WO2004094623A2 - Proteines d'adhesion cellulaire et de matrice extracellulaire - Google Patents

Proteines d'adhesion cellulaire et de matrice extracellulaire Download PDF

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WO2004094623A2
WO2004094623A2 PCT/US2004/008971 US2004008971W WO2004094623A2 WO 2004094623 A2 WO2004094623 A2 WO 2004094623A2 US 2004008971 W US2004008971 W US 2004008971W WO 2004094623 A2 WO2004094623 A2 WO 2004094623A2
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polynucleotide
seq
polypeptide
amino acid
sequence
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WO2004094623A3 (fr
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Anita Swarnakar
Shanya D. Becha
Uyen K. Tran
Joseph P. Marquis
Vicki S. Elliott
Thomas W. Richardson
Narinder K. Chawla
Jonathan T. Wang
Kristin D. Favero
Mili Gera
Xin Jiang
Alan A. Jackson
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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, cell adhesion and extracellular matrix proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and cell proliferative disorders, including cancer.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and cell adhesion and extracellular matrix proteins.
  • the surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the ECM.
  • the interaction of the cell with its surroundings profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. Families of cell adhesion molecules include the cadherins, integrins, lectins, neural cell adhesion proteins, and some members of the proline-rich proteins.
  • Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms.
  • cadherin molecules cooperate to form focal contacts, or adhesion plaques, between adjacent epithelial cells.
  • the cadherin family includes the classical cadherins and protocadherins.
  • Classical cadherins include the E-cadherin, N-cadherin, and P-cadherin subfamilies.
  • E-cadherin is present on many types of epithelial cells and is especially important for embryonic development.
  • N-cadherin is present on nerve, muscle, and lens cells and is also critical for embryonic development.
  • P-cadherin is present on cells of the placenta and epidermis.
  • cadherins are involved in a variety of cell-cell interactions (Suzuki, S.T. (1996) J. Cell Sci. 109:2609-2611).
  • the intracellular anchorage of cadherins is regulated by their dynamic association with catenins, a family of cytoplasmic signal transduction proteins associated with the actin cytoskeleton.
  • the anchorage of cadherins to the actin cytoskeleton appears to be regulated by protein tyrosine phosphorylation, and the cadherins are the target of phosphorylation-induced junctional disassembly (Aberle, H. et al. (1996) J. Cell. Biochem. 61:514-523).
  • Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to the internal cytoskeleton. Integrins are composed of two noncovalently associated transmembrane glycoprotein subunits called ⁇ and ⁇ . At least 8 different ⁇ subunits ( ⁇ l- ⁇ 8) and at least 12 different subunits have been identified ( ⁇ l- ⁇ 8, ⁇ L, ⁇ M, ocX, and ⁇ llb). Individual ⁇ subunits are capable of associating with different ⁇ subunits, suggesting a possible mechanism for specifying integrin function and ligand binding affinity. Members of the ⁇ subunit family are generally of 90- 110 kilodaltons (kD) in molecular weight and share about 40-48% amino acid sequence homology.
  • kD kilodaltons
  • cysteines distributed among four repeating units are also conserved. Some variation in these conserved features is observed among some of the more divergent ⁇ subunit family members. Members of the subunit family are generally 150-200 kilodaltons in molecular weight and are not as well conserved as the ⁇ subunit family. All contain seven repeating domains of 24-45 amino acids spaced about 20-35 amino acids apart. The N-termini each contain 3-4 divalent cation binding sites. (For review, see Pigott, R. and C. Power (1994) The Adhesion Molecule Facts Book, Academic Press, San Diego, CA, pp. 9-12.)
  • Integrins function as receptors that specifically recognize and bind to ECM proteins such as fibronectin, fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand factor, and collagen. Some integrins recognize a specific motif, the RGD sequence, at the C-termini of the ECM proteins they bind. Integrins also bind to immunoglobulin superfamily proteins such as ICAM-1, -2, and -3 an VCAM-1.
  • Hemopexin is a serum glycoprotein that binds heme and transports it to the liver for breakdown and iron recovery, after which the free hemopexin returns to the circulation.
  • Structurally hemopexin consists of two similar halves of approximately two hundred amino acid residues connected by a histidine-rich hinge region. Hemopexin-like domains have been found in other types of proteins, including vitronectin (Hunt, L.T. et al. (1987) Protein Seq. Data Anal. 1:21-26; Stanley, K.K. (1986) FEBS Lett. 199:249-253). Vitronectin is a cell adhesion and spreading factor found in plasma and other tissues. Like hemopexin, vitronectin has two hemopexin-like domains.
  • Somatomedin B is a cysteine-rich serum factor, which is proteolytically derived from the N-terminal extremity of vitronectin.
  • FAK focal adhesion kinase
  • MLK mitogen-activated protein kinase
  • RTKs growth factor receptor tyrosine kinases
  • Integrins have also been shown to play a vital role in "anoikis," a term describing programmed cell death caused by loss of cell anchorage (Frisch, S.M. and E. Ruoslahti (1997) Curr. Opin. Cell Biol. 9:701-706).
  • a number of diseases have been attributed to integrin defects. (See Pigott and Power, supra).
  • leukocyte adhesion deficiency is an inherited disorder characterized by the impaired migration of neutrophils to sites of extravascular inflammation. LAD is caused by abnormal splicing of and a missense mutation in the RNA encoding the ⁇ 2 subunit.
  • defects in platelet integrin are correlated with Glanzmann's thrombasthemia, a bleeding disorder characterized by insufficient platelet aggregation.
  • Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K. and M.E. Taylor (1993) Annu. Rev. Cell Biol. 9:237-264). This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L. (1991) J. Cell. Biochem. 45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).
  • Lectins are further classified into subfamilies based on carbohydrate-binding specificity and other criteria.
  • the galectin subfamily includes lectins that bind ⁇ -galactoside carbohydrate moieties in a thiol-dependent manner (reviewed in Hadari, Y.R. et al. (1998) J. Biol. Chem. 270:3447-3453).
  • Galectins are widely expressed and developmentally regulated.
  • Galectins contain a characteristic carbohydrate recognition domain (CRD).
  • the CRD comprises about 140 amino acids and contains several stretches of about 1 - 10 amino acids which are highly conserved among all galectins.
  • a particular 6-amino acid motif within the CRD contains conserved tryptophan and arginine residues which are critical for carbohydrate binding.
  • the CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation. Secondary structure predictions indicate that the CRD forms several ⁇ -sheets.
  • Galectins play a number of roles in diseases and conditions associated with cell-cell and cell- matrix interactions. For example, certain galectins associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice. Moreover, anti-galectin antibodies inhibit processes associated with cell transformation, such as cell aggregation and anchorage-independent growth (see, for example, Su, Z.-Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7252-7257).
  • Selectins comprise a specialized lectin subfamily involved primarily in inflammation and leukocyte adhesion (Reviewed in Lasky, supra). Selectins mediate the recruitment of leukocytes from the circulation to sites of acute inflammation and are expressed on the surface of vascular endothelial cells in response to cytokine signaling. Selectins bind to specific ligands on the leukocyte cell membrane and enable the leukocyte to adhere to and migrate along the endothelial surface. Binding of selectin to its ligand leads to polarized rearrangement of the actin cytoskeleton and stimulates signal transduction within the leukocyte (Brenner, B. et al. (1997) Biochem. Biophys. Res. Commun.
  • NCAPs Neural cell adhesion proteins
  • NCAPS genes encoding NCAPS are linked with neurological diseases, including hereditary neuropathy, Charcot-Marie-Tooth disease, Dejerine-Sottas disease, X-linked hydrocephalus, MASA syndrome (mental retardation, aphasia, shuffling gait and adducted thumbs), and spastic paraplegia type I.
  • expression of NCAP is not restricted to the nervous system.
  • LI for example, is expressed in melanoma cells and hematopoietic tumor cells where it is implicated in cell spreading and migration, and may play a role in tumor progression (Montgomery, A.M. et al. (1996) J. Cell Biol. 132:475-485).
  • NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et al., supra). They are generally linked to the plasma membrane through a transmembrane domain and/or a glycosyl-phosphatidylinositol (GPI) anchor. The GPI linkage can be cleaved by GPI phospholipase C. Most NCAPs consist of an extracellular region made up of one or more irnmunoglobulin domains, a membrane spanning domain, and an intracellular region. Many NCAPs contain post-translational modifications including covalently attached oligosaccharide, glucuronic acid, and sulfate.
  • GPI glycosyl-phosphatidylinositol
  • NCAPs fall into three subgroups: simple-type, complex-type, and mixed-type.
  • Simple-type NCAPs contain one or more variable or constant immunoglobulin domains, but lack other types of domains.
  • Members of the simple-type subgroup include Schwann cell myelin protein (SMP), li bic system- associated membrane protein (LAMP), opiate-binding cell-adhesion molecule (OBCAM), and myelin-associated glycoprotein (MAG).
  • SMP Schwann cell myelin protein
  • LAMP li bic system- associated membrane protein
  • OBCAM opiate-binding cell-adhesion molecule
  • MAG myelin-associated glycoprotein
  • the complex-type NCAPs contain fibronectin type III domains in addition to the immunoglobulin domains.
  • the complex-type subgroup includes neural cell-adhesion molecule (NCAM), axonin-1, Fll, Bravo, and LI.
  • NCAM neural cell-ad
  • NCAPs contain a combination of immunoglobulin domains and other motifs such as tyrosine kinase and epidermal growth factor-like domains.
  • This subgroup includes Trk receptors of nerve growth factors such as nerve growth factor (NGF) and neurotropin 4 (NT4), Neu differentiation factors such as glial growth factor II (GGFII) and acetylcholine receptor-inducing factor (ARIA), and the semaphorin/collapsin family such as semaphorin B and collapsin.
  • NGF nerve growth factor
  • NT4 neurotropin 4
  • Neu differentiation factors such as glial growth factor II (GGFII) and acetylcholine receptor-inducing factor (ARIA)
  • semaphorin/collapsin family such as semaphorin B and collapsin.
  • Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been proposed to have roles in protein-protein interactions and are suggested to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J.A.
  • NCAP subfamily includes cell adhesion proteins expressed on distinct subpopulations of brain neurons.
  • Members of the NCAP-LON subgroup possess three immunoglobulin domains and bind to cell membranes through GPI anchors.
  • Kilon (a kindred of NCAP-LON), for example, is expressed in the brain cerebral cortex and hippocampus (Funatsu, N. et al. (1999) J. Biol. Chem. 274:8224-8230). lmmunostaining localizes Kilon to the dendrites and soma of pyramidal neurons.
