Cell adhesion and extracellular matrix proteins

TECHNICAL FIELD

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.

BACKGROUND OF THE INVENTION

Cell Adhesion 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. These proteins share multiple repeats of a cadherin-specific motif, and the repeats form the folding units of the cadherin extracellular domain. 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. Recent studies report that protocadherins 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 α and β. At least 8 different β subunits (β1-β8) and at least 12 different α subunits have been identified (α1-α8, αL, αM, αX, and αIIb). Individual α subunits are capable of associating with different β subunits, suggesting a possible mechanism for specifying integrin function and ligand binding affinity. Members of the β subunit family are generally of 90-110 kilodaltons (kD) in molecular weight and share about 40-48% amino acid sequence homology. About 56 cysteines distributed among four repeating units are also conserved. Some variation in these conserved features is observed among some of the more divergent β subunit family members. Members of the α subunit family are generally 150-200 kilodaltons in molecular weight and are not as well conserved as the β 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, Calif., 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. For example, binding of integrin to its extracellular ligand may stimulate changes in intracellular calcium levels or protein kinase activity (Sjaastad, M. D. and Nelson, W. J. (1997) BioEssays 19:47-55). At least ten cell surface receptors of the integrin family recognize the ECM component fibronectin, which is involved in many different biological processes including cell migration and embryogenesis (Johansson, S. et al. (1997) Front. Biosci. 2:D126-D146). Integrins also bind to immunoglobulin superfamily proteins such as ICAM-1, -2, and -3 and VCAM-1.

Most integrins have been shown to activate focal adhesion kinase (FAK), a protein tyrosine kinase that is linked to Ras signaling pathways that modify the cytoskeleton and stimulate the mitogen-activated protein kinase (MAPK) cascade (Hanks, S. K. and T. R. Polte (1997) BioEssays 19:137-145). Integrins can also influence growth factor signaling through direct interaction with growth factor receptor tyrosine kinases (RTKs) (Miyamoto, S. et al. (1996) J. Cell Biol. 135:1633-1642). 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). For example, leukocyte adhesion deficiency (LAD) 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 β2 subunit. Additionally, 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. A. (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, in particular, includes lectins that bind β-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 β-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, or LEC-CAMs, 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. 231:802-807; Hidari, K. I. et al. (1997) J. Biol. Chem. 272:28750-28756). Members of the selectin family possess three characteristic motifs: a lectin or carbohydrate recognition domain; an epidermal growth factor-like domain; and a variable number of short consensus repeats (scr or “sushi” repeats) which are also present in complement regulatory proteins.

Neural cell adhesion proteins (NCAPs) play roles in the establishment of neural networks during development and regeneration of the nervous system (Uyemura, K. et al. (1996) Essays Biochem. 31:37-48; Brummendorf, T., and F. G. Rathjen (1996) Curr. Opin. Neurobiol. 6:584-593). NCAP participates in neuronal cell migration, cell adhesion, neurite outgrowth, axonal fasciculation, pathfinding, synaptic target-recognition, synaptic formation, myelination and regeneration. NCAPs are expressed on the surfaces of neurons associated with learning and memory. Mutations in 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. In some cases, expression of NCAP is not restricted to the nervous system. L1, 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 immunoglobulin domains, a membrane spanning domain, and an intracellular region. Many NCAPs contain post-translational modifications including covalently attached oligosaccharide, glucuronic acid, and sulfate. 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), limbic system-associated membrane protein (LAMP), opiate-binding cell-adhesion molecule (OBCAM), and myelin-associated glycoprotein (MAG). 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, F11, Bravo, and L1 . Mixed-type 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.

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. (2000) Curr. Opin. Neurobiol. 10:88-94).

An NCAP subfamily, the NCAP-LON subgroup, 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). Immunostaining localizes Kilon to the dendrites and soma of pyramidal neurons. Kilon has three C2 type immunoglobulin-like domains, six predicted glycosylation 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. In magnocellular neurons, 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. Leukocyte function-associated antigen 1 (LFA-1) is expressed on the surface of all white blood cells and is a receptor for intercellular adhesion molecules (ICAM) 1 and 2 which are members of the immunoglobulin superfamily. The interaction of LFA-1 with ICAMs 1 and 2 provides essential accessory adhesion signals in many immune interactions, including those between T and B lymphocytes and cytotoxic T cells and their targets. In addition, both ICAMs are expressed at low levels on resting vascular endothelium. 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).

