1, IL-6, IL-8, TNF- ⁇ , and platelet-activating factor, which stimulate production of prostaglandins and leukotrienes that mediate inflammation and septic shock; 2) activation of the complement cascade; and 3) activation of the coagulation cascade.
  • THP-1 is a promonocyte ceU line that was isolated from the peripheral blood of a 1 -year-old male with acute monocytic leukemia.
  • PMA is a broad activator of the protein kinase C-dependent pathways.
  • THP-1 differentiates into a macrophage-like ceU that displays many characteristics of peripheral human macrophages.
  • BRCA1 and BRCA2 are known to greatly predispose a woman to breast cancer and may be passed on from parents to chUdren (Gish, supra).
  • this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial ceUs.
  • epidermal growth factor EGF
  • EGFR epidermal growth factor
  • EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis.
  • EGF has effects on ceU functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation.
  • the abundance of erbB receptors, such as HER- 2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S.S. et al. (1994) Am. J. Clin. Pafhol. 102:S13-S24).
  • CeU lines derived from human mammary epithelial ceUs at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these ceU lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba, I.I. et al. (1998) Chn. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial ceUs at various stages of malignant transformation. Lung Cancer
  • Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred. Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and hver.
  • Non SmaU CeU Lung Carcinoma (NSCLC) group includes squamous ceU carcinomas, adenocarcinomas, and large cell carcinomas and accounts for about 70% of aU lung cancer cases.
  • Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands.
  • Squamous ceU carcinomas typicaUy arise in proximal airways.
  • SCLC SmaU Cell Lung Carcinoma
  • Lung cancer ceUs accumulate numerous genetic lesions, many of which are associated with cytologically visible chromosomal aberrations.
  • the high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease.
  • Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earhest genetic lesions leading to lung cancer. Deletions at chromosome arms 9 ⁇ and 17 ⁇ are also common.
  • Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2.
  • thrombospondin-1 thrombospondin-1, fibronectin, interceUular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentially expressed in lung cancers.
  • Wang et al. 2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentiaUy overexpresssed in squamous ceU carcinoma compared with normal lung epithelium.
  • the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 and cytokeratin 13.
  • Ovarian Cancer Ovarian Cancer
  • Ovarian cancer is the leading cause of death from a gynecologic cancer.
  • the majority of ovarian cancers are derived from epithehal ceUs, and 70% of patients with epitheUal ovarian cancers present with late-stage disease. As a result, the long-term survival rate for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate. Genetic variations involved in ovarian cancer development include mutation of p53 and microsateUite instabUity. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors. Prostate Cancer
  • Prostate cancer is a common malignancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
  • cancer ceUs arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer.
  • prostatic cancers Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous cell and transitional ceU carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.
  • prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype.
  • the initial step in tumor progression involves the hyperprohferation of normal luminal and/or basal epithehal ceUs. Androgen responsive ceUs become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent ceUs evolve from the hyperplastic population. These ceUs represent a more advanced form of prostate tumor that may become invasive and potentiaUy become metastatic to the bone, brain, or lung. A variety of genes maybe differentiaUy expressed during tumor progression.
  • LOV loss of heterozygosity
  • FISH Fluorescence in situ hybridization
  • PSA prostate specific antigen
  • PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithehal ceUs.
  • the quantity of PSA correlates with the number and volume of the prostatic epithehal ceUs, and consequently, the levels of PSA are an exceUent indicator of abnormal prostate growth.
  • Men with prostate cancer exhibit an early hnear increase in PSA levels foUowed by an exponential increase prior to diagnosis.
  • PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer.
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF ⁇ Tumor Growth Factor alpha
  • TGF- ⁇ family of growth factors are generaUy expressed at increased levels inhuman cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold, L.I. (1999) Crit. Rev. Oncog. 10:303-360).
  • FinaUy there are human ceU lines representing both the androgen-dependent stage of prostate cancer (LNCap) as well as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proved useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of cell treatments on these expressed genes (Chung, T.D. (1999) Prostate 15:199-207).
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of ceU prohferative disorders including cancer, developmental disorders, neurological disorders, autoimmune/inflammatory disorders, reproductive disorders, and disorders of the placenta.
  • Various embodiments of the invention provide purified polypeptides, proteins associated with ceU growth, differentiation, and death, referred to cohectively as 'CGDD' and individuaUy as 'CGDD-1,' 'CGDD-2,' 'CGDD-3,' 'CGDD-4,' 'CGDD-5,' 'CGDD-6,' 'CGDD-7,' 'CGDD-8,' 'CGDD-9,' 'CGDD-10,' 'CGDD-11,' 'CGDD-12,' 'CGDD-13,' 'CGDD-14,' 'CGDD-15,' 'CGDD- 16,' 'CGDD-17,' 'CGDD-18,' 'CGDD-19,' and 'CGDD-20' and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions.
  • Embodiments also provide methods for utilizing the purified proteins associated with ceU growth, differentiation, and death and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utilizing the purified proteins associated with ceU growth, differentiation, and death and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
  • An embodiment 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:l- 20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-20.
  • StiU another embodiment 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:1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1 -20. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:21-40.
  • Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a 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:1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
  • Another embodiment provides a ceU transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment 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: 1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU 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.
  • Yet another embodiment 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:1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -20.
  • StiU yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, 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 can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, 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).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleot
  • 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 specificaUy 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.
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • StUl yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, b) a polynucleotide comprising a naturally occuning polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, 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).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polyn
  • 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.
  • the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
  • compositions 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 ID NO: 1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and a pharmaceutically acceptable excipient
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
  • inventions provide a method of treating a disease or condition associated with decreased or abnormal expression of functional CGDD, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment 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:1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
  • the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting agonist activity in the sample.
  • Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • Yet another embodiment 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.
  • Still yet another embodiment 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:1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
  • the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional CGDD, comprismg administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specifically 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:1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20.
  • 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 specifically binds to the polypeptide.
  • Yet another embodiment 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 ID NO: 1-20, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-20, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-20.
  • 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.
  • StiU yet another embodiment 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 ID NO:21-40, the method comprising a) contacting a sample comprising the target polynucleotide with 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.
  • Another embodiment 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:21-40, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, hi) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
  • 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:21-40, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:21-40, 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 can comprise a fragment of a polynucleotide selected from the 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 amount 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 full length polynucleotide and polypeptide embodiments of the 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 polypeptide embodiments 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 embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 hsts the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA libraries for polynucleotide embodiments.
  • 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 polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • a host ceU includes a plurality of such host cells
  • an antibody is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
  • CGDD refers to the amino acid sequences of substantiaUy purified CGDD obtained from any species, particularly a mammahan 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, 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.
  • an "aUelic 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 allelic variants of its naturaUy occurring form.
  • Common mutational changes which give rise to allelic 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 functional characteristic of CGDD. Included within this definition are polymorphisms which may or may not be readUy detectable using a particular ohgonucleotide probe of the polynucleotide encoding CGDD, and improper or unexpected hybridization to aUehc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding CGDD.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a sUent change and result in a functionaUy equivalent CGDD.
  • Dehberate amino acid substitutions may be made on the basis of one or more sim arities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipafhic nature of the residues, as long as the biological or imrnunological 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 hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oligopeptide, a peptide, a polypeptide, or a 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 limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
  • PCR polymerase chain reaction
  • antagonists refers to a molecule which inhibits or attenuates the biological activity of CGDD. Antagonists may include proteins such as antibodies, anticalins, 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 well 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 oligopeptide 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 Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobuhn, 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 ehcit 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 hbraries.
  • 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 maybe replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer hfetime 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 specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • intramer 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 (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • the term “spiegehner” refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; ohgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or ohgonucleotides having modified bases such as 5-mefhyl cytosine, 2'-deoxyuracU, 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 capability of the natural, recombinant, or synthetic CGDD, or of any ohgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement,
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides 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 stabhizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardfs ' solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the 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.