  • Kilon has three C2 type immunoglobujin-like domains, six predicted giycosylation sites, and a GPI anchor. Expression of Kilon is developmentally regulated. It is expressed at higher levels in adult brain in comparison to embryonic and early postnatal brains. Confocal microscopy shows the presence of Kilon in dendrites of hypothalamic magnocellular neurons secreting neuropeptides, oxytocin or arginine vasopressin (Miyata, S. et al. (2000) J. Comp. Neurol. 424:74-85). Arginine vasopressin regulates body fluid homeostasis, extracellular osmolarity and intravascular volume.
  • Oxytocin induces contractions of uterine smooth muscle during child birth and of myoepithelial cells in mammary glands during lactation.
  • Kilon is proposed to play roles in the reorganization of dendritic connections during neuropeptide secretion.
  • the co-ordinated function of effector and accessory cells in the immune system is assisted by adhesion molecules on the cell surface that stabilize interactions between different cell types.
  • LFA-1 Leukocyte function-associated antigen 1
  • ICAM intercellular adhesion molecules
  • ICAM-1 and 2 are members of the immunoglobulin superfamily.
  • ICAM-1 intercellular adhesion molecules 1 and 2
  • ICAM-1 is expressed at low levels on resting vascular endofhelium.
  • ICAM-1 is strongly upregulated by cytokine stimulation and plays a key role in the arrest of leukocytes in blood vessels at sites of inflammation and injury.
  • a third ligand for LFA-1 expressed in resting leukocytes is ICAM-3.
  • ICAM-3 is closely related to ICAM-1 and is constitutively expressed on all leukocytes. It consists of five immunoglobulin domains and binds LFA-1 through its two N-terminal domains (Fawcett, J. et al. (1992) Nature 360:481-484).
  • PRPs proline-rich proteins
  • PRPs are defined by a high frequency of proline, ranging from 20-50% of the total amino acid content. Some PRPs have short domains which are rich in proline. These proline-rich regions are associated with protein-protein interactions.
  • PRPs proline-rich synapse-associated proteins
  • PSD postsynaptic density
  • ProSAP family Members of the ProSAP family contain six to seven ankyrin repeats at the N-terminus, followed by an SH3 domain, a PDZ domain, and ( seven proline-rich regions and a SAM domain at the C terminus.
  • Another member of the PRP family is the HLA-B-associated transcript 2 protein (B AT2) which is rich in proline and includes short tracts of polyproline, polyglycine, and charged amino acids.
  • BAT2 also contains four RGD (Arg-Gly-Asp) motifs typical of integrins (Banerji, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87:2374-2378).
  • Toposome is a cell-adhesion glycoprotein isolated frommesenchyme-blastula embryos. Toposome precursors including vitellogenin promote cell adhesion of dissociated blastula cells.
  • MAM domain a domain of about 170 amino acids found in the extracellular region of diverse proteins. These proteins all share a receptor-like architecture comprising a signal peptide, followed by a large N-terminal extracellular domain, a transmembrane region, and an intracellular domain (PROSITE document PDOC00604 MAM domain signature and profile).
  • MAM domain proteins include zonadhesin, a sperm-specific membrane protein that binds to the zona pellucida of the egg; neuropilin, a cell adhesion molecule that functions during the formation of certain neuronal circuits, and Xenopus laevis thyroid hormone induced protein B, which contains four MAM domains and is involved in metamorphosis (Brown, D.D. et al. (1996) Proc. Natl. Acad. Sci. USA 93:1924- 1929).
  • the WSC domain was originally found in the yeast WSC (cell-wall integrity and stress response component) proteins which act as sensors of environmental stress.
  • the WSC domains are extracellular and are thought to possess a carbohydrate binding role (Ponting, C.P. et al. (1999) Curr. Biol. 9:S1-S2).
  • a WSC domain has recently been identified in polycystin-1, a human plasma membrane protein. Mutations in polycystin-1 are the cause of the commonest form of autosomal dominant polycystic kidney disease (Ponting, C.P. et al. (1999) Curr. Biol. 9:R585-R588).
  • LRR Leucine rich repeats
  • LRR motifs are short motifs found in numerous proteins from a wide range of species. LRR motifs are of variable length, most commonly 20-29 amino acids, and multiple repeats are typically present in tandem. LRR motifs are important for protein/protein interactions and cell adhesion, and LRR proteins are involved in cell/cell interactions, morphogenesis, and development (Kobe, B. and J. Deisenhofer (1995) Curr. Opin. Struct. Biol. 5:409-416).
  • the human ISLR (immunoglobulin superfamily containing leucine-rich repeat) protein contains a C2-type immunoglobulin domain as well as LRR motifs.
  • the ISLR gene is linked to the critical region for Bardet-Biedl syndrome, a developmental disorder of which the most common feature is retinal dystrophy (Nagasawa, A. et al. (1999) Genomics 61:37-43).
  • the sterile alpha motif (SAM) domain is a conserved protein binding domain, approximately 70 amino acids in length, and is involved in the regulation of many developmental processes in eukaryotes.
  • SAM domain can potentially function as a protein interaction module through its ability to formhomo- or hetero-oligomers with other SAM domains (Schultz, J. et al. (1997) Protein Sci. 6:249-253).
  • Vinculin is a cellular adhesion molecule that is involved in the attachment of actin microfilaments to the plasma membrane of eukaryotic cells.
  • This protein is composed of approximately 1000 amino acid residues and is characterized by an acidic N-terminal domain consisting of either two (in C. elegans) or three (in vertebrates) repeats of a 110 amino acid region. A proline-rich region is followed by a basic C-terminal domain.
  • Two signature patterns are found in the N-terminal domain, one which seems to be involved in protein-protein interactions and one based on the repeated region (PROSITE document PDOC00568 Vinculin family signatures).
  • Synapsins are a family of proteins that coat synaptic vesicles and bind to actin filaments as well as other components of the cytoskeleton.
  • Synapsins I and II each exist as two alternately spliced variants termed IA and IB or IIA and IIB and differ from each other in their C-termini.
  • Two conserved domains within these proteins are an octapeptide containing a phosphorylated serine residue and a second stretch of 11 highly conserved residues (PROSITE document PDOC00345 Synapsin signatures).
  • Osteonectin domain signatures are derived from three extracellular proteins (SPARC or osteonectin, SCI, and QR1) which contain a region of 240 highly-conserved amino acid residues in their C-termini. Two signature patterns were developed based on this conserved region, one based on a cysteine-rich region and the other based on a stretch of 11 highly conserved residues (PROSITE document PDOC00535 Osteonectin domain signatures).
  • Extracellular Matrix Proteins Extracellular Matrix Proteins
  • the extracellular matrix is a complex network of glycoproteins, polysaccharides, proteoglycans, and other macromolecules that are secreted from the cell into the extracellular space.
  • the ECM remains in close association with the cell surface and provides a supportive meshwork that profoundly influences cell shape, motility, strength, flexibility, and adhesion. In fact, adhesion of a cell to its surrounding matrix is required for cell survival except in the case of metastatic tumor cells, which have overcome the need for cell-ECM anchorage. This phenomenon suggests that the ECM plays a critical role in the molecular mechanisms of growth control and metastasis. (Reviewed in Ruoslahti, E. (1996) Sci. Am. 275:72-77.) Furthermore, the ECM dete ⁇ nines the structure and physical properties of connective tissue and is particularly important for morphogenesis and other processes associated with embryonic development and pattern formation.
  • the coUagens comprise a family of ECM proteins that provide structure to bone, teeth, skin, ligaments, tendons, cartilage, blood vessels, and basement membranes. Multiple collagen proteins have been identified. Three collagen molecules fold together in a triple helix stabilized by interchain disulfide bonds. Bundles of these triple helices then associate to form fibrils. Collagen primary structure consists of hundreds of (Gly-X-Y) repeats where about a third of the X and Y residues are Pro. Glycines are crucial to helix formation as the bulkier amino acid sidechains cannot fold into the triple helical conformation. Because of these strict sequence requirements, mutations in collagen genes have severe consequences.
  • Osteogenesis imperfecta patients have brittle bones that fracture easily; in severe cases patients die in utero or at birth.
  • Ehlers-Danlos syndrome patients have hyperelastic skin, hypermobile joints, and susceptibility to aortic and intestinal rupture.
  • Chondrodysplasia patients have short stature and ocular disorders.
  • Alport syndrome patients have hematuria, sensorineural deafness, and eye lens deformation. (Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, Inc., New York, NY, pp. 2105-2117; and Creighton, T.E. (1984) Proteins. Structures and Molecular Principles. W.H. Freeman and Company, New York, NY, pp. 191-197.)
  • Elastin and related proteins confer elasticity to tissues such as skin, blood vessels, and lungs.
  • Elastin is a highly hydrophobic protein of about 750 amino acids that is rich in proline and glycine residues.
  • Elastin molecules are highly cross-linked, forming an extensive extracellular network of fibers and sheets.
  • Elastin fibers are surrounded by a sheath of microfibrils which are composed of a number of glycoproteins, including fibrillin. Mutations in the gene encoding fibrillin are responsible for Marian's syndrome, a genetic disorder characterized by defects in connective tissue. In severe cases, the aortas of afflicted individuals are prone to rupture. (Reviewed in Alberts, B. et al.
  • the fibulin proteins connect elastic fibers and are thought to promote the formation and stabilization of the fibers.
  • Members of the fibulin family contain epidermal growth factor-like motifs as well as an RGD cell attachment sequence (Midwood, K.S. and J.E. Schwarzbauer (2002) Current Biology 12:R279-R281).
  • Fibronectin is a large ECM glycoprotein found in all vertebrates. Fibronectin exists as a dimer of two subunits, each containing about 2,500 amino acids. Each subunit folds into a rod-like structure containing multiple domains. The domains each contain multiple repeated modules, the most common of which is the type IH fibronectin repeat.
  • the type III fibronectin repeat is about 90 arnino acids in length and is also found in other ECM proteins and in some plasma membrane and cytoplasmic proteins.
  • some type III fibronectin repeats contain a characteristic tripeptide consisting of Arginine-Glycine- Aspartic acid (RGD). The RGD sequence is recognized by the integrin family of cell surface receptors and is also found in other ECM proteins.
  • Laminin is a major glycoprotein component of the basal lamina which underlies and supports epithelial cell sheets.
  • Larrrinin is one of the first ECM proteins synthesized in the developing embryo.
  • Larninin is an 850 kilodalton protein composed of three polypeptide chains joined in the shape of a cross by disulfide bonds.
  • Laminin is especially important for angiogenesis and, in particular, for guiding the formation of capillaries.
  • Many proteinaceous ECM components are proteoglycans.
  • Proteoglycans are composed of uhbranched polysaccharide chains (glycosaminoglycans) attached to protein cores.
  • Common proteoglycans include aggrecan, betaglycan, decorin, perlecan, serglycin, and syndecan-1.