Cell adhesion proteins also include some members of the proline-rich proteins (PRPs). 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. One family of PRPs are the proline-rich synapse-associated proteins (ProSAPs) which have been shown to bind to members of the postsynaptic density (PSD) protein family and subtypes of the somatostatin receptor (Yao, L. et al. (1999) J. Biol. Chenm 274:

27463-27466; Zitzer, H. et al. (1999) J. Biol. Chem. 274:32997-33001). 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. Several groups of ProSAPs are important structural constituents of synaptic structures in human brain (Zitzer et al., supra). Another member of the PRP family is the HLA-B-associated transcript 2 protein (BAT2) 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 from mesenchyme-blastula embryos. Toposome precursors including vitellogenin promote cell adhesion of dissociated blastula cells.

There are additional specific domains characteristic of cell adhesion proteins. One such domain is the 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).

Leucine rich repeats (LRR) 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. The SAM domain can potentially function as a protein interaction module through its ability to form homo- or hetero-oligomers with other SAM domains (Schultz, J. et al. (1997) Protein Sci. 6:249-253).

Extracellular Matrix Proteins

The extracellular matrix (ECM) 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 determines 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 collagens 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, N.Y., pp. 2105-2117; and Creighton, T. E. (1984) Proteins, Structures and Molecular Principles, W.H. Freeman and Company, New York, N.Y., 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 Marfan'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. (1994) Molecular Biology of the Cell, Garland Publishing, New York, N.Y., pp. 984-986.) The fibulin proteins connect elastic fibers and are though to promote the formation and stabilization of the fiber. 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 III fibronectin repeat. The type III fibronectin repeat is about 90 amino acids in length and is also found in other ECM proteins and in some plasma membrane and cytoplasmic proteins. Furthermore, 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. Disruption of both copies of the gene encoding fibronectin causes early embryonic lethality in mice. The mutant embryos display extensive morphological defects, including defects in the formation of the notochord, somites, heart, blood vessels, neural tube, and extraembryonic structures. (Reviewed in Alberts et al., supra, pp. 986-987.)

Laminin is a major glycoprotein component of the basal lamina which underlies and supports epithelial cell sheets. Laminin is one of the first ECM proteins synthesized in the developing embryo. Laminin 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. (Reviewed in Alberts et al., supra, pp. 990-991.)

Many proteinaceous ECM components are proteoglycans. Proteoglycans are composed of unbranched polysaccharide chains (glycosaminoglycans) attached to protein cores. Common proteoglycans include aggrecan, betaglycan, decorin, perlecan, serglycin, and syndecan-1. Some of these molecules not only provide mechanical support, but also bind to extracellular signaling molecules, such as fibroblast growth factor and transforming growth factor β, suggesting a role for proteoglycans in cel-cell communication. (Reviewed in Alberts et al., supra, pp. 973-978.) Likewise, the 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).

Dentin phosphoryn (DPP) is a major component of the dentin ECM. DPP is a proteoglycan that is synthesized and expressed by 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.

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). Hemomucin is 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 TIGR/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. For example, in the C-terminal domain of versican, 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 in human gliomas, and appears to play a role in determining the invasive potential of brain tumor cells (Gary, S. C. et al. (1998) Curr. Opin. Neurobiol. 8:576-581). There is close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin (Kohda, D. et al. (1996) Cell 86:767-775).

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. For example, 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, C. G. (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. For example, 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. Cell 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 immunoglobulins. 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).

Other ECM proteins are members of the type A domain of von Willebrand 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-like 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 extracellular matrix protein that is expressed in a broad range of mammalian tissues and organs. Matrilin-2 is thought to play a role in ECM assembly by bridging collagen fibrils and the aggrecan network (Deak, F. et al. (1997) J. Biol. Chem. 272:9268-9274).