  • 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 helical 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 imrnunological 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 imrnunological 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 a polynucleotide encoding CGDD which can be 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/arnino acid residue.
  • a fragment may comprise from about 5 to about 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:21-40 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:21-40, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO :21-40 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amphfication technologies and in analogous methods that distinguish SEQ ID NO:21-40 from related polynucleotides.
  • the precise length of a fragment of SEQ ID NO:21-40 and the region of SEQ ID NO:21-40 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 ID NO:1-20 is encoded by a fragment of SEQ ID NO:21-40.
  • a fragment of SEQ ID NO: 1-20 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:1-20.
  • a fragment of SEQ ID NO:1-20 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO: 1-20.
  • the precise length of a fragment of SEQ ID NO: 1-20 and the region of SEQ ID NO: 1-20 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
  • a “fuU length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • Homology refers to sequence simUarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned 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 alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5:151- 153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191).
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to ahgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also avaUable is a tool caUed “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences” can be accessed and used interactively at ncbi.nlm.nih.gov/gori7bl2.html. The "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2
  • Percent identity may be measured over the length of an entire defined 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 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 identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence ahgnment are weU-known. Some ahgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detaU above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • percent similarity and “% similarity” refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (A ⁇ ril-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example:
  • Gap x drop-off 50
  • 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
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome rephcation, 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 ability.
  • 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 annealing 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 annealing 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 annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 jug/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.
  • Such wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (Tute 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.
  • An equation for calculating T m and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D.W. RusseU (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).
  • 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 readtiy apparent to those of ordinary skill 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 simUar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acids 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 present in solution and another nucleic acid immobilized on a sohd support (e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • sohd support e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • 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 signaling molecules, which may affect ceUular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or ohgopeptide fragment of CGDD which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or ohgopeptide 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 plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of CGDD. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or imrnunological properties of CGDD.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, ohgonucleotide, 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-like or RNA-like material.
  • PNA peptide nucleic acid
  • operably linked 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 linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked 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 oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the ceU.
  • Post-translational modification of an CGDD may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synfheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic milieu of CGDD.
  • Probe refers to nucleic acids encoding CGDD, their complements, or fragments thereof, which are used to detect identical, ahehc or related nucleic acids.
  • Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemUuminescent agents, and enzymes.
  • Primmers are short nucleic acids, usuaUy DNA ohgonucleotides, 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 amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer 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. Methods for preparing and using probes and primers are described in, for example,
  • 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).
  • Ohgonucleotides 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 ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kUobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (avaUable to the pubUc 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 (avaUable to the pubhc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides 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 pubhc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of ahgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments.
  • ohgonucleotides 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 partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
  • 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 linked 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 maybe 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 imrnunological 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 stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cof actors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aU occurrences of the nitrogenous base thymine are replaced with uracU, 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 bodUy fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a cell; 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 about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, shdes, 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 cell 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 limited to, bacteriophage or viral infection, electroporation, heat shock, hpofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of dehberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C et al. (2002) Science 295:868-872).
  • a recombinant viral vector such as a lentiviral vector
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction 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.
  • 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 "aUelic” (as defined above), "splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but wiU generally have a greater or lesser number of polynucleotides due to alternate splicing during mRNA processing.
  • the conesponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotides that vary from one species to another. The resulting polypeptides wUl 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 or sequence similarity 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 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 greatersequence identity or sequence simUarity over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include 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 proliferative disorders including cancer, developmental disorders, neurological disorders, autoirnmune/inflammatory disorders, reproductive disorders, and disorders of the placenta.
  • CGDD ceU growth, differentiation, and death
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte 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 ID) as shown.
  • Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
  • Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ID 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 probabUity scores for the matches 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 applicable, 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 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites.
  • Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were apphed.
  • Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabhsh that the claimed polypeptides are proteins associated with ceU growth, differentiation, and death.
  • SEQ ID NO:l is 73% identical, fromresidue N84 to residue P366, to human M-phase inducer phosphatase 3 isoform cdc25Cdm (GenBank ID gl 1932149) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabUity score is 1.3E-102, which indicates the probability of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ID NO: 1 has homology to CeU division cycle 25C, a member of the Cdc25 famUy of protein tyrosine phosphatases that positively regulate the cell division cycle, which dephosphorylates the cychn B-dependent protein kinase CDC2 and thereby triggers entry into mitosis.
  • SEQ ID NO:l also contains a domain with homology to Rhodanese (sulfur transferase), as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein famUies/domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:l is a cell division-cycle phosphatase.
  • SEQ ID NO:5 is 99% identical, fromresidue Ml to residue Y1752, to human (AF509326) abnormal spindles (GenBank ID g23884551) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probabUity of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ID NO:5 also has homology to proteins that are smaU molecule-binding proteins, that may participate in coordinating mitotic spindle functions with mitotic entry, which activity may be regulated by calmodulin binding, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:5 also contains IQ calmoduhn-binding domains and short calmoduhn-binding domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PFAM and SMART databases of conserved protein famihes/domains. (See Table 3.) Data from BLAST analyses against the PRODOM database, provide further corroborative evidence that SEQ ID NO:5 is a calmoduhn-binding molecule.
  • HMM hidden Markov model
  • SEQ ID NO:13 is 96% identical, fromresidue Ml to residue S243, to human cyclin E2 (GenBank ID g4008085) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabUity score is 5. le-193, which indicates the probabUity of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:13 also has homology to nuclear cyclin E2, a Gl -specific CDK kinase regulatory subunit that interacts with CDK2 and CDK3 and is overexpressed in transformed ceUs, as dete ⁇ nined by BLAST analysis using the PROTEOME database.
  • BLAST Basic Local Ahgnment Search Tool
  • SEQ ID NO: 13 also contains a cyclin, N-terminal domain and a domain present in cyclins, TFIIB and retinoblastoma as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM/SMART databases of conserved protein famUies/domains.
  • HMM hidden Markov model
  • SEQ ID NO:19 is a splice variant of human TACC1 (transforming acidic coiled-coU 1) (GenBank ID g3435157) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.4e-46, which indicates the probabUity of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO: 19 also has homology to proteins that are involved in microtubule-centrosome interactions, that may contribute to genetic instabUity, and that may promote malignant ceU growth, as determined by BLAST analysis using the PROTEOME database. Data from BLAST analysis against the PRODOM database (see Table 3) provides further corroborative evidence that SEQ ID NO:19 is a member of the TACC family of transforming proteins.
  • BLAST Basic Local Ahgnment Search Tool
  • SEQ ID NO:2-4, SEQ ID NO:6-12, SEQ ID NO:14-18, and SEQ ID NO:20 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO: 1-20 are described in Table 7.
  • the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 hsts the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence inbasepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:21-40 or that distinguish between SEQ ID NO:21-40 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotides.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The S anger 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 FL_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 apphed, and YYYYY is the number of the prediction generated by the algorithm, and N JA3 ..., 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.
  • a polynucleotide sequence identified as FlXXXXXX_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-stxetching" algorithm was apphed, 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 hsts examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those fuU length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID).
  • Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB 1 SNP).
  • Column 7 shows the allele found in the EST sequence.
  • Columns 8 and 9 show the two aUeles found at tiie SNP site.
  • Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST.
  • Columns 11- 14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n/a (not avaUable) indicates that the allele frequency was not determined for the population.
  • the invention also encompasses CGDD variants.
  • CGDD variants can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the CGDD amino acid sequence, and can contain at least one functional or structural characteristic of CGDD.
  • Various embodiments also encompass polynucleotides which encode CGDD.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:21-40, which encodes CGDD.
  • the polynucleotide sequences of SEQ ID NO:21-40 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base Ihymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses variants of a polynucleotide encoding CGDD.
  • a variant polynucleotide wiU have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polynucleotide sequence identity to a polynucleotide encoding CGDD.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:21-40 which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:21-40.
  • Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CGDD.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding CGDD.
  • a sphce variant may have portions which have significant sequence identity to a polynucleotide encoding CGDD, but wUl generaUy have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing.