  • glycoproteins tenascin-C and tenascin-R are expressed in developing and lesioned neural tissue and provide stimulatory and anti-adhesive (inhibitory) properties, respectively, for axonal growth (Faissner, A. (1997) Cell Tissue Res. 290:331-341).
  • DPP Dentin phosphoryn
  • odontoblasts Gu, K. et al. (1998) Eur. J. Oral Sci. 106:1043- 1047). DPP is believed to nucleate or modulate the formation of hydroxyapatite crystals.
  • Amelogenin is an extracellular matrix protein that plays a role in the biomineralization of tooth enamel. This protein participates in the regulation of crystallite formation during tooth enamel development and thus is thought to play a major role in the structural organization and minerahzation of developing tooth enamel (Li, W. et al. (2001) Matrix Biol. 19:755-60).
  • Mucins are highly glycosylated glycoproteins that are the major structural component of the mucus gel. The physiological functions of mucins are cytoprotection, mechanical protection, maintenance of viscosity in secretions, and cellular recognition.
  • MUC6 is a human gastric mucin that is also found in gall bladder, pancreas, seminal vesicles, and female reproductive tract (Toribara, N.W. et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, N.W. et al. (1993) J. Biol. Chem. 268:5879-5885). Hemomucinis a novel Drosophila surface mucin that may be involved in the induction of antibacterial effector molecules (Theopold, U et al. (1996) J. Biol. Chem. 217:12708-12715).
  • Olfactomedin was originally identified as the major component of the mucus layer surrounding the chemosensory dendrites of olfactory neurons. Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs. The TIGPv/myocilin protein, an olfactomedin-related protein expressed in the eye, is associated with the pathogenesis of glaucoma (Kulkarni, N.H. et al. (2000) Genet. Res. 76:41-50).
  • Ankyrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular functions.
  • ANK repeats are composed of about 33 amino acids that form a helix-turn- helix core preceded by a protruding "tip.” These tips are of variable sequence and may play a role in protein-protein interactions.
  • the helix-turn-helix region of the ANK repeats stack on top of one another and are stabilized by hydrophobic interactions (Yang, Y. et al. (1998) Structure 6:619-626).
  • Sushi repeats also called short consensus repeats (SCR), are found in a number of proteins that share the common feature of binding to other proteins.
  • the sushi domain is important for heparin binding.
  • Sushi domains contain basic amino acid residues, which may play a role in binding (Oleszewski, M. et al. (2000) J. Biol. Chem 275:34478- 34485).
  • Link, or X-link, modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration.
  • the Link module superfamily includes CD44, cartilage link protein, and aggrecan.
  • This family also includes BEHAB (brain enriched hyaluronan-binding)/brevican, a component of the brain ECM that is dramatically upregulated inhuman gliomas, and appears to play a role in deterrrjining the invasive potential of brain tumor cells (Gary, S.C. et al. (1998) Curr. Opin. Neurobiol. 8:576-581).
  • Multidomain or mosaic proteins play an important role in the diverse functions of the extracellular matrix (Engel, J. et al. (1994) Development (Camb.):S35-S42).
  • ECM proteins are frequently characterized by the presence of one or more domains which may contain a number of potential intracellular disulfide bridge motifs.
  • domains which match the epidermal growth factor (EGF) tandem repeat consensus are present within several known extracellular proteins that promote cell growth, development, and cell signaling.
  • This signature sequence is about forty amino acid residues in length and includes six conserved cysteine residues, and a calcium-binding site near the N-terminus of the signature sequence.
  • the main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet.
  • Subdomains between the conserved cysteines vary in length (Davis, CG. (1990) New Biol. 5:410-419).
  • Post-translational hydroxylation of aspartic acid or asparagine residues has been associated with EGF-like domains in several proteins (Prosite PDOC00010).
  • a number of proteins that contain calcium-binding EGF-like domain signature sequences are involved in growth and differentiation.
  • Examples include bone morphogenic protein 1, which induces the formation of cartilage and bone; crumbs, which is a Drosophila epithelial development protein; Notch and a number of its homologs, which are involved in neural growth and differentiation, and transforming growth factor beta-1 binding protein (Expasy PROSITE document PDOC00913; Soler, C. and G. Carpenter, in Nicola, N.A. (1994) The Cytokine Facts Book. Oxford University Press, Oxford, UK, pp. 193-197). EGF-like domains mediate protein-protein interactions for a variety of proteins.
  • EGF-like domains in the ECM glycoprotein fibulin- 1 have been shown to mediate both self-association and binding to fibronectin (Tran, H. et al. (1997) J. Biol. Chem. 272:22600-22606).
  • Point mutations in the EGF-like domains of ECM proteins have been identified as the cause of human disorders such as Marfan syndrome and pseudochondroplasia (Maurer, P. et al. (1996) Curr. Opin. CeU Biol. 8:609-617).
  • the CUB domain is an extracellular domain of approximately 110 amino acid residues found mostly in developmentally regulated proteins.
  • the CUB domain contains four conserved cysteine residues and is predicted to have a structure similar to that of immunoglobuUns.
  • Vertebrate bone morphogenic protein 1, which induces cartilage and bone formation, and fibropellins I and III from sea urchin, which form the apical lamina component of the ECM, are examples of proteins that contain both CUB and EGF domains (PROSITE PDOC00908).
  • ECM proteins are members of the type A domain of von WiUebrand factor (vWFA)- like module superfamily, a diverse group of proteins with a module sharing high sequence similarity.
  • the vWFA-like module is found not only in plasma proteins but also in plasma membrane and ECM proteins (Colombatti, A. and P. Bonaldo (1991) Blood 77:2305-2315). Crystal structure analysis of an integrin vWFA-Hke module shows a classic "Rossmann" fold and suggests a metal ion-dependent adhesion site for binding protein ligands (Lee, J.-O. et al. (1995) Cell 80:631-638).
  • This family includes the protein matrilin-2, an extraceUular matrix protein that is expressed in a broad range of mammaUan tissues and organs.
  • Matrilin-2 is thought to play a role in ECM assembly by bridging coUagen fibrils and the aggrecan network (Deak, F. et al. (1997) J. Biol. Chem. 272:9268-9274).
  • the thrombospondins are multimeric, calcium-binding extraceUular glycoproteins found widely in the embryonic extraceUular matrix. These proteins are expressed in the developing nervous system or at specific sites in the adult nervous system after injury.
  • Thrombospondins contain multiple EGF-type repeats, as weU as a motif known as the thrombospondin type 1 repeat (TSR).
  • TSR thrombospondin type 1 repeat
  • the TSR is approximately 60 amino acids in length and contains six conserved cysteine residues. Motifs within TSR domains are involved in mediating ceU adhesion through binding to proteoglycans and sulfated glycoHpids. Thrombospondin- 1 inhibits angiogenesis and modulates endothelial ceU adhesion, motility, and growth.
  • TSR domains are found in a diverse group of other proteins, most of which are expressed in the developing nervous system and have potential roles in the guidance of ceU and growth cone migration.
  • TSR proteins that contain TSRs include the F-spondin gene family, the semaphorin 5 family, UNC-5, and SCO-spondin.
  • the TSR superfarnuy includes the ADAMTS proteins which contain an ADAM (A Disintegrin and MetaUoproteinase) domain as weU as one or more TSRs.
  • ADAM Disintegrin and MetaUoproteinase
  • the ADAMTS proteins have roles in regulating the turnover of cartilage matrix, regulation of blood vessel growth, and possibly development of the nervous system. (Reviewed in Adams, J.C. and R.P. Tucker (2000) Dev. Dyn. 218:280-299.)
  • Fibrinogen the principle protein of vertebrate blood clotting, is a hexamer consisting of two sets of three different chains (alpha, beta, and gamma).
  • the C-terminal domain of the beta and gamma chains comprises about 270 amino acid residues and contains four cysteines involved in two disulfide bonds. This domain has also been found in mammaUan tenascin-X, an ECM protein that appears to be involved in ceU adhesion (Prosite PDOC00445).
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a plurality of molecules spatiaUy 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 appUcations, 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.
  • Atherosclerosis and the associated coronary artery disease and cerebral stroke represent the most common cause of death in bonUzed countries. Although certain key risk factors have been identified, a full molecular characterization that elucidates the causes and provide care for this complex disease has not been achieved. Molecular characterization of growth and regression of atherosclerotic vascular lesions requires identification of the genes that contribute to features of the lesion including growth, stability, dissolution, rupture and, most lethaUy, induction of occlusive vessel thrombus. Vascular lesions principaUy involve the vascular endotheUum and the surrounding smooth muscle tissue. Development of atherosclerosis is understood to be induced by the presence of circulating
  • Upoprotein Lipoproteins, such as the cholesterol-rich low-density Upoprotein (LDL), accumulate in the extraceUular space of the vascular intirna, and undergo modification. Oxidation of LDL (Ox- LDL) occurs most avidly in the sub-endotheUal space where circulating antioxidant defenses are less effective. Mononuclear phagocytes enter the tima, differentiate into macrophages, and ingest modified Upids including Ox-LDL. During Ox-LDL uptake, macrophages produce cytokines (e.g. tumor necrosis factor ⁇ (TNF- ⁇ ) and interleukin-1 (IL-1)) and growth factors (e.g.
  • TNF- ⁇ tumor necrosis factor ⁇
  • IL-1 interleukin-1
  • M-CSF M-CSF, VEGF, and PDGF-BB
  • eUcit further ceUular events that modulate atherogenesis
  • atherogenesis such as smooth muscle ceU proUferation and production of extraceUular matrix by vascular endotheUum.
  • these macrophages may activate genes in endotheUum and smooth muscle tissue involved in inflammation and tissue differentiation, including superoxide dismutatse (SOD), IL-8, and ICAM-1.
  • SOD superoxide dismutatse
  • IL-8 ICAM-1.
  • the vascular endotheUum influences not only the three classicaUy interacting components of hemostasis: the vessel, the blood platelets and the clotting and fibrinolytic systems of plasma, but also the natural sequelae: inflammation and tissue repair.
  • Two principal modes of endotheUal behavior may be differentiated, best defined as an anti- and a prothrombotic state.
  • endotheUum mediates vascular dilatation (formation of nitric oxide (NO), PGI 2 , adenosine, hyperpolarising factor), prevents platelet adhesion and activation (production of adenosine, NO and PGI 2 , removal of ADP), blocks thrombin formation (tissue factor pathway inhibitor, activation of protein C via fhrombomoduUn, activation of antithrombin III) and mitigates fibrin deposition (t- and scuplasminogen activator production).
  • Adhesion and transmigration of inflammatory leukocytes are attenuated, e.g. by NO and IL-10, and oxygen radicals are efficiently scavenged (urate, NO, glutathione, SOD).