  • a splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding CGDD over its entire length; however, portions of the splice 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 encoding
  • CGDD CGDD.
  • a polynucleotide comprising a sequence of SEQ ID NO:21, a polynucleotide comprising a sequence of SEQ ID NO:22 and a polynucleotide comprising a sequence of SEQ ID NO:23 are sphce variants of each other.
  • a polynucleotide comprising a sequence of SEQ ID NO:32 and a polynucleotide comprising a sequence of SEQ ID NO:35 are sphce variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:33 and a polynucleotide comprising a sequence of SEQ ID NO:34 are sphce variants of each other.
  • Any one of the sphce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CGDD.
  • polynucleotides which encode CGDD and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring CGDD under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides 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 utilized 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 polynucleotides which encode CGDD and CGDD derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding CGDD or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO:21-40 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
  • Hybridization conditions including annealing and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are well 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 (Apphed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Apphed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wney VCH, New York NY, pp. 856-853).
  • machines such as the MICROLAB 2200 liquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then
  • the nucleic acids encoding CGDD maybe extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • one method which may be employed restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Apphc. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Trigha, T. et al.
  • a third method involves PCR amphfication of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Apphc. 1:111-119).
  • multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers may be designed using commerciaUy avaUable software, such as OLIGO 4.06 primer analysis software (National Biosciences, Madison MM) 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
  • hbraries When screening for full length cDNAs, it is preferable to use hbraries that have been size-selected to include larger cDNAs. In addition, random-primed hbraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an ohgo d(T) library does not yield a full-length cDNA. Genomic hbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commerciaUy avaUable may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capiUary 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/hght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed 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 limited amounts in a particular sample.
  • 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 polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express CGDD.
  • the polynucleotides of the 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 limited 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 ohgonucleotides may be used to engineer the nucleotide sequences.
  • ohgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce 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.
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library 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.
  • polynucleotides encoding CGDD may be synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • CGDD itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or sohd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (Apphed Biosystems).
  • AdditionaUy the amino acid sequence of CGDD, or any part thereof, may be altered during direct synthesis andor combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance liquid chromatography (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 (Creighton, supra, pp. 28-53).
  • the polynucleotides 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 polynucleotides encoding CGDD.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding CGDD. Such signals include the ATG initiation codon and adjacent sequences, e.g.
  • Methods which are weU known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding CGDD and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).
  • a variety of expression vector/host systems may be utihzed to contain and express polynucleotides encoding CGDD.
  • 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., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems (Sambrook and Russell, supra; Ausubel et al., supra; Van Heeke, G.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31 :219-226; Verma, I.M. and N. Somia (1997) Nature 389:239- 242).
  • the invention is not limited by the host ceU employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding CGDD.
  • routine cloning, subcloning, and propagation of polynucleotides encoding CGDD can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Joha CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRIPT Stratagene, La Joha CA
  • PSPORT1 plasmid Invitrogen.
  • 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 (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem 264:5503-5509).
  • 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 polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
  • Plant systems may also be used for expression of CGDD. Transcription of polynucleotides 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 maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ.
  • constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw HU1, New York NY, pp. 191-196).
  • mammalian ceUs a number of viral-based expression systems may be utilized.
  • polynucleotides encoding CGDD may be hgated 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 ceUs (Logan, J.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host ceUs.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs Human artificial chromosomes
  • CGDD complementary metal-oxide-semiconductor
  • ceU lines can be transformed into ceU lines using expression vectors which may contain viral origins of rephcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs may be aUowed 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 allows growth and recovery of ceUs which successfully 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 lines. These include, but are not hmited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ' and apr ' cells, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • ⁇ ls and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • Additional selectable genes have been described, e.g., tipB and hisD, which alter ceUular requirements for metabohtes (Hartman, S.C. and R.C.
  • Visible markers e.g., anfhocyanins, green fluorescent proteins (GFP; BD 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 (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
  • 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 polynucleotides 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 well.
  • host ceUs that contain the polynucleotide encoding CGDD and that express CGDD may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amphfication, 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.
  • Imrnunological methods for detecting and measuring the expression of CGDD using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated ceU sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on CGDD is preferred, but a competitive binding assay may be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CGDD include ohgolabehng, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide.
  • polynucleotides encoding CGDD, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commerciaUy avaUable kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemUuminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with polynucleotides encoding CGDD may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly 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 ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, 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 avaUable 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 polynucleotides encoding CGDD may be hgated 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 commerciaUy avaUable antibody may facUitate the screening of peptide libraries for inhibitors of CGDD activity.
  • Heterologous protein and peptide moieties may also facUitate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin 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 immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hernagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy avaUable 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 following purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16).
  • a variety of commerciaUy avaUable kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled CGDD maybe 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 CGDD
  • One or more test compounds may be screened for specific binding to CGDD.
  • 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to CGDD.
  • Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or smaU molecules.
  • variants of CGDD can be used to screen for binding of test compounds, such as antibodies, to CGDD, a variant of CGDD, or a combination of CGDD and/or one or more variants CGDD.
  • a variant of CGDD can be used to screen for compounds that bind to a variant of CGDD, but not to CGDD having the exact sequence of a sequence of SEQ ID NO: 1-20.
  • CGDD variants used to perfomi such screening can have a range of about 50% to about 99% sequence identity to CGDD, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to CGDD can be closely related to the natural ligand of CGDD, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural ligand of a receptor CGDD (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to CGDD can be closely related to the natural receptor to which CGDD binds, at least a fragment of the receptor, or a fragment of the receptor including aU or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for CGDD which is capable of propagating a signal, or a decoy receptor for CGDD which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) C r. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328- 336).
  • the compound can be rationally designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGi (Taylor, P.C et al. (2001) Cun. Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to CGDD, fragments of CGDD, or variants of CGDD.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of CGDD.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of CGDD.
  • an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of CGDD.
  • anticalins can be screened for specific binding to CGDD, fragments of
  • CGDD CGDD
  • Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Skena, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity. In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit
  • CGDD involves producing appropriate cells which express CGDD, either as a secreted protein or on the cell membrane.
  • Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells 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 solid 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.
  • the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a sohd support.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
  • examples of such assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Nati. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982- 10988).
  • a polypeptide compound such as a ligand
  • CGDD CGDD, fragments of CGDD, or variants of CGDD 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 plurality of test compounds may be screened.
  • polynucleotides encoding CGDD or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • mouse ES cells such as the mouse 129/SvJ cell line
  • the ES ceUs are transformed 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) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceU 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 lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, 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 lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • CGDD CGDD
  • a mammal inbred to overexpress CGDD may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • THERAPEUTICS Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CGDD and proteins associated with cell growth, differentiation, and death.
  • examples of tissues expressing CGDD can be found in Table 6 and can also be found in Example XI.
  • CGDD appears to play a role in ceU proliferative disorders including cancer, developmental disorders, neurological disorders, autoimmune/inflammatory disorders, reproductive disorders, and disorders of the placenta.
  • 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 limited to, a ceU prohferative 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, colon, gaU bladder, ganglia, gastrointestinal tract, heart, kidney
  • 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 limited to, those described above.
  • a composition comprising a substantially purified CGDD in conjunction with a suitable pharmaceutical earner may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CGDD including, but not limited 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 limited to, those hsted 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.
  • disorders include, but are not hmited to, those ceU proliferative disorders including cancer, developmental disorders, neurological disorders, autoimmune/inflammatory disorders, reproductive disorders, and disorders of the placenta described above.
  • an antibody which specificaUy binds CGDD may be used directly as an antagonist or indirectly as a targeting or delivery 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 limited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments 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 skUl 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 hbraries 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 limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • neutralizing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have apphcation in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others may be immunized by injection with CGDD or with any fragment or ohgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase imrnunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oU emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium paiyum are especiaUy preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to CGDD have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wUl consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantiaUy 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 maybe produced.
  • Monoclonal antibodies to CGDD maybe prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the hterature (Oriandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter,
  • Antibody fragments which contain specific binding sites for CGDD may also be generated.
  • such fragments include, but are not limited 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 hbraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989)
  • 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 estabhshed specificities are well 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).
  • Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for CGDD.
  • K a is defined as the molar concentration of CGDD-antibody complex divided by the molar concentrations of free antigen and free antibody under equihbrium conditions.
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular CGDD epitope, represents a true measure of affinity.
  • High-af ⁇ nity antibody preparations with K a 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 6 to 10 7 L/mole are preferred for use in immunopurification and simUar 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; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John WUey & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of CGDD-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generaUy avaUable (Catty, supra; Cohgan et al., supra).
  • 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 oligonucleotides) to the coding or regulatory regions of the gene encoding CGDD.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
  • antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CGDD (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Allergy Chn. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) FASEB J. 9:1288- 1296).
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno- associated virus vectors (Miller, A.D.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- linked 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. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • hepatitis B or C virus HBV, HCV
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodiumfalciparum and Trypanosoma cruzi.
  • the expression of CGDD from an appropriate population of transduced ceUs may alleviate the clinical manifestations caused by the genetic deficiency.
  • diseases or disorders caused by deficiencies in CGDD are treated by constructing mammalian 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 delivery, (hi) hposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (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 limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (BD Clontech, Palo Alto CA).
  • CGDD may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (n) an inducible promoter (e.g., the tetracycline-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.V. and H.M. Blau (1998) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • TRANSFECTION KIT avaUable from Invitrogen
  • aUow one with ordinary skUl in the art to dehver 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 mammahan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to CGDD expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding CGDD under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (hi) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commercially avaUable (Stratagene) and are based onpubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad.
  • the vector is propagated in an appropriate vector producing ceU line (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. Mffler (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R.
  • VSVg vector producing ceU line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus 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 skUled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71 :7020- 7029; Bauer, G.
  • an adenovirus-based gene therapy delivery system is used to dehver polynucleotides encoding CGDD to ceUs which have one or more genetic abnormalities with respect to the expression of CGDD.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary sk l in the art.
  • Rephcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas
  • adenoviral vectors for gene therapy
  • Adenovirus vectors for gene therapy 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).
  • a herpes-based, gene therapy delivery system is used to dehver polynucleotides encoding CGDD to target ceUs which have one or more genetic abnormalities with respect to the expression of CGDD.
  • the use of herpes simplex virus (HS V)-based vectors may be especiaUy valuable for introducing 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 skill in the art.
  • a rephcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to dehver a reporter gene to the eyes of primates (Liu, X. et al.
  • HSV-1 virus vector has also been disclosed in detaU in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 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 cell 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.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to dehver polynucleotides encoding CGDD to target ceUs.
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • RNA rephcation a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA rephcates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • alphaviruses will aUow the introduction of CGDD into a variety of cell types.
  • the specific transduction of a subset of ceUs in a population may require the sorting of cells prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, perfo ⁇ ning alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weU known to those with ordinary skUl in the art.
  • Ohgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple hehx pairing is useful because it causes inhibition of the ability of the double hehx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNAhave been described in the literature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Pubhshing, 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
  • Ribozymes 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 specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding CGDD.
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding CGDD. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU lines, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stability and half-life. Possible modifications include, but are not limited 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 linkages within the backbone of the molecule.
  • RNA interference RNA interference
  • PTGS post-transcriptional gene silencing
  • RNAi is a post-transcriptional mode of gene sUencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene.
  • dsRNA double-stranded RNA
  • PTGS can also be accomphshed by use of DNA or DNA fragments as weU. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T. (2000; Nature 404:804-808).
  • PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
  • RNAi can be induced in mammalian ceUs by the use of small interfering RNA also known as siRNA.
  • siRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
  • siRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs.
  • the use of siRNA for inducing RNAi in mammalian ceUs is described by Elbasbir, S.M. et al. (2001 ; Nature 411 :494-498).
  • siRNA can be generated indirectly by introduction of dsRNA into the targeted ceU.
  • siRNA can be synthesized directly and introduced into a ceU by transfection methods and agents described herein or known in the art (such as hposome-mediated transfection, viral vector methods, or other polynucleotide deUvery/ ntroductory methods).
  • Suitable siRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3 ' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred.
  • Regions to be avoided for target siRNA sites include the 5 ' and 3 ' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
  • the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be ehminated from consideration.
  • the selected siRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy avaUable methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).
  • long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
  • This can be accomphshed using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958).
  • shRNAs can be dehvered to target ceUs using expression vectors known in the art.
  • siRNA An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion).
  • PSILENCER1.0-U6 circular plasmid
  • shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene- specific sUencing.
  • the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis.
  • Expression levels of the mRNA of a targeted gene can be determined, for example, by northern analysis methods using the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein.
  • Expression levels of the protein encoded by the targeted gene can be determined, for example, by microarray methods; by polyacrylamide gel electrophoresis; and by Western analysis using standard techniques known in the art.
  • 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 maybe effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, 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 therapeutically useful, and in the treatment of disorders associated with decreased CGDD expression or activity, a compound which specifically promotes expression of the polynucleotide encoding CGDD may be therapeuticaUy useful.
  • 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 library of naturaUy-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 library of chemical compounds created combinatorially 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 permeabihzed cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding CGDD are assayed by any method commonly known in the art.
  • 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; Amdt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun.
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides
  • vectors into ceUs or tissues are avaUable and equaUy suitable for use in vivo, in vitro, and ex vivo.
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by hposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
  • any of the therapeutic methods described above may be apphed 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, celluloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • Such compositions may consist of CGDD, antibodies to CGDD, and mimetics, agonists, antagonists, or inhibitors of CGDD.
  • compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generaUy aerosohzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol dehvery of fast-acting formulations is weU-known in the art.
  • 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 capability of those skilled in the art.
  • Speciahzed forms of compositions may be prepared for direct intraceUular dehvery of macromolecules comprising CGDD or fragments thereof.
  • macromolecules comprising CGDD or fragments thereof.
  • hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular delivery of the macromolecule.
  • CGDD or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV 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 therapeuticaUy 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 administration. 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 amehorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU 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 little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of
  • the exact dosage wUl be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration 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, drug combinations), 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-life 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 administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generaUy avaUable to practitioners in the art. Those skilled in the art wUl employ different formulations for nucleotides than for proteins or their inhibitors. SimUarly, dehvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc.
  • 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 utilize 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 estabhshes the parameters for diagnosing disease.
  • polynucleotides encoding CGDD may be used for diagnostic purposes.
  • the polynucleotides which may be used include ohgonucleotides, 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 maybe 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 polynucleotides, 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 amphfication wUl determine whether the probe identifies only naturaUy occurring sequences encoding CGDD, aUehc 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 ID NO:21-40 or from genomic sequences including promoters, enhancers, and introns of the CGDD gene.
  • Means for producing specific hybridization probes for polynucleotides encoding CGDD include the cloning of polynucleotides encoding CGDD or CGDD derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commerciaUy avaUable, 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 radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding CGDD may be used for the diagnosis of disorders associated with expression of CGDD.
  • a ceU prohferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycyfhemia 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, colon, gaU bladder, gangha, gastrointestinal tract, heart, kidney, hver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis
  • MCTD mixed connective tissue disease
  • Polynucleotides 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-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered CGDD expression. Such qualitative or quantitative methods are weU known in the art.
  • polynucleotides encoding CGDD may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to 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 polynucleotides 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 clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabhshed. This may be accomphshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CGDD, under conditions suitable for hybridization or amphfication.
  • 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 estabhsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to dete ⁇ riine 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 clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive teatment earlier, thereby preventing the development or further progression of the cancer.
  • ohgonucleotides designed from the sequences encoding CGDD may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
  • Ohgomers wiU 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.
  • Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oligonucleotide primers derived from polynucleotides 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 limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from polynucleotides encoding CGDD are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodUy fluids, and the like.