  • prothrombotic, proinflammatory state is characterised by vaso-constriction, platelet and leukocyte activation and adhesion (externaUsation, expression and upregulation of, for example, von Willebrand factor, platelet activating factor, P-selectin, ICAM-1, IL-8, MCP-1, and TNF- ⁇ ), promotion of thrombin formation, coagulation and fibrin deposition at the vascular waU (expression of tissue factor, PAI-1, and phosphatidyl serine) and, in platelet-leukocyte coaggregates, additional inflammatory interactions via attachment of platelet CD40-Ugand to endotheUal, monocyte and B-ceU CD40.
  • BRCA1 and BRCA2 are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra).
  • this type of hereditary breast cancer accounts for only about 5% to 9% ofbreast cancers, while the vast majority ofbreast cancer is due to non-inherited mutations that occur in breast epitheUal cells.
  • 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 ceU motiUty, chemotaxis, secretion and differentiation.
  • the abundance of erbB receptors, such as HER- 2/neu, HER-3, and HER-4, and their Ugands 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. Pathol. 102:S13-S24).
  • markers ofbreast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed in human breast carcinoma ceUs; Drgl or RTP, a gene whose expression is (Uminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S 100 protein famny, aU of which are down-regulated in mammary carcinoma ceUs relative to normal mammary epitheUal ceUs (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al.
  • CeU lines derived from human mammary epitheUal ceUs at various stages ofbreast cancer provide a useful model to study the process of maUgnant 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) Clin. Cancer Res. 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epitheUal ceUs at various stages of maUgnant transformation. Ovarian Cancer
  • Ovarian cancer is the leading cause of death from a gynecologic cancer.
  • the majority of ovarian cancers are derived from epitheUal 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 instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and ceU proUferative disorders, including cancer.
  • Various embodiments of the invention provide purified polypeptides, ceU adhesion and extraceUular matrix proteins, referred to coUectively as 'CADECM' and individuaUy as 'CADECM- 1,' 'CADECM-2,' 'CADECM-3,' 'CADECM-4,' 'CADECM-5,' 'CADECM-6,' 'CADECM-7,' 'CADECM-8,' 'CADECM-9,' 'CADECM-10,' 'CADECM-11,' 'CADECM-12,' 'CADECM-13,' 'CADECM-14,' 'CADECM-15,' 'CADECM-16,' 'CADECM-17,' 'CADECM-18,' 'CADECM-19,' 'CADECM-20,' 'CADECM-21,' 'CADECM-22,' 'CADECM-23,' 'CADECM-24,' 'CADECM-25,' 'CAD
  • Embodiments also provide methods for utilizing the purified ceU adhesion and extraceUular matrix proteins 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 ceU adhesion and extraceUular matrix proteins 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- 30, 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:1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-30.
  • 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:1-30, 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 -30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-30.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:31-60.
  • StiU 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:1-30, 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:1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30.
  • Another embodiment provides a ceU transformed with the recombinant polynucleotide.
  • Yet another embodiment provides a transgenic organism comprising the recombinant polynucleot
  • 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: 1-30, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least
  • 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:1-30, 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: 1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30.
  • 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:31-60, 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:31-60, 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:31-60, 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:31-60, 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.
  • StiU 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:31-60, 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:31-60, 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 polynu
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction ampUfication, 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:1-30, 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:1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and a pharmaceutically acceptable excipient
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30.
  • inventions provide a method of treating a disease or condition associated with decreased or abnormal expression of functional CADECM, 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: 1-30, 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:1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30.
  • 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 CADECM, comprising administering to a patient in need of such treatment the composition.
  • StiU 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: 1-30, 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: 1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30.
  • 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 CADECM, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-30, 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:1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30.
  • 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:1-30, 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:1-30, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-30.
  • 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:31-60, 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:31-60, 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:31-60, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of n), and v) an RNA equivalent of i
  • 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:31-60, 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:31-60, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of n), 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 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 Usts 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 Ubraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDN A Ubraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with appUcable 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.
  • AU pubUcations mentioned herein are cited for the purpose of describing and disclosing the ceU lines, protocols, reagents and vectors which are reported in the pubUcations and which might be used in connection with various embodiments of the invention. None herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
  • DEFINITIONS "CADECM” refers to the amino acid sequences of substantiaUy purified CADECM obtained from any species, particularly a marnmaUan 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 CADECM.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of CADECM either by directly interacting with CADECM or by acting on components of the biological pathway in which CADECM participates.
  • AUeUc variant is an alternative form of the gene encoding CADECM.
  • AUeUc variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
  • a gene may have none, one, or many aUeUc variants of its naturaUy occurring form.
  • Common mutational changes which give rise to aUeUc 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 CADECM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as CADECM or a polypeptide with at least one functional characteristic of CADECM. Included within this definition are polymorphisms which may or may not be readUy detectable using a particular oUgonucleotide probe of the polynucleotide encoding CADECM, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding CADECM.
  • 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 CADECM.
  • DeUberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophiUcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of
  • 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 hydrophiUcity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having sirnUar hydrophiUcity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oUgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules.
  • amino acid sequence is recited to refer to a sequence of a naturaUy occurring protein molecule
  • amino acid sequence and Uke 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. AmpUfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid ampUfication technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of CADECM.
  • Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of CADECM either by directly interacting with CADECM or by acting on components of the biological pathway in which CADECM participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic deterrninant.
  • Antibodies that bind CADECM polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oUgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole limpet hemocyanin (KLH).
  • the coupled peptide is then used to immunize the animal.
  • the term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eUcit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oUgonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Ubraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-Uke molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2 -OH group of a ribonucleotide may be replaced by 2 -F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specificaUy cross-linked to their cognate Ugands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense”
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone linkages such as phosphorofhioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-mefhoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyura l, or 7-deaza-2'- deoxyguanosine.
  • PNA peptide nucleic acid
  • 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 CADECM, or of any oUgopeptide 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, 3'-TCA-5'.
  • 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 CADECM or fragments of CADECM may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe In hybridizations, the probe maybe deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry ilk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry ilk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (AppUed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (Accelrys, Burlington MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • 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 heUcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by giycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
  • a "fragment” is a unique portion of CADECM or a polynucleotide encoding CADECM 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/amino acid residue. For example, 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 ma 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:31-60 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:31-60, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:31-60 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and ampUfication technologies and in analogous methods that distinguish SEQ ID NO :31-60 from related polynucleotides.
  • the precise length of a fragment of SEQ ID NO:31-60 and the region of SEQ ID NO:31 -60 to which the fragment corresponds are routinely deterrninable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:1-30 is encoded by a fragment of SEQ ID NO:31-60.
  • a fragment of SEQ ID NO:1-30 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:1-30.
  • a fragment of SEQ ID NO:1-30 can be used as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO: 1-30.
  • the precise length of a fragment of SEQ ID NO:1-30 and the region of SEQ ID NO:1-30 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 “fuU length” polynucleotide sequence encodes a "full length” polypeptide sequence.
  • “Homology” refers to sequence similarity 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 aUgned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize aUgnment 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 MEG ALIGN version 3.12e sequence aUgnment 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 "weighted" residue weight table is selected as the default.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aUgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences” tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or maybe 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 arnino 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 aUgned using a standardized algorithm.
  • Methods of polypeptide sequence aUgnment are weU-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail 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 % sirnuarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aUgned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • 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.
  • Human artificial chromosomes 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 repUcation, 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 still retains its original binding abiUty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive 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 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (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 weU 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
  • RNA:DNA hybridizations Useful variations on these wash conditions wiU be readily apparent to those of ordinary skiU in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary simUarity between the nucleotides. Such simUarity 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 immobnized on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • a soUd support e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed.
  • Immuno response can refer to conditions associated with inflarnmation, 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 oUgopeptide fragment of CADECM which is capable of eUciting an immune response when introduced into a Uving organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oUgopeptide fragment of CADECM which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a pluraUty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • array element refers 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 CADECM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CADECM.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oUgonucleotide, 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 oUgonucleotide 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 Ufespan in the ceU.
  • Post-translational modification of an CADECM may involve Upidation, giycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic miUeu of CADECM.
  • Probe refers to nucleic acids encoding CADECM, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acids.
  • Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Ugands, chemUuminescent agents, and enzymes.
  • Primmers are short nucleic acids, usuaUy DNA oUgonucleotides, which maybe 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 ampUfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. 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.
  • 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).
  • OUgonucleotides 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 oUgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 lobases. 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 pubUc from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming Ubrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oUgonucleotides 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 (avaUable to the pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturaUy occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accompUshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and RusseU (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, 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 may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionucUdes; enzymes; fluorescent, chemuuminescent, 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 CADECM, nucleic acids encoding CADECM, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 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, sUdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. 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, Upofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of repUcation either as an autonomously repUcating 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 deUberate 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).
  • 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. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and RusseU (supra).
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "aUeUc” (as defined above), “spUce,” “species,” or “polymorphic” variant.
  • a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing during mRNA processing.
  • the corresponding 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 wiU generaUy have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence simUarity 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 greater sequence identity or sequence simUarity over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include new human ceU adhesion and extraceUular matrix proteins (CADECM), the polynucleotides encoding CADECM, and the use of these compositions for the diagnosis, treatment, or prevention of immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and ceU proUferative disorders, including cancer.
  • CADECM ceU adhesion and extraceUular matrix proteins
  • 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, fuU 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 probability 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 appUcable, 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 giycosylation sites.
  • Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appUed.
  • SEQ ID NO:8 is 100% identical, fromresidue Ml to residue S326, and 80% identical, fromresidue E314 to residue L363, to human vitronectin (GenBank ID gl4326449) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabUity score is 1.1 e-204, which indicates the probabUity of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:8 also has homology to proteins that are locaUzed to the extraceUular region (excluding ceU waU), mediate adhesion and migration, and are vitronectin proteins as determ ned by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:8 also contains hemopexin domains and somatomedin B domains 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.)
  • HMM hidden Markov model
  • SEQ ID NO:14 is 99% identical, fromresidue Ml to residue Q525, to human cadherin-19 (GenBank ID gl0803406) as determined by BLAST. (See Table 2.) The BLAST probabUity score is 2.3e-282. SEQ ID NO:14 also has homology to proteins that are locaUzed to the plasma membrane, have adhesion function, and are cadherins as determined by BLAST analysis using the PROTEOME database. SEQ ID NO: 14 also contains cadherin domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein famuies/domains.
  • HMM hidden Markov model
  • SEQ ID NO: 14 is a cadherin.
  • SEQ ID NO: 18 is 99% identical, from residue 125 to residue Q135, and 99% identical, fromresidue A144 to residue A287, to rat ankyrin binding ceU adhesion molecule neurofascin (GenBank ID gl 842429) as determined by BLAST. (See Table 2.) The BLAST probabUity score is 1.2e-132.
  • SEQ ID NO:18 also has homology to proteins that are locaUzed to the plasma membrane, are members of the immunoglobulin superfamUy, are neuronal ceU adhesion molecules, and are overexpressed in brain tumors, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO: 18 also contains an immunoglobulin C-2 type domain, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based SMART database of conserved protein famiUes/domains.
  • HMM hidden Markov model
  • SEQ ID NO: 18 also contains an Ig superfamUy from SCOP domain, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based INCY database of conserved protein famuies/domains. (See Table 3.) Data from BLAST analyses against the PRODOM and DOMO databases provide further corroborative evidence that SEQ ID NO:18 is a neuronal ceU adhesion molecule.
  • HMM hidden Markov model
  • SEQ ID NO:26 is 80% identical, fromresidue P16 to residue K1402, to mouse nidogen-2 (GenBank ID g23592218) as determined by BLAST. (See Table 2.) The BLAST probability score is 0.0.
  • SEQ ID NO:26 also has homology to proteins that are locaUzed to the extraceUular matrix (cuticle and basement membrane; excluding ceU waU); and specificaUy to nidogen-2, a basement membrane protein that locaUzes to elastic fibers of blood vessel waUs; has an mtegrin-binding RGD sequence; may facilitate stabilization of basement membrane networks; binds perlecan (HSPG2), laminin-1, and coUagens; and supports ceU attachment in some ceU lines, as determined by BLAST analysis using the PROTEOME database.
  • HSPG2 binds perlecan
  • laminin-1 laminin-1
  • coUagens supports ceU attachment in some ceU lines, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:26 also contains an EGF-like, low-density Upoprotein receptor repeat class B, thyroglobuUn type-1 repeat, calcium-binding EGF-like, and low-density Upoprotein-receptor YWTD domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM/SMART databases of conserved protein famiUes/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:26 is an extraceUular matrix protein.
  • HMM hidden Markov model
  • SEQ ID NO:27 is 100% identical, fromresidue V36 to residue A288, to human mDC-SIGNIA type 111 isoform (GenBank ID gl5281077) as determined BLAST. (See Table 2.) The BLAST probabUity score is 9.2e-138. SEQ ID NO:27 also has homology to proteins that are locaUzed to the plasma membrane, possess ICAM binding affinity, and are C-type lectins, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:27 also contains a C- type lectin domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein farnUies/domains. (See Table 3.) Data fromBLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:27
  • SEQ ID NO:27 is a C-type lectin.
  • SEQ ID NO:l-7, SEQ ID NO:9-13, SEQ ID NO:15-17, SEQ ID NO:19-25, and SEQ ID NO:28-30 were analyzed and annotated in a simUar manner.
  • the algorithms and parameters for the analysis of SEQ ID NO:1-30 are described in Table 7.
  • the full 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 Usts 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 full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:31-60 or that distinguish between SEQ ID NO:31-60 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 Ubraries or from pooled cDNA Ubraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as
  • FL_XXXXXXX_Nj_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 appUed, and YYYYY is the number of the prediction generated by the algorithm, and N w ... > ⁇ 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 F XXXXXX_gMAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was appUed, 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 "NM,” “NP,” 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 following Table Usts 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 Ubraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA Ubrary is the Incyte cDNA Ubrary 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 Ubraries 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 (CB1 SNP).
  • Column 7 shows the allele found in the EST sequence.
  • Columns 8 and 9 show the two aUeles found at the 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 CADECM variants.
  • CADECM 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 CADECM amino acid sequence, and can contain at least one functional or structural characteristic of CADECM.
  • Various embodiments also encompass polynucleotides which encode CADECM.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:31-60, which encodes CADECM.
  • the polynucleotide sequences of SEQ ID NO:31-60 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses variants of a polynucleotide encoding CADECM.
  • a variant polynucleotide wUl 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 CADECM.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:31-60 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:31-60.
  • Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CADECM.
  • a polynucleotide variant of the invention is a spUce variant of a polynucleotide encoding CADECM.
  • a spUce variant may have portions which have significant sequence identity to a polynucleotide encoding CADECM, but wUl generaUy have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing during mRNA processing.
  • a spUce 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 CADECM over its entire length; however, portions of the spUce 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 CADECM.
  • a polynucleotide comprising a sequence of SEQ ID NO:31 is a spUce variant of a polynucleotide comprising a sequence of SEQ ID NO:35; and a polynucleotide comprising a sequence of SEQ ID NO:33 and a polynucleotide comprising a sequence of SEQ ID NO:34 are spUce variants of one other; a polynucleotide comprising a sequence of SEQ ID NO:32, a polynucleotide comprising a sequence of SEQ ID NO:47 and a polynucleotide comprising a sequence of SEQ ID NO:50 are spUce variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:42 and a polynucleotide comprising a sequence of SEQ ID NO:57 are spUce variants of each other; a polynucleotide comprising
  • Any one of the spUce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CADECM. Any one of the spUce variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CADECM.
  • polynucleotides which encode CADECM and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occu ⁇ ing CADECM under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding CADECM 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-Ufe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of polynucleotides which encode CADECM and CADECM derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide maybe inserted into any of the many avaUable expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding CADECM 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:31-60 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. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.” Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppUed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with niachines such as the MICROLAB 2200 Uquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppUed Biosystems).
  • Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppUed 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, WUey VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding CADECM may be 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.
  • restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods AppUc. 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 (TrigUa, T. et al.
  • a third method, capture PCR involves PCR ampUfication of DNA fragments adjacent to known sequences inhuman and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppUc. 1:111-119).
  • multiple restriction enzyme digestions and Ugations maybe 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, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C
  • Genomic Ubraries does not yield a full-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • CapUlary electrophoresis systems which are commercially 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/Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display maybe computer contioUed.
  • CapUlary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode CADECM may be cloned in recombinant DNA molecules that direct expression of CADECM, 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 CADECM.
  • the polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter CADECM-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 oUgonucleotides may be used to engineer the nucleotide sequences.
  • oUgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter giycosylation patterns, change codon preference, produce spUce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDENG (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
  • DNA shuffling is a process by which a Ubrary 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.
  • polynucleotides encoding CADECM 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).
  • CADECM 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 soUd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp.
  • AdditionaUy the amino acid sequence of CADECM, or any part thereof, may be altered during direct synthesis and/or 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 Uquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
  • 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 CADECM. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding CADECM.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).
  • Methods which are weU known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding CADECM 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 utilized to contain and express polynucleotides encoding CADECM. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., 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.
  • 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.
  • Expression vectors derived fro 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)
  • a number of cloning and expression vectors maybe selected depending upon the use intended for polynucleotides encoding CADECM.
  • routine cloning, subcloning, and propagation of polynucleotides encoding CADECM can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (invitrogen).
  • PBLUESCRIPT Stratagene, La JoUa 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 CADECM 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 CADECM.
  • 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 pastoi'is.
  • such vectors direct either the secretion or intracettular 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 CADECM. Transcription of polynucleotides encoding CADECM ma 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
  • plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. CeU Differ. 17:85-105).
  • These 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 Hffl, New York NY, pp. 191-196).
  • a number of viral-based expression systems may be utilized.
  • polynucleotides encoding CADECM may be Ugated 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 CADECM in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
  • RSV Rous sarcoma virus
  • S V40 or EBV-based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to deUver larger fragments of
  • DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and dehvered via conventional deUvery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
  • stable expression of CADECM in ceU lines is preferred.
  • polynucleotides encoding CADECM can be transformed into ceU lines using expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate 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 aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ' ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to mefhotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD which alter ceUular requirements for metabohtes
  • Visible markers e.g., anthocyanins, 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 CADECM is inserted within a marker gene sequence
  • transformed ceUs containing polynucleotides encoding CADECM can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding CADECM under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
  • CADECM may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, 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. Immunological methods for detecting and measuring the expression of CADECM using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), racuoirnmunoassays (RIAs), and fluorescence activated ceU sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs racuoirnmunoassays
  • FACS fluorescence activated ceU sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on CADECM is prefe ⁇ ed, but a competitive binding assay may be employed.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CADECM include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide.
  • polynucleotides encoding CADECM, or any fragments thereof may be 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.
  • 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 Uke.
  • Host ceUs transformed with polynucleotides encoding CADECM may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture. The protein produced by a transformed ceU may be secreted or retained intracettularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode CADECM may be designed to contain signal sequences which direct secretion of CADECM through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its abiUty 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, giycosylation, phosphorylation, Upidation, 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 ceUular rnachinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are avaUable from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture CoUection
  • Manassas VA Manassas VA
  • natural, modified, or recombinant polynucleotides encoding CADECM may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric CADECM protein containing a heterologous moiety that can be recognized by a commerciaUy avaUable antibody may facilitate the screening of peptide Ubraries for inhibitors of CADECM activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commerciaUy avaUable affinity matrices.
  • Such moieties include, but are not limited to, glutafhione 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 immobUized glutafhione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable in ⁇ munoaffinity 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 CADECM encoding sequence and the heterologous protein sequence, so that CADECM maybe 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 CADECM may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • CADECM, fragments of CADECM, or variants of CADECM may be used to screen for compounds that specifically bind to CADECM.
  • One or more test compounds may be screened for specific binding to CADECM. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to CADECM. Examples of test compounds can include antibodies, anticaUns, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
  • variants of CADECM can be used to screen for binding of test compounds, such as antibodies, to CADECM, a variant of CADECM, or a combination of CADECM and/or one or more variants CADECM.
  • a variant of CADECM can be used to screen for compounds that bind to a variant of CADECM, but not to CADECM having the exact sequence of a sequence of SEQ ID NO: 1-30.
  • CADECM variants used to perform such screening can have a range of about 50% to about 99% sequence identity to CADECM, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to CADECM can be closely related to the natural ligand of CADECM, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (CoUgan, 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 CADECM (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 CADECM can be closely related to the natural receptor to which CADECM binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for CADECM which is capable of propagating a signal, or a decoy receptor for CADECM which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Curr. Opin. CeU 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) Curr. Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having simUar or, alternatively, different specificities can be screened for specific binding to CADECM, fragments of CADECM, or variants of CADECM.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of CADECM.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of CADECM.
  • 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 CADECM.
  • anticalins can be screened for specific binding to CADECM, fragments of CADECM, or variants of CADECM.
  • Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol.
  • 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 lipocaUns, a site which can be re- engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the 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.
  • screening for compounds which specifically bind to, stimulate, or inhibit CADECM involves producing appropriate cells which express CADECM, either as a secreted protein or on the ceU membrane.
  • Preferred cells can include ceUs from mammals, yeast, Drosoph ⁇ la, or E. coli.