  • 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 oligonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in silico 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).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as hfe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu
  • Methods which may also be used to quantify the expression of CGDD include radiolabeling or biotinylating nucleotides, coamphfication of a control nucleic acid, and interpolating results from standard curves (Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C et al. (1993) Anal. Bioche 212:229-236).
  • the speed of quantitation of multiple samples may be accelerated by ranning the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • ohgonucleotides or longer fragments derived from any of the polynucleotides 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 (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No.
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or ceU type.
  • ttie hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microanay.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, cell lines, 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 hne.
  • 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 preclinical 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). If a test compound has a signature simUar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • the toxicity of a test compound can be 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 conesponding 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.
  • Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or ceU type.
  • proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a 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 isoelectric 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 visuahzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or sUver 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 partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed 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 interest. 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 contacting the microarray with 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 maybe 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 reliable 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 treated 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 treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual protems 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.
  • Microanays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.
  • 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 potentially 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 (BACs), bacterial PI constructions, or single chromosome cDNA hbraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA hbraries
  • nucleic acid sequences may be used to develop genetic linkage maps, for example, which conelate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) 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.
  • OMIM Online Mendelian Inheritance in Man
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammahan 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 localized by genetic linkage 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 (Gatti, R.A. et al. (1988) Nature 336:577-580).
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • CGDD its catalytic or immunogenic fragments, or ohgopeptides thereof can be used for screening hbraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a sohd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between CGDD and the agent being tested may be measured.
  • WO84/03564 large numbers of different smaU test compounds are synthesized on a sohd 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 drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a sohd support.
  • competitive drug screening assays in which neutralizing antibodies capable of binding CGDD specificaUy compete with a test compound for binding CGDD.
  • 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 cunently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs are derived from cDNA hbraries described in the LIFESEQ database (Incyte, Palo Alto CA). Some tissues are homogenized and lysed in guanidinium isothiocyanate, whUe others are homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates are centrifuged over CsCl cushions or extracted with chloroform. RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods. Phenol extraction and precipitation of RNA are repeated as necessary to increase RNA purity.
  • TRIZOL Invitrogen
  • RNA is treated with DNase.
  • poly(A)+ RNA is isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA is isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
  • Stratagene is provided with RNA and constructs the corresponding cDNA hbraries. Otherwise, cDNA is synthesized and cDNA hbraries are constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription is initiated using ohgo d(T) or random primers. Synthetic ohgonucleotide adapters are hgated to double stranded cDNA, and the cDNA is digested with the appropriate restriction enzyme or enzymes.
  • the cDNA is size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs are ligated into compatible restriction enzyme sites of the polyhnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid
  • Plasmids obtained as described in Example I are recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids are purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaifhersburg MD); and QIAWELL 8 Plasmid,
  • plasmid DNA is amplified from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem 216:1-14). Host ceU lysis and thermal cycling steps are carried out in a single reaction mixture. Samples are processed and stored in 384- weU plates, and the concentration of amplified plasmid DNA is quantified fluorometricaUy 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 II are sequenced as follows. Sequencing reactions are processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (HamUton) Uquid transfer system. cDNA sequencing reactions are prepared using reagents provided by Amersham Biosciences or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides are carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Apphed 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 are identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences are selected for extension using the techniques disclosed in Example VIII.
  • Polynucleotide sequences derived from Incyte cDNAs are validated by removing vector, linker, 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 are then queried against a selection of pubhc databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus noiyegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte, Palo Alto CA); hidden Markov model ( ⁇ MM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM (Haft, D.H.
  • pubhc databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.
  • the queries are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences are assembled to produce fuU length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences are used to extend Incyte cDNA assemblages to full length. Assembly is performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages are screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the fuU length polynucleotide sequences are translated to derive the corresponding full length polypeptide sequences.
  • a polypeptide may begin at any of the methionine residues of the fuU length translated polypeptide.
  • FuU length polypeptide sequences are subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, bidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
  • GenBank protein databases Genpept
  • PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM bidden Markov model
  • Prosite bidden Markov model-based protein famUy databases
  • HMM-based protein famUy databases such as PFAM, INCY, and TIGRFAM
  • HMM-based protein domain databases such as SMART.
  • FuU length polynucleotide sequences are also
  • Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between ahgned sequences.
  • Table 7 summarizes tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides apphcable 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, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probabUity values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabUity value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karhn (1998) Cun. 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 of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once is set to 30 kb.
  • the encoded polypeptides are analyzed by querying against PFAM models for proteins associated with cell growth, differentiation, and death. Potential proteins associated with ceU growth, differentiation, and death are also identified by homology to Incyte cDNA sequences that have been annotated as proteins associated with ceU growth, differentiation, and death. These selected Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri pubhc databases.
  • Genscan-predicted sequences are 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 is also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription.
  • Incyte cDNA coverage is avaUable, this information is used to correct or confirm the Genscan predicted sequence.
  • FuU length polynucleotide sequences are obtained by assembling Genscan- predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial cDNA sequences are extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III are mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster is analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible sphce variants that are subsequently confirmed, edited, or extended to create a fuU length sequence. Sequence intervals in which the entire length of the interval is present on more than one sequence in the cluster are identified, and intervals thus identified are considered to be equivalent by transitivity.
  • Partial DNA sequences are extended to full length with an algorithm based on BLAST analysis.
  • First, partial cDNAs assembled as described in Example III are queried against pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program.
  • the nearest GenBank protein homolog is then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein is generated by using the resultant high-scoring segment pahs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pahs
  • GenBank protein homolog the chimeric protein, or both are used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences are therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences are examined to determine whether they contain a complete gene.
  • SHGC Stanford Human Genome Center
  • WIGR Whitehead Institute for Genome Research
  • 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.
  • Analogous computer techniques applying BLAST are used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte). 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 simUar.
  • 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 normalized value between 0 and 100, and is calculated as foUows: the BLAST score is multiphed 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 pah with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quahty in a BLAST ahgnment. 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.
  • polynucleotides encoding CGDD are analyzed with respect to the tissue sources from which they are derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA sequence is derived from a cDNA hbrary 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 structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ ceUs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognafhic system; unclassified/mixed; or urinary tract.
  • the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding CGDD.
  • cDNA sequences and cDNA library/tissue information are found in the LIFESEQ database (Incyte, Palo Alto CA).
  • Full length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using ohgonucleotide primers designed from this fragment.
  • One primer is synthesized to initiate 5' extension of the known fragment, and the other primer is synthesized to initiate 3' extension of the known fragment.
  • the initial primers are 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 hairpin structures and primer-primer dimerizations is avoided.
  • Selected human cDNA hbraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed.
  • PCR is performed in 96-weU plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2- mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pah 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+ are 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 6: 68°C, 5 min; Step 7: storage at 4°C
  • the concentration of DNA in each weU is 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 is scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l ahquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose gel to determine which reactions are successful in extending the sequence.
  • the extended nucleotides are desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to rehgation into pUC 18 vector
  • the digested nucleotides are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are excised, and agar digested with Agar ACE (Promega).
  • Extended clones were rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fUl-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed cells are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb hquid media.
  • the ceUs are lysed, and DNA is amphfied by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing parameters: 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 is quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamphfied using the same conditions as described above.
  • Samples are dUuted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • SNPs single nucleotide polymorphisms
  • Preliminary filters remove the majority of basecall errors by requiring a minimum Phred quality score of 15, and remove sequence alignment enors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants.
  • An automated procedure of advanced chromosome analysis is apphed to the original chromatogram ffles in the vicinity of the putative SNP.
  • Clone error filters use statistically generated algorithms to identify enors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering enor fUters use statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences.
  • a final set of filters removes duplicates and SNPs found in immunoglobulins or T-cell receptors.
  • Certain SNPs are selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprises 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprises 194 individuals (97 male, 97 female), all African Americans.
  • the Hispanic population comprises 324 individuals (162 male, 162 female), all Mexican Hispanic.