  • Cells expressing CADECM or ceU membrane fractions which contain CADECM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either CADECM 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 CADECM, either in solution or affixed to a solid support, and detecting the binding of CADECM 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 soUd 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 Ugands (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. Natl. 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
  • CADECM, fragments of CADECM, or variants of CADECM may be used to screen for compounds that modulate the activity of CADECM.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for CADECM activity, wherein CADECM is combined with at least one test compound, and the activity of CADECM in the presence of a test compound is compared with the activity of CADECM in the absence of the test compound. A change in the activity of CADECM in the presence of the test compound is indicative of a compound that modulates the activity of CADECM.
  • test compound is combined with an in vitro or ceU-free system comprising CADECM under conditions suitable for CADECM activity, and the assay is performed.
  • a test compound which modulates the activity of CADECM may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraUty of test compounds maybe screened.
  • polynucleotides encoding CADECM or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
  • 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 CADECM 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.
  • ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding CADECM can also be used to create "knockin" humanized animals
  • pigs pigs
  • transgenic animals mice or rats
  • knockin technology a region of a polynucleotide encoding CADECM 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.
  • a mammal inbred to overexpress CADECM e.g., by secreting CADECM in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol.
  • CADECM appears to play a role in immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and ceU proUferative disorders, including cancer. In the treatment of disorders associated with increased CADECM expression or activity, it is desirable to decrease the expression or activity of CADECM. , In the treatment of disorders associated with decreased CADECM expression or activity, it is desirable to increase the expression or activity of CADECM.
  • CADECM 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 CADECM.
  • disorders include, but are not Umited to, an immune system disorder, such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Gushing' s disease, Addison's disease/adult respiratory distress syndrome, aUergies, ankylosing spondylitis, amyloidosis,
  • an immune system disorder
  • composition comprising a substantiaUy purified CADECM in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADECM including, but not limited to, those provided above.
  • an agonist which modulates the activity of CADECM may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADECM including, but not limited to, those Usted above.
  • an antagonist of CADECM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CADECM. Examples of such disorders include, but are not limited to, those immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and ceU proUferative disorders, including cancer described above.
  • an antibody which specificaUy binds CADECM may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express CADECM.
  • a vector expressing the complement of the polynucleotide encoding CADECM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CADECM 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 skiU in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of CADECM may be produced using methods which are generaUy known in the art.
  • purified CADECM may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind CADECM.
  • Antibodies to CADECM may also be generated using methods that are weU known in the art.
  • Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • neutraUzing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have appUcation 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 CADECM or with any fragment or oUgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological 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 BaciUi Calmette-Guerin
  • Corynebacteriumparvum are especiaUy preferable. It is preferred that the oUgopeptides, peptides, or fragments used to induce antibodies to
  • CADECM have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natural protein. Short stretches of CADECM amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to CADECM may be 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. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120).
  • chimeric antibodies such as the spUcing 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. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce CADECM-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries (Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin Ubraries or panels of highly specific binding reagents as disclosed in the Uterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299). Antibody fragments which contain specific binding sites for CADECM 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 Ubraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
  • Various 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 estabUshed specificities are weU known in the art.
  • Such irnmunoassays typicaUy involve the measurement of complex formation between CADECM and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering CADECM epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of CADECM-antibody complex divided by the molar concentrations of free antigen and free antibody under equUibrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular CADECM epitope represents a true measure of affinity.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the CADECM-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are prefe ⁇ ed for use in immunopurification and simUar procedures which ultimately require dissociation of CADECM, 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 quaUty and suitability of such preparations for certain downstream appUcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of CADECM-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quaUty and usage in various appUcations are generaUy avaUable (Catty, supra; CoUgan et al., supra).
  • polynucleotides encoding CADECM 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 oUgonucleotides) to the coding or regulatory regions of the gene encoding CADECM.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
  • antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CADECM (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 CUn. 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.
  • polynucleotides encoding CADECM may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) co ⁇ ect 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. (1993) CeU 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene
  • conditionaUy lethal gene product e.g., in the case of cancers which result from unregulated ceU proUferation
  • a protein which affords protection against intraceUular parasites e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci.
  • CADECM hepatitis B or C virus
  • fungal parasites such as Candida ⁇ lbicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodium f ⁇ lciparum and Trypanosoma cruzi.
  • diseases or disorders caused by deficiencies in CADECM are treated by constructing mammaUan expression vectors encoding CADECM and introducing these vectors by mechanical means into CADECM-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ti) ballistic gold particle deUvery, (in) Uposome-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 CADECM 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).
  • CADECM 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), (U) 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) Cu ⁇ . Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • CommerciaUy avaUable Uposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, avaUable from Invitrogen
  • aUow one with ordinary skUl in the art to deUver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A. J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammaUan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to CADECM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding CADECM under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (Hi) a Rev- responsive element (RRE) along with additional retrovirus as-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy avaUable (Stratagene) and are based onpubUshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • 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 VS Vg (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 AD. MUler (1988) J. Virol. 62:3802-3806; Dun, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al.
  • VPCL 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 skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71 :7020- 7029; Bauer, G. et al.
  • an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding CADECM to ceUs which have one or more genetic abnormaUties with respect to the expression of CADECM.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skill in the art.
  • RepUcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No.
  • Adenovirus vectors for gene therapy hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).
  • a herpes-based, gene therapy deUvery system is used to deUver polynucleotides encoding CADECM to target ceUs which have one or more genetic abnormaUties with respect to the expression of CADECM.
  • herpes simplex virus (HSV)-based vectors may be especiaUy valuable for introducing CADECM to ceUs of the central nervous system, for which HS V has a tropism.
  • the construction and packaging of herpes-based vectors are weU known to those with ordinary sk l in the art.
  • a repUcation-competent herpes simplex virus (HSV) type 1- based vector has been used to deUver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
  • the construction of a HSV-1 virus vector has also been disclosed in detaU in U.S. Patent No.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding CADECM to target ceUs.
  • SFV Semliki Forest Virus
  • alphavirus RNA repUcation a subgenomic RNA is generated that normaUy encodes the viral capsid proteins.
  • This subgenomic RNA repUcates 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).
  • enzymatic activity e.g., protease and polymerase.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the ability to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic repUcation of alphaviruses can be altered to suit the needs of the gene therapy appUcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses wiU aU ow the introduction of CADECM into a variety of ceU types.
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weU known to those with ordinary skill in the art.
  • OUgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heUx base-pairing methodology.
  • Triple heUx pairing is useful because it causes inhibition of the ab ity of the double heUx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the Uterature (Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura PubUshing, Mt. Kisco NY, pp. 163-177).
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding CADECM.
  • 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 oUgonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oUgonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding CADECM. 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-Ufe. 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' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • RNAi RNA interference
  • PTGS post-transcriptional gene sUencing
  • RNAi is a post-transcriptional mode of gene sUencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specificaUy 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 deUvery and/or viral vector deUvery methods described herein or known in the art.
  • RNAi can be induced in mammaUan ceUs by the use of smaU interfering RNA also known as siRNA.
  • siRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNAby 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 mammaUan ceUs is described by Elbashir, 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 Uposome-mediated transfection, viral vector methods, or other polynucleotide deUvery/introductory 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-bmding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
  • UTRs untranslated regions
  • 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 eliminated 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 sUencing 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 deUvered to target ceUs using expression vectors known in the art.
  • siRNA An example of a suitable expression vector for deUvery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once deUvered to the target tissue, 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 CADECM.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oUgonucleotides, 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 specifically inhibits expression of the polynucleotide encoding CADECM may be therapeutically useful, and in the treatment of disorders associated with decreased CADECM expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding CADECM may be therapeutically 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 CADECM is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabUized cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding CADECM 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 CADECM.
  • the amount of hybridization maybe quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28. ⁇ 15) or a human cell line such as HeLa ceU (Clarke, MX. 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 oUgonucleotides) 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 oUgonucleotides
  • 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. DeUvery by transfection, by Uposome 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).
  • compositions which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA).
  • Such compositions may consist of CADECM, antibodies to CADECM, and mimetics, agonists, antagonists, or inhibitors of CADECM.
  • 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, subUngual, or rectal means.
  • routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subUngual, or rectal means.
  • Compositions for pulmonary administration may be prepared in Uquid or dry powder form. These compositions are generaUy aerosoUzed immediately prior to inhalation by the patient. In the case of smaU molecules (e.g. traditional low molecular weight organic drugs), aerosol deUvery 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.
  • SpeciaUzed forms of compositions may be prepared for direct intraceUular deUvery of macromolecules comprising CADECM or fragments thereof.
  • Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule.
  • CADECM 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 initiaHy either in ceU culture assays, e.g., of neoplastic ceUs, or in 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 CADECM or fragments thereof, antibodies of CADECM, and agonists, antagonists or inhibitors of CADECM, which ameUorates 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 Uttle or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • the exact dosage wUl be determined by the practitioner, in Ught 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 combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of deUvery is provided in the Uterature and generaUy avaUable to practitioners in the art.
  • wiU employ different formulations for nucleotides than for proteins or fheir inhibitors.
  • deUvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc.
  • antibodies which specificaUy bind CADECM may be used for the diagnosis of disorders characterized by expression of CADECM, or in assays to monitor patients being treated with CADECM or agonists, antagonists, or inhibitors of CADECM.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CADECM include methods which utilize the antibody and a label to detect CADECM 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.
  • CADECM CADECM
  • ELISAs RIAs
  • FACS fluorescence-activated cell sorting
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • normal or standard values for CADECM expression are estabUshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to CADECM under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of CADECM expressed in subject, control, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • polynucleotides encoding CADECM may be used for diagnostic purposes.
  • the polynucleotides which may be used include oUgonucleotides, 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 CADECM may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of CADECM, and to monitor regulation of CADECM levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding CADECM or closely related molecules maybe used to identify nucleic acid sequences which encode CADECM.
  • the specificity of the probe determine 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 ampUfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding CADECM, aUeUc 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 CADECM 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:31-60 or from genomic sequences including promoters, enhancers, and introns of the CADECM gene.
  • Means for producing specific hybridization probes for polynucleotides encoding CADECM include the cloning of polynucleotides encoding CADECM or CADECM 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 radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaUne phosphatase coupled to the probe via avidin/biotin coupling systems, and the Uke.
  • Polynucleotides encoding CADECM may be used for the diagnosis of disorders associated with expression of CADECM.
  • an immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVf), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectoderma
  • an immune system disorder such as acquired immunodefic
  • Polynucleotides encoding CADECM maybe used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Uke assays; and in microa ⁇ ays utilizing fluids or tissues from patients to detect altered CADECM expression.
  • Such quaUtative or quantitative methods are weU known in the art.
  • polynucleotides encoding CADECM may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding CADECM 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 CADECM 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 estabUshed. This may be accomphshed by confining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CADECM, under conditions suitable for hybridization or ampUfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earUer, thereby preventing the development or further progression of the cancer.
  • oUgonucleotides designed from the sequences encoding CADECM may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OUgomers wiU preferably contain a fragment of a polynucleotide encoding CADECM, or a fragment of a polynucleotide complementary to the polynucleotide encoding CADECM, and wUl be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from polynucleotides encoding CADECM 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
  • oUgonucleotide primers derived from polynucleotides encoding CADECM are used to ampUfy 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 oUgonucleotide primers are fluorescently labeled, which aUows detection of the amplirners in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy, sequence database analysis methods, termed in siUco SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASS ARRAY 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 inmonogenic 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 Ufe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, whUe 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-Upoxygenase 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 (1999) Mol. Med.
  • CADECM CADECM
  • Methods which may also be used to quantify the expression of CADECM include radiolabeling or biotinylating nucleotides, coampUfication 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. Biochem. 212:229-236).
  • the speed of quantitation of multiple samples maybe accelerated by running the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dUutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oUgonucleotides 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 maybe selected for a patient based on his/her pharmacogenomic profile.
  • CADECM may be used as elements on a microa ⁇ ay.
  • 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. 5,840,484; hereby expressly incorporated by reference herein).
  • 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.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraUty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU 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 line.
  • 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 well 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.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • 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 CADECM to quantify the levels of CADECM expression.
  • the antibodies are used as elements on a microa ⁇ ay, 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 a ⁇ ay 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 co ⁇ elation 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 maybe more rehable 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 proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microa ⁇ ays may be prepared, used, and analyzed using methods known in the art (Brennan,
  • nucleic acid sequences encoding CADECM maybe 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 maybe 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 Ubraries (Ha ⁇ ington, J. J. et al.
  • nucleic acid sequences may be used to develop genetic tinkage maps, for example, which co ⁇ elate 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 MendeUan Inheritance in Man (OMIM) World Wide Web site. Correlation between the location tif the gene encoding CADECM 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 MendeUan Inheritance in Man
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
  • physical mapping techniques such as linkage analysis using estabhshed chromosomal markers
  • linkage analysis using estabhshed chromosomal markers may be used for extending genetic maps.
  • the placement of a gene on the chromosome of another mammaUan 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.
  • 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.
  • CADECM its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries 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 soUd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between CADECM and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT appUcation WO84/03564).
  • This method large numbers of different smaU test compounds are synthesized on a soUd substrate.
  • the test compounds are reacted with CADECM, or fragments thereof, and washed.
  • Bound CADECM is then detected by methods weU known in the art.
  • Purified CADECM can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutraUzing antibodies can be used to capture the peptide and immobilize it on a soUd support.
  • nucleotide sequences which encode CADECM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs are derived from cDNA Ubraries described in the LIFESEQ database (Incyte, Palo Alto CA). Some tissues are homogenized and lysed in guanidiniurn isothiocyanate, whUe others are homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL
  • RNA is treated with DNase.
  • poly(A)+ RNA is isolated using oUgo d(T)-c ⁇ upled 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
  • cDNA Ubraries are synthesized and cDNA Ubraries 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 usmg oUgo d(T) or random primers. Synthetic oUgonucleotide adapters are Ugated 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 Ugated into compatible restriction enzyme sites of the polylrnker of a suitable plasmid, e.g., PBLUESCRiPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2- TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo Alto CA), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof.
  • a suitable plasmid e.g., PBLUESCRiPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2- TOP
  • Plasmids obtained as described in Example I are recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU 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, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids are resuspended in 0.1 ml of distilled water and stored, with or without lyophiUzation, at 4°C
  • plasmid DNA is ampUfied 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 ampUfied 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 (AppUed 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 (AppUed 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 (AppUed 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 vahdated 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 pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences fro Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte, Palo Alto CA); hidden Markov model (HMM)-based protein famUy databases such as PFAM, F CY, and TIGRFAM (Haft, D.H.
  • pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PRO
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. US A 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) Cu ⁇ . 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 fuU 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 conesponding fuU length polypeptide sequences.
  • a polypeptide may begin at any of the methionine residues of the full 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, hidden Markov model (HMM)-based protein family 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 hidden Markov model
  • Prosite Prosite
  • HMM-based protein family databases such as PFAM, INCY, and TIGRFAM
  • HMM-based protein domain databases such as SMART.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASER
  • Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aUgnment program (DNASTAR), which also calculates the percent identity between aUgned 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 appUcable, 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. Kartin (1997) J. Mol. Biol. 268:78-94; Burge, C and S. KarUn (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • Genscan is a FASTA database 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 ceU adhesion and extraceUular matrix proteins. Potential ceU adhesion and extraceUular matrix proteins are also identified by homology to Incyte cDNA sequences that have been annotated as ceU adhesion and extraceUular matrix proteins.
  • Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri pubUc databases. Where necessary, the 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 pubUc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When 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 pubUc cDNA sequences using the assembly process described in Example III. Alternatively, fuU length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
  • Partial cDNA sequences 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 spUce 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 fuU length with an algorithm based on BLAST analysis.
  • First, partial cDNAs assembled as described in Example III are queried against pubUc databases such as the GenBank primate, rodent, mammaUan, 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 pairs (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 pairs
  • GenBank protein homolog the chimeric protein, or both are used as probes to search for homologous genomic sequences from the pubUc 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.
  • sequences used to assemble SEQ ID NO:31-60 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- aterman algorithm. Sequences from these databases that matched SEQ ID NO:31-60 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data avaUable from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the clustered sequences have been previously mapped. Inclusion of a mapped sequence in a cluster results in the assignment of aU sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • 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.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound (S ambrook and RusseU, supra, ch. 7 ; Ausubel et al., supra, ch. 4).
  • 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 simUarity between two sequences and the length of the sequence match.
  • the product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quaUty in a BLAST aUgnment. 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 CADECM 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 JU).
  • Each cDNA sequence is derived from a cDNA Ubrary 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; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Ubraries in each category is counted and divided by the total number of Ubraries 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 Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding CADECM.
  • cDNA sequences and cDNA Ubrary/tissue information are found in the LIFESEQ database (Incyte, Palo Alto CA). VIII. Extension of CADECM Encoding Polynucleotides
  • FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide 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 Ubraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed. High fideUty ampUfication is obtained by PCR using methods weU known in the art. 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 ⁇ SO ⁇ and 2- mercaptoefhanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pair PCI A and PCI B: Step 1: 94°C 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C
  • the parameters for primer pair T7 and SK+ 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
  • the plate is scanned in a Ffuoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • Ffuoroskan 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 aUquot 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 reUgation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to reUgation 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 reUgated using T4 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs are selected on antibiotic-containing media, and mdividual colonies are picked and cultured overnight at 37 °C in 384-weU plates in LB/2x carb Uquid media.
  • the ceUs are lysed, and DNA is ampUfied 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 reamptified using the same conditions as described above.
  • Samples are diluted 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 (AppUed Biosystems).
  • SNPs single nucleotide polymorphisms
  • LIFESEQ database Incyte
  • Sequences from the same gene are clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene.
  • An algorithm consisting of a series of filters is used to distinguish SNPs from other sequence variants. Preliminary filters remove the majority of basecall errors by requiring a minimum Phred quality score of 15, and remove sequence alignment e ⁇ ors and e ⁇ ors resulting from improper trimrning of vector sequences, chimeras, and spUce variants.
  • Clone e ⁇ or filters use statisticaUy generated algorithms to identify e ⁇ ors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering e ⁇ or filters use statistically generated algorithms to identify e ⁇ ors 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 Venezuelan, 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.
  • Hybridization probes derived from SEQ ID NO:31-60 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pahs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments. OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 Ci of
  • [ ⁇ _ 32 p] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aUquot 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 transfe ⁇ ed to NYTRAN PLUS nylon membranes (Schleicher & SchueU, DurhamNH). Hybridization is carried out for 16 hours at 40 °C To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared. XI. Microarrays
  • the linkage or synthesis of array elements upon a microa ⁇ ay can be achieved utilizing photoUthography, 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 soUd with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach. Oxford University Press, London). Suggested substrates include silicon, silica, glass sUdes, glass chips, and sUicon wafers.
  • a procedure analogous to a dot or slot blot may also be used to a ⁇ ange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical a ⁇ ay 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 oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers 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 a ⁇ ay element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • 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) ceUulose 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 GEMBRIGHT 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 labehng) 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 a ⁇ ay elements.
  • Each a ⁇ ay 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. AmpUfied a ⁇ ay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified a ⁇ ay elements are immobilized on polymer-coated glass slides.
  • Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma-Aldrich, St. Louis MO) in 95% ethanol.
  • Coated sUdes are cured in a 110°C oven.
  • a ⁇ ay elements are appUed to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l of the a ⁇ ay 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 a ⁇ ay element sample per sUde.
  • Microa ⁇ ays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microa ⁇ ays 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 microa ⁇ ays 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 microa ⁇ ay surface and covered with an 1.8 cm 2 coverslip.
  • the a ⁇ ays are transfe ⁇ ed to a waterproof chamber having a cavity just shghtly larger than a microscope slide.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a comer of the chamber.
  • the chamber containing the a ⁇ ays is incubated for about 6.5 hours at 60° C.
  • a ⁇ ays 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 a ⁇ ay using a 20X microscope objective (Nikon, Inc., MelvUle NY).
  • the slide containing the a ⁇ ay 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 a ⁇ ay 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 a ⁇ ay 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 caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the a ⁇ ay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be co ⁇ elated with a weight ratio of hybridizing species of 1 : 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 usmg 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 co ⁇ ected 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). Array 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 of SEQ ID NO:40 was down-regulated in treated breast carcinoma cells versus untreated human mammary epitheUal ceUs (HMEC) as determined by microarray analysis.
  • HMEC human mammary epitheUal ceUs
  • the gene expression profile of this nonmaUgnant mammary epitheUal ceU line HMEC was compared to the gene expression profiles of seven breast carcinoma lines at different stages of tumor progression.
  • AU ceUs were grown under optimal growth conditions in the presence of growth factors and nutrients, with the seven breast carcinoma ceU Unes being treated with mammary epithelium growth medium (MEGM).
  • MEGM mammary epithelium growth medium
  • expression of SEQ ID NO:40 was down-regulated in breast carcinoma ceUs versus untreated human mammary epitheUal ceUs (HMEC) as determined by microarray analysis.
  • HMEC human mammary epitheUal ceUs
  • the gene expression profile of this nonmaUgnant mammary epitheUal ceU line HMEC was compared to the gene expression profiles of the same seven breast carcinoma ceU Unes Usted above.
  • AU ceUs were grown in basal media in the absence of growth factors and hormones for 24 hours prior to comparison
  • the expression of SEQ ID NO:40 was decreased at least two-fold in one of the seven breast carcinoma ceU Unes tested, the BT-474.