  • the Asian population comprises 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies are first analyzed in the Caucasian population; in some cases those SNPs which show no allelic variance in this population are not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes
  • Hybridization probes derived from SEQ ID NO:21-40 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 Ci of
  • [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An ahquot 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 II, 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 NYTRAN PLUS nylon membranes (Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuahzed using autoradiography or an alternative imaging means and compared. XI. Microarrays
  • the linkage or synthesis of array elements upon a microarray can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing; see, e.g., BaldeschweUer et al., supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays : A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass shdes, glass chips, and silicon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using avaUable methods and machines weU known to those of ordinary skUl in the art and may contain any appropriate number of elements (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 ohgomers thereof may comprise the elements of the microanay. Fragments or ohgomers 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 microanay may be assessed.
  • microarray preparation and usage is described in detaU below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(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 Biosciences).
  • 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 labeling) 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 (BD Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • SpeedVAC SpeedVAC
  • Sequences of the present invention are used to generate anay elements.
  • Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • Amplified anay elements are then purified using SEPHACRYL-400 (Amersham Biosciences). Purified anay elements are immobilized on polymer-coated glass slides. Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distUled water washes between and after treatments.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l of the anay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capUlary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of anay element sample per slide.
  • Microanays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distUled water. Non-specific binding sites are blocked by incubation of microanays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C followed by washes in 0.2% SDS and distilled water as before. Hybridization 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 aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
  • the anays are transfened to a waterproof chamber having a cavity just slightly larger than a microscope slide.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the anays 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 lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the anay using a 20X microscope objective (Nikon, Inc., MelvUle NY).
  • the slide containing the anay is placed on a computer-controlled 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 light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier 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 anay is typically 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 cahbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the anay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be conelated with a weight ratio of hybridizing species of l:100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 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 conected 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 conesponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Anay elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed. Expression
  • SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 showed tissue-specific expression as determined by microarray analysis.
  • RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microanay.
  • SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 can be used as a tissue marker for spinal cord nerve and hver tissues.
  • expression of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 was upregulated in stimulated THP-1 ceUs versus untreated THP-1 ceUs as determined by microarray analysis.
  • THP-1 ceUs were stimulated in vitro with soluble PMA and ionomycin for 0.5, 1, 2, 4, and 8 hours. These treated ceUs were compared to untreated THP-1 ceUs kept in culture in the absence of stimuli.
  • Expression of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 was increased at least two-fold at aU time points tested.
  • THP-1 differentiates into a macrophage-like ceU that displays many characteristics of peripheral human macrophages.
  • SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 can be used for one or more of the following: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • RPMI 6666 is a B ceU lymphoblast ceU line derived from the peripheral blood of a male with Hodgkin's disease.
  • RPMI 666 ceUs are immature lymphocyte-producing immunoglobulins and present ceU-associated Epstein-Barr virus (EBV) particles.
  • EBV Epstein-Barr virus
  • RPMI 6666 ceUs have been used to study ceU signaling in human B ceUs and identify factors produced by those cells.
  • the RPMI 6666 ceUs were stimulated in vitro with E. coli LPS for 0.5, 1, 2, 4, and 8 hours.
  • SEQ ID NO:25 can be used for one or more of the foUowing: i) monitoring treatment of autoimmune/inflammatory disorders, ii) diagnostic assays for autoimmune/inflammatory disorders, and hi) developing therapeutics and/or other treatments for autoimmune/iimammatory disorders.
  • MCF-10A is a breast mammary gland (luminal ductal characteristics) ceU line that was isolated from a female with fibrocystic breast disease. MCF-10A expresses cytoplasmic keratins, epithehal sialomucins, and milkfat globule antigens. PMA is a broad activator of the protein kinase C-dependent pathways. Ionomycin is a calcium ionophore that permits the entry of calcium into the ceU, increasing the cytosolic calcium concentration.
  • BT-20 is a breast carcinoma ceU line derived in vitro from the cells emigrating out of thin slices of a tumor mass.
  • BT-20 cells were stimulated in vitro with soluble PMA and ionomycin for 0.5, 1, 2, 4, and 8 hours.
  • Treated cells were compared to untreated ceUs kept in culture in the absence of stimuli.
  • Expression of SEQ ID NO:25 was decreased at least two-fold i the 4 and 8 hour time points. Therefore, in various embodiments, SEQ ID NO:25 can be used for one or more of the foUowing: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and hi) developing therapeutics and/or other treatments for breast cancer.
  • DU-145 is a prostate carcinoma ceU line isolated from a metastatic site in the brain of a male with widespread metastatic prostate cancer. DU-145 has no detectable sensitivity to hormones. DU-145 ceUs were stimulated in vitro with soluble PMA and ionomycin for 4, 8, 14, and 24 hours. Treated cells were compared to untreated ceUs kept in culture in the absence of stimuli. Expression of SEQ ID NO:25 was decreased at least two-fold in aU time points for the treated cells.
  • expression of SEQ ID NO:25 was down-regulated in treated versus untreated human prostate adenocarcinoma ceU lines as determined by microarray analysis.
  • PC-3 is a prostate adenocarcinoma ceU line isolated from a metastatic site in the bone of a male with grade IV prostate adenocarcinoma.
  • PC-3 ceUs were stimulated in vitro with soluble PMA and ionomycin for 4, 8, 14, and 24 hours. Treated ceUs were compared to untreated ceUs kept in culture in the absence of stimuli. Expression of SEQ ID NO:25 was decreased at least two-fold in the 24 hour time point.
  • SEQ ID NO:25 was up-regulated in diseased versus normal human prostate ceU lines as determined by microanay analysis.
  • the gene expression profiles of prostate carcinoma ceU hnes CA-HPV-10, LNCaP, PC-3, DU-145, and MDAPCa2b were compared to that of nontumorigenic primary prostate epithehal PrEC ceUs.
  • Expression of SEQ ID NO:25 was increased at least two-fold in the DU-145 and PC-3 prostate carcinoma ceUs. Therefore, in various embodiments, SEQ ID NO:25 can be used for one or more of the foUowing: i) monitoring treatment of prostate cancer, ii) diagnostic assays for prostate cancer, and hi) developing therapeutics and/or other treatments for prostate cancer.
  • SEQ ID NO:25 showed tissue-specific expression as determined by microanay analysis.
  • RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microanay.
  • SEQ ID NO:25 Since tiiese hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another. The expression of SEQ ID NO:25 was increased by at least two-fold in thymus as compared to the reference sample. Therefore, SEQ ID NO:25 can be used as a tissue marker for thymus.
  • SEQ ID NO:30 showed differential expression in immune system ceUs exposed to various stimuli, as determined by microarray analysis.
  • Jurkat cells were stimulated in vitro with soluble PMA and ionomycin for 0.5, 1 , 2, and 4 hours. These treated ceUs were compared to untreated Jurkat ceUs kept in culture in the absence of stimuli.
  • SEQ ID NO:30 was found to be upregulated by at least two-fold in ceUs treated for 0.5, 1, 2, and 4 hours.
  • SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • PBMCs from the blood of 4 healthy volunteer donors were stimulated with LPS in the presence or absence of IL-10, for 2, 4, 24, and 72 hours.
  • matching PBMCs were also treated for the same duration with IL-10 alone to monitor the effects of this factor in the absence of stimulation. The treated PBMCs were compared to matching PBMCs kept in culture for 24 hours in the presence of medium alone.
  • SEQ ID NO:30 was found to be upregulated by at least two-fold in ceUs treated with LPS alone for 2, 4, and 24 hours; with IL-10 alone for 2 and 4 hours; and with LPS + IL-10 for 2 and 4 hours. Therefore, in various embodiments, SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • PHA blasts derived from the PBMCs of 5 healthy volunteer donors were stimulated for 9 days in the presence of PHA and IL-2. These T ceU blasts were washed and stimulated for 1 and 6 hours in the presence of anti-CD3 monoclonal antibody, anti-CD28 antibody, or a combination of both. These reactivated T ceUs were compared to matching untreated PHA blasts.