  • SEQ ID NO:40 can be used for one or more of the foUowing: i) monitoring treatment ofbreast cancer, u) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer.
  • SEQ ID NO:44 showed tissue-specific expression as determined by microa ⁇ ay 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 microarray.
  • SEQ ID NO:44 was increased by at least two-fold in ventricular heart tissue as compared to the reference sample. Therefore, SEQ ID NO:44 can be used as a tissue marker for ventricular heart tissue.
  • expression of SEQ ID NO:47 was down-regulated in breast carcmoma ceUs versus human mammary epitheUal ceUs (HMEC) as determined by microarray analysis. The gene expression profile of this nonmaUgnant mammary epitheUal ceU line HMEC was compared to the gene expression profiles ofbreast carcinoma Unes at different stages of tumor progression.
  • AU ceUs were grown in basal media in the absence of growth factors and hormones for 24 hours prior to comparison.
  • the expression of SEQ ID NO:47 was decreased at least two-fold in five out of seven breast carcinoma ceU Unes tested (BT-20, BT-483, MCF-7, MCF- 10A, and MDA-MB-468).
  • the expression of SEQ ID NO:47 was down-regulated in breast carcinoma ceUs versus untreated human mammary epitheUal ceUs (HMEC) as determined by microa ⁇ ay analysis.
  • HMEC human mammary epitheUal ceUs
  • the gene expression profile of this nonmaUgnant mammary epitheUal ceU line HMEC was compared to the gene expression profiles of seven breast carcinoma Unes at different stages of tumor progression.
  • SEQ ID NO:47 can be used for one or more of the foUowing: i) monitoring treatment ofbreast cancer, U) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer.
  • SEQ ID NO:50 showed differential expression in a number of breast cancer ceU Unes, as determined by microa ⁇ ay analysis.
  • the gene expression profile of a nonmaUgnant mammary epitheUal ceU Une (HMEC) was compared to the gene expression profiles of breast carcinoma Unes at different stages of tumor progression.
  • aU ceUs were grown under optimal growth conditions, in the presence of growth factors and nutrients; breast carcinoma ceU lines were also treated with mammary epitheUum growth medium (MEGM).
  • MEGM mammary epitheUum growth medium
  • the expression level of SEQ ID NO:50 was decreased by at least 2-fold in MCF7, BT-474, BT-483 and MDA-MB-468 ceU Unes, when compared to the expression levels of SEQ ID NO:50 in HMEC ceUs.
  • aU ceUs were grown in basal media in the absence of growth factors and hormones for 24 hours prior to comparison.
  • the expression level of SEQ ID NO:50 was decreased by at least 2- fold in MCF-10A, MCF7, BT-20, BT-483 and MDA-MB-468 ceU Unes, when compared to the expression levels of SEQ ID NO:50 in HMEC ceUs.
  • the ceU Unes examined included the above Usted ceU Unes and the foUowing additional ceU Unes: a) T-47D, a breast carcinoma ceU Une isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast; b) Sk- BR-3, a breast adenocarcinoma ceU Une isolated from a maUgnant pleural effusion of a 43 -year-old female; c) MDA-mb-231, a breast tumor ceU Une isolated from the pleural effusion of a 51 -year-old female; and d) MDA-mb-435S, a spindle-shaped strain that evolved from the parent Une (435) isolated by R.
  • SEQ ID NO:50 was examined in several breast cancer Unes, detaUed above, and compared to the expression level detected in MCF-10A fibroblastic ceUs.
  • the expression of SEQ ID NO:50 decreased at least 3-fold in T-47D ceUs, in comparison to the gene expression levels detected in MCF-10A ceUs. Therefore, in various embodiments, SEQ ID NO:50 can be used for one or more of the foUowing: i) monitoring treatment ofbreast cancer, U) diagnostic assays for breast, and Ui) developing therapeutics and/or other treatments breast cancer.
  • expression of SEQ ID NO:59 and SEQ ID NO:60 was up-regulated in diseased tissue versus normal tissue as determined by microa ⁇ ay analysis.
  • a normal ovary from a female donor was compared to an ovarian tumor from the same donor (Huntsman Cancer Institute, Salt Lake City, UT).
  • Expression of SEQ ID NO:59 and SEQ ID NO:60 was increased at least twofold in one out of two samples examined for SEQ ID NO:59 and SEQ ID NO:60.
  • SEQ ID NO:59 and SEQ ID NO:60 can be used for one or more of the foUowing: i) monitoring treatment of ovarian cancer, U) diagnostic assays for ovarian cancer, and Ui) developing therapeutics and/or other treatments for ovarian cancer.
  • Sequences complementary to the CADECM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring CADECM.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CADECM.
  • a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oUgonucleotide is designed to prevent ribosomal binding to the CADECM-encoding transcript.
  • CADECM expression and purification of CADECM 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 tip-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express CADECM upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • CADECM in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU Unes with recombinant Autographica calif omica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica calif omica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CADECM 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 Spodopterafi'ugiperda (Sf9) insect ceUs inmost cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, Y. et al. (1996) Hum. Gene Ther. 7:1937- 1945).
  • CADECM 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 CADECM obtained by these methods can be used cUrectly in the assays shown in Examples XVII and XVIII, where appUcable. XIV. Functional Assays
  • CADECM function is assessed by expressing the sequences encoding CADECM at physiologically elevated levels in mammaUan ceU culture systems.
  • cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT 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 Une, for example, an endotheUal or hematopoietic ceU Une, using either Uposome 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 reUable 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.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Hght scatter and 90 degree side hght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU 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 Or erod, M.G. (1994; Flow Cvtometry, Oxford, New York NY).
  • CADECM The influence of CADECM on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding CADECM 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 immunoglobuUn G (IgG).
  • Transfected ceUs are efficiently separated fromnontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods well known by those of skUl in the art. Expression of mRNA encoding CADECM and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques.
  • the CADECM amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or inhydrophiUc regions are weU described in the art (Ausubel et al., supra, ch. 11).
  • oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hy ⁇ j-oxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti- CADECM activity by, for example, binding the peptide or CADECM to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Media containing CADECM are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of CADECM (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/CADECM binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotiope, such as urea or thiocyanate ion), and CADECM is coUected.
  • CADECM or 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).
  • 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 CADECM, washed, and any weUs with labeled CADECM complex are assayed. Data obtained using different concentrations of CADECM are used to calculate values for the number, affinity, and association of CADECM with the candidate molecules.
  • molecules interacting with CADECM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially avaUable kits based on the two-hybrid system, such as the MATCHMAKER system (BD Clontech).
  • CADECM may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) US . Patent No. 6,057, 101).
  • An assay for CADECM activity measures the expression of CADECM on the ceU surface.
  • cDNA encoding CADECM is transfected into a non-leukocytic ceU Une.
  • CeU surface proteins are labeled with biotin (de la Fuente, MA. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed usmg CADECM-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of CADECM expressed on the ceU surface.
  • an assay for CADECM activity measures the amount of ceU aggregation induced by overexpression of CADECM.
  • cultured ceUs such as NIH3T3 are transfected with cDNA encoding CADECM contained within a suitable mammaUan expression vector under control of a strong promoter.
  • Cotransfection with cDNA encoding a fluorescent marker protem, such as Green Fluorescent Protein (CLONTECH) is useful for identifying stable transfectants.
  • the amount of ceU agglutination, or clumping, associated with transfected ceUs is compared with that associated with untransfected ceUs.
  • the amount of ceU agglutination is a direct measure of CADECM activity. , ⁇ _schreib compassion
  • an assay for CADECM activity measures the disruption of cytoskeletal filament networks upon overexpression of CADECM in cultured ceU Unes (Rezniczek, G. A. et al.
  • cDNA encoding CADECM is subcloned into a mammaUan expression vector that drives high levels of cDNA expression.
  • This construct is transfected into cultured ceUs, such as rat kangaroo PtK2 or rat bladder carcinoma 804G ceUs.
  • Actin fUaments and intermediate filaments such as keratin and vimentin are visuahzed by immunofluorescence microscopy using antibodies and techniques weU known in the art.
  • the configuration and abundance of cyoskeletal filaments can be assessed and quantified using confocal imaging techniques.
  • the bundling and coUapse of cytoskeletal filament networks is indicative of CADECM activity.
  • ceU adhesion activity in CADECM is measured in a 96-weU plate in which weUs are first coated with CADECM by adding solutions of CADECM of varying concentrations to the weUs. Excess CADECM is washed off with saline, and the weUs incubated with a solution of 1 % bovine serum albumin to block non-specific ceU binding. AUquots of a ceU suspension of a suitable ceU type are then added to the weUs and incubated for a period of time at 37 °C Non-adherent ceUs are washed off with saline and the ceUs stained with a suitable ceU stain such as Coomassie blue.
  • a suitable ceU stain such as Coomassie blue.
  • the intensity of staining is measured using a variable wavelength multi-weU plate reader and compared to a standard curve to dete ⁇ nine the number of ceUs adhering to the CADECM coated plates.
  • the degree of ceU staining is proportional to the ceU adhesion activity of CADECM in the sample.
  • ABI FACTURA A program that removes vector sequences and masks Applied Biosystems, Foster City, CA. ambiguous bases in nucleic acid sequences.
  • fastx score 100 or greater
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) Genome Res. 8:186-194.
  • TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
  • HMM hidden Markov model

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Abstract

Plusieurs formes de réalisation de la présente invention concernent des protéines d'adhésion cellulaire et de matrice extracellulaire humaines (PACME) et des polynucléotides qui codent ces PACME. Les formes de réalisation de l'invention comprennent également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. D'autres formes de réalisation comprennent des méthodes de diagnostic, de traitement ou de prévention des maladies associées à l'expression aberrante des PACME.
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US20130004955A1 (en) * 2009-10-26 2013-01-03 Externautics S.P.A. Ovary Tumor Markers and Methods of Use Thereof
US8921058B2 (en) 2009-10-26 2014-12-30 Externautics Spa Prostate tumor markers and methods of use thereof
US20170227543A1 (en) * 2009-10-26 2017-08-10 Externautics Spa Breast Tumor Markers And Methods Of Use Thereof

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US20130004955A1 (en) * 2009-10-26 2013-01-03 Externautics S.P.A. Ovary Tumor Markers and Methods of Use Thereof
US8921058B2 (en) 2009-10-26 2014-12-30 Externautics Spa Prostate tumor markers and methods of use thereof
US20170227543A1 (en) * 2009-10-26 2017-08-10 Externautics Spa Breast Tumor Markers And Methods Of Use Thereof
US10288617B2 (en) * 2009-10-26 2019-05-14 Externautics Spa Ovary tumor markers and methods of use thereof

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