  • SEQ ID NO:30 was found to be upregulated by at least two-fold in ceUs treated with anti-CD3 monoclonal antibody for 1 and 6 hours, as weU as in ceUs treated with anti-CD3 + anti-CD28 monoclonal antibodies for 1 and 6 hours.
  • SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, h) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • THP-1 ceUs were stimulated in vitro with soluble PMA and ionomycin for 0.5, 1, 2, 4, and 8 hours. These treated ceUs were compared to untreated THP-1 ceUs kept in culture in the absence of stimuli.
  • SEQ ID NO:30 was found to be upregulated by at least two- fold in ceUs treated for 0.5, 1, 2, 4, and 8 hours. Therefore, in various embodiments, SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • to study the response of promonocytes and monocytes to LPS to study the response of promonocytes and monocytes to LPS.
  • SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and ih) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • SEQ ID NO:30 was found to be downregulated by at least two-fold in matched tumorous versus normal lung tissues in four out often donors tested. Therefore, in various embodiments, SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, h) diagnostic assays for lung cancer, and hi) developing therapeutics and/or other treatments for lung cancer.
  • CeU lines compared included: a) PrEC, a primary prostate epithehal ceU line isolated from a normal donor, b) DU 145, a prostate carcinoma ceU line isolated from a metastatic site in the brain of 69-year old male with widespread metastatic prostate carcinoma, c) LNCaP, a prostate carcinoma ceU line isolated from a lymph node biopsy of a 50-year-old male with metastatic prostate carcinoma, and d) PC-3, a prostate adenocarcinoma ceU line isolated from a metastatic site in the bone of a 62-year-old male with grade IV prostate adenocarcinoma.
  • SEQ ID NO:30 was found to be upregulated by at least two-fold in the DU 145 ceU line and downregulated by at least two-fold in the LNCaP ceU line. Therefore, in various embodiments, SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of prostate cancer, ii) diagnostic assays for prostate cancer, and hi) developing therapeutics and/or other treatments for prostate cancer.
  • HAECs were treated with TNF- ⁇ for 1, 2, 4, 6, 8, 10, 24, and 48 hours. These TNF- ⁇ treated ceUs were compared to untreated HAECs.
  • SEQ ID NO:30 was found to be upregulated by at least two-fold in ceUs treated for 2, 4, 6, 8, 10, 24, and 48 hours. Therefore, in various embodiments, SEQ ID NO:30 can be used for one or more of the following: i) monitoring treatment of immune disorders and related diseases and conditions, h) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • HUVECs were grown to 85% confluency and then treated with 10 ng/ml TNF- ⁇ for 0.33, 0.66, 1, 2, 4, 8, 24, and 48 hours.
  • TNF- ⁇ -treated ceUs were compared to untreated HUVECs coUecled at 85% confluency (0 hour).
  • SEQ ID NO:30 was found to be upregulated by at least two-fold in ceUs treated for 0.66, 1, 2, 4, 8, and 24 hours. Therefore, in various embodiments, SEQ ID NO:30 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, h) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • SEQ ID NO:30 showed tissue-specific expression as determined by microanay analysis. RNA samples isolated from a variety of normal human tissues were compared to
  • Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uterus (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and mdividuaUy hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another.
  • the expression of SEQ ID NO:30 was increased by at least two-fold in blood leukocytes as compared to the reference sample.
  • SEQ ID NO:30 can be used as a tissue marker for blood leukocytes.
  • SEQ ID NO:32 and SEQ ID NO:35 were upregulated by at least two-fold in matched tumorous versus normal lung tissues in the same four out of ten donors tested. Therefore, in various embodiments, SEQ ID NO:32 and/or 35 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, h) diagnostic assays for lung cancer, and hi) developing therapeutics and/or other treatments for lung cancer.
  • SEQ ID NO:32 and SEQ ID NO:35 were upregulated by at least two-fold in matched tumorous versus normal ovarian tissues in the same donor tested.
  • SEQ ID NO:32 and/or SEQ ID NO:35 can be used for one or more of the foUowing: i) monitoring treatment of ovarian cancer, ii) diagnostic assays for ovarian cancer, and hi) developing therapeutics and/or other treatments for ovarian cancer.
  • XII Complementary Polynucleotides
  • Sequences complementary to the CGDD-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring CGDD.
  • ohgonucleotides comprising from about 15 to 30 base pahs
  • essentiaUy the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CGDD.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oligonucleotide 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 limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express CGDD upon induction with isopropyl beta-D- fhiogalactopyranoside (IPTG).
  • IPTG isopropyl beta-D- fhiogalactopyranoside
  • Expression of CGDD in eukaryotic ceUs is achieved by infecting insect or mammalian ceU hnes with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • 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 transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requhes additional genetic modifications to baculovirus (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum Gene Ther. 7:1937- 1945).
  • CGDD is synthesized as a fusion protein with, e.g., glutathione S-transferase (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 glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • 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 et al. (supra, ch. 10 and 16). Purified CGDD obtained by these methods can be used directly in the assays shown in Examples XVII and XVIII, where applicable. XIV. Functional Assays
  • CGDD function is assessed by expressing the sequences encoding CGDD at physiologicaUy elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endofhehal or hematopoietic ceU line, using either hposome 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 nontransfected ceUs and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; BD Clontech), CD64, or a CD64-GFP fusion protein.
  • Flow cytometry (FCM) an automated, laser optics-based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other ceUular properties.
  • 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 light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intraceUular 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 nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods weU known by those of skill in the art. Expression of mRNA encoding CGDD and other genes of interest can be analyzed by northern analysis or microarray techniques. XV. Production of CGDD Specific Antibodies
  • the CGDD amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skUl in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophihc regions are weU described in the art (Ausubel et al, supra, ch. 11).
  • ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma-
  • CGDD CGDD
  • An immunoaffinity column is constructed by covalently coupling anti-CGDD antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • activated chromatographic resin such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • 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.
  • CGDD CGDD
  • biologicaUy active fragments thereof are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem J. 133:529-539).
  • 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.
  • 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 all interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • CGDD activity is demonstrated by measuring the induction of terminal differentiation or ceU cycle progression when CGDD is expressed at physiologicaUy elevated levels in mammalian ceU culture systems.
  • cDNA is subcloned into a mammahan 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 cytomegalovirus promoter.
  • 5-10 g of recombinant vector are transiently transfected into a human ceU line, preferably of endofhehal or hematopoietic origin, using either hposome 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 nontransfected ceUs and is a rehable 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
  • Flow cytometry detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU cycle progression or terminal differentiation.
  • an in vitro assay for CGDD activity measures the transformation of normal human fibroblast ceUs overexpressing antisense CGDD RNA (Garkavtsev, I. and K. Riabowol (1997) Mol. CeU Biol. 17:2014-2019).
  • cDNA encoding CGDD is subcloned into the pLNCX retroviral vector to enable expression of antisense CGDD RNA.
  • the resulting construct is transfected into the ecotropic BOSC23 virus-packaging cell line. Virus contained in the BOSC23 culture supernatant is used to infect the amphotropic CAK8 virus-packaging ceU line.
  • 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 transformed cells: 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 ability to induce tumors when injected into immunodeficient mice.
  • the activity of CGDD is proportional to the extent of transformation of Hs68 ceUs.
  • CGDD can be expressed in a mammalian ceU line by transforming the ceUs with a eukaryotic expression vector encoding CGDD.
  • Eukaryotic expression vectors are commerciaUy avaUable, and the techniques to introduce them into ceUs are weU known to those skilled in the art.
  • ceUs are fractionated as described by Jiang, H.P. et al. (1992; Proc. Natl. Acad. Sci. 89:7856-7860).
  • ceUs peUeted by low-speed centrifugation are resuspended in buffer (10 mM TRIS-HCl, pH 7.4/ 10 mM NaCl 3 mM MgCl 2 / 5 mM EDTA with 10 ug/ml aprotinin, 10 ug/ml leupeptin, 10 ug/ml pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride) and homogenized.
  • the homogenate is centrifuged at 600 x g for 5 minutes.
  • the particulate and cytosol fractions are separated by ultracentrifugation of the supernatant at 100,000 x g for 60 minutes.
  • the nuclear fraction is obtained by resuspending the 600 x g peUet in sucrose solution (0.25 M sucrose/ 10 mM TRIS-HCl, pH 7.4/ 2 mM MgCl 2 ) and recentrifuged at 600 x g. Equal amounts of protein from each fraction are apphed to an SDS/10% polyacrylamide gel and blotted onto membranes. Western blot analysis is performed using CGDD anti-serum. The locahzation of CGDD is assessed by the intensity of the conesponding band in the nuclear fraction relative to the intensity in the other fractions. Alternatively, the presence of CGDD in ceUular fractions is examined by fluorescence microscopy using a fluorescent antibody specific for CGDD.
  • CGDD activity may be demonstrated as the ability to interact with its associated Ras superfamUy protein, in an in vitro binding assay.
  • the candidate Ras superfamUy proteins are expressed as fusion proteins with glutathione S-transferase (GST), and purified by affinity chromatography on glutathione-Sepharose.
  • GST glutathione S-transferase
  • the Ras superfamUy proteins are loaded with GDP by incubating 20 mM Tris buffer, pH 8.0, containing 100 mM NaCl, 2 mM EDTA, 5 mM MgC12, 0.2 mM DTT, 100 ⁇ M AMP-PNP and 10 ⁇ M GDP at 30°C for 20 minutes.
  • CGDD is expressed as a FLAG fusion protein in a baculovirus syste Extracts of these baculovirus ceUs containing CGDD-FLAG fusion proteins are precleared with GST beads, then incubated with GST- Ras superfamUy fusion proteins. The complexes formed are precipitated by glutathione-Sepharose and separated by SDS-polyacrylamide gel electrophoresis. The separated proteins are blotted onto nitroceUulose membranes and probed with commerciaUy avaUable anti-FLAG antibodies. CGDD activity is proportional to the amount of CGDD-FLAG fusion protein detected in the complex.
  • the ability of CGDD to suppress tumorigenesis can be measured by designing an antisense sequence to the 5' end of the gene and transfecting NIH 3T3 cells with a vector transcribing this sequence.
  • an assay for CGDD activity measures the effect of injected CGDD on the degradation of maternal transcripts.
  • Procedures for oocyte collection from Swiss albino mice, injection, and culture are as described in Stutz et al., (supra).
  • a decrease in the degradation of maternal RNAs as compared to control oocytes is indicative of CGDD activity.
  • CGDD activity is measured as the ability of purified CGDD to bind to RNAse as measured by the assays described in Example XVII.
  • an assay for CGDD activity measures syncytium formation in COS ceUs transfected with an CGDD expression plasmid, using the two-component fusion assay described in Mi (supra).
  • This assay takes advantage of the fact that human interleukin 12 (IL-12) is a heterodimer comprising subunits with molecular weights of 35 kD (p35) and 40 kD ( ⁇ 40).
  • IL-12 human interleukin 12
  • COS ceUs transfected with expression plasmids carrying the gene for p35 are mixed with COS ceUs cotransfected with expression plasmids carrying the genes for p40 and CGDD.
  • the level of IL-12 activity in the resulting conditioned medium corresponds to the activity of CGDD in this assay.
  • Syncytium formation may also be measured by light microscopy (Mi et al, supra).
  • An alternative assay for CGDD activity measures ceU prohferation as the amount 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. Where applicable, varying amounts of CGDD hgand are added to the transfected ceUs.
  • 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 cyclin 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 123 I- cyclin-ubiquitin formed is quantified by PHOSPHORIMAGER analysis. The amount of cychn-ubiquitin formation is proportional to the activity of CGDD in the reaction.
  • 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. Mammalian cells are transfected with plasmid containing cDNA encoding CGDD by methods weU known in the art.
  • CeUs are then incubated with radiolabeled nucleotide for various lengths of time. Chromosomal DNA is coUected, and radioactivity is detected using a scintillation counter. Incorporation of radiolabel into chromosomal DNA is proportional to the degree of stimulation of the ceU cycle. To determine if CGDD promotes apoptosis, chromosomal DNA is coUected as above, and analyzed using polyacrylamide gel electrophoresis, by methods weU known in the art. Fragmentation of DNA is quantified by comparison to untransfected control ceUs, and is proportional to the apoptotic activity of CGDD.
  • cyclophilin activity of CGDD is measured using a chymotrypsin-coupled assay to measure the rate of cis to trans interconversion (Fischer, G. et al. (1984) Biomed. Biochi Acta 43:1101-1111).
  • the chymotrypsin is used to estimate the trans-substrate cleavage activity at Xaa-Pro peptide bonds, wherein the rate constant for the cis to trans isomerization can be obtained by measuring the rate constant of the substrate hydrolysis at the slow phase.
  • Samples are incubated in the presence or absence of the irnmunosuppressant drugs CsA or FK506, reactions initiated by addition of chymotrypsin, and the fluorescent reaction measured.
  • cyclophUin activity of CGDD is monitored by a quantitative immunoassay that measures its affinity for stereospecific binding to the irnmunosuppressant drug cyclosporin (Quesniaux, V.F. et al. (1987) Eur. J. Immunol 17:1359-1365).
  • the cyclophilin- cyclosporin complex is coated on a sohd phase, with binding detected using anti-cyclophilin rabbit antiserum enhanced by an antiglobuhn-enzyme conjugate.
  • activity of CGDD is monitored by a binding assay developed to measure the non-covalent binding between FKBPs and irnmunosuppressant drugs in the gas phase using electrospray ionization mass spectrometry (Trepanier, D.J. et al. (1999) Ther. Drug Monit. 21:274- 280).
  • electrospray ionization ions are generated by creating a fine spray of highly charged droplets in the presence of a strong electric field; as the droplet decreases in size, the charge density on the surface increases.
  • Ions are electrostaticaUy directed into a mass analyzer, where ions of opposite charge are generated in spatiaUy separate sources and then swept into capUlary inlets where the flows are merged and where reactions occur.
  • charge states of bound versus unbound CGDD/immunosuppressive drug complexes By comparing the charge states of bound versus unbound CGDD/immunosuppressive drug complexes, relative binding affinities can be estabhshed and correlated with in vitro binding and immunosuppressive activity.
  • Rhodanese activity of CGDD is measured with capillary zone electrophoresis (Glatz, Z. et al. (1999) J. Chromatogr. A. 838:139-148).
  • the determination of rhodanese activity of MDDT is performed in a 75-micron fused-silica capUlary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capUlary temperature of 25 °C and direct detection at 200 nm.
  • Short-end injection or long-end injection procedures are used for sample application. Enzymatic reactions are canied out directly in thermostatted autosampler vials and the formation of SCN- is monitored by sequential capUlary zone electrophoretic runs.

Abstract

L'invention concerne divers modes de réalisation, qui mettent en oeuvre des protéines humaines associées à la croissance, à la différenciation et à la mort cellulaire (CGDD), et des polynucléotides identifiant et codant CGDD. On décrit également des modes de réalisation qui mettent en oeuvre des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. D'autres modes de réalisation mettent en oeuvre des méthodes de diagnostic, de traitement ou de prévention de troubles associés à l'expression aberrante de CGDD.
PCT/US2004/009388 2003-03-24 2004-03-23 Proteines associees a la croissance, a la differenciation et a la mort cellulaire WO2004085625A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2009023959A1 (fr) * 2007-08-22 2009-02-26 University Health Network Peptides de liaison à la calmoduline

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US20070048301A1 (en) * 2002-11-26 2007-03-01 Bodary-Winter Sarah C Compositions and methods for the treatment of immune related diseases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070048301A1 (en) * 2002-11-26 2007-03-01 Bodary-Winter Sarah C Compositions and methods for the treatment of immune related diseases

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009023959A1 (fr) * 2007-08-22 2009-02-26 University Health Network Peptides de liaison à la calmoduline

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