The thrombospondins are multimeric, calcium-binding extracellular glycoproteins found widely in the embryonic extracellular 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 well as a motif known as the thrombospondin type 1 repeat (TSR). The TSR is approximately 60 amino acids in length and contains six conserved cysteine residues. Motifs within TSR domains are involved in mediating cell adhesion through binding to proteoglycans and sulfated glycolipids. Thrombospondin-1 inhibits angiogenesis and modulates endothelial cell 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 cell and growth cone migration. Proteins that contain TSRs include the F-spondin gene family, the semaphorin 5 family, UNC-5, and SCO-spondin. The TSR superfamily includes the ADAMTS proteins which contain an ADAM (A Disintegrin and Metalloproteinase) domain as well as one or more TSRs. 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 mammalian tenascin-X, an ECM protein that appears to be involved in cell adhesion (Prosite PDOC00445).

Expression Profiling

Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.

One area in particular in which microarrays find use is in gene expression analysis. 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. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, 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 specifically related to a particular genetic predisposition, condition, disease, or disorder.

Ovarian Cancer

Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rates 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 microsatellite instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.

Peroxisome Proliferator-activated Receptor Gamma (PPARγ) Agonist

Thiazolidinediones (TZDs) act as agonists for the peroxisome-proliferator-activated receptor gamma (PPARγ), a member of the nuclear hormone receptor superfamily. TZDs reduce hyperglycemia, hyperinsulinemia, and hypertension, in part by promoting glucose metabolism and inhibiting gluconeogenesis. Roles for PPARγ and its agonists have been demonstrated in a wide range of pathological conditions including diabetes, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers such as breast, prostate, liposarcoma, and colon cancer.

The mechanism by which TZDs and other PPARγ agonists enhance insulin sensitivity is not fully understood, but may involve the ability of PPARγ to promote adipogenesis. When ectopically expressed in cultured preadipocytes, PPARγ is a potent inducer of adipocyte differentiation. TZDs, in combination with insulin and other factors, can also enhance differentiation of human preadipocytes in culture (Adams et al. (1997) J. Clin. Invest. 100:3149-3153). The relative potency of different TZDs in promoting adipogenesis in vitro is proportional to both their insulin sensitizing effects in vivo, and their ability to bind and activate PPARγ in vitro. Interestingly, adipocytes derived from omental adipose depots are refractory to the effects of TZDs. It has therefore been suggested that the insulin sensitizing effects of TZDs may result from their ability to promote adipogenesis in subcutaneous adipose depots (Adams et al., supra). Further, dominant negative mutations in the PPARγ gene have been identified in two non-obese subjects with severe insulin resistance, hypertension, and overt non-insulin dependent diabetes mellitus (NIDDM) (Barroso et al. (1998) Nature 402:880-883).

NIDDM is the most common form of diabetes mellitus, a chronic metabolic disease that affects 143 million people worldwide. NIDDM is characterized by abnormal glucose and lipid metabolism that result from a combination of peripheral insulin resistance and defective insulin secretion. NHDDM has a complex, progressive etiology and a high degree of heritability. Numerous complications of diabetes including heart disease, stroke, renal failure, retinopathy, and peripheral neuropathy contribute to the high rate of morbidity and mortality.

At the molecular level, PPARγ functions as a ligand activated transcription factor. In the presence of ligand, PPARγ forms a heterodimer with the retinoid X receptor (RXR) which then activates transcription of target genes containing one or more copies of a PPARγ response element (PPRE). Many genes important in lipid storage and metabolism contain PPREs and have been identified as PPARγ targets, including PEPCK, aP2, LPL, ACS, and FAT-P (Auwerx, J. (1999) Diabetologia 42:1033-1049). Multiple ligands for PPARγ have been identified. These include a variety of fatty acid metabolites; synthetic drugs belonging to the TZD class, such as Pioglitazone and Rosiglitazone (BRL49653); and certain non-glitazone tyrosine analogs such as GI262570 and GW1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenous ligand for PPARγ.

Expression of PPARγ is very high in adipose but barely detectable in skeletal muscle, the primary site for insulin stimulated glucose disposal in the body. PPARγ is also moderately expressed in large intestine, kidney, liver, vascular smooth muscle, hematopoietic cells, and macrophages. The high expression of PPARγ in adipose suggests that the insulin sensitizing effects of TZDs may result from alterations in the expression of one or more PPARγ regulated genes in adipose tissue. Identification of PPARγ target genes will contribute to better drug design and the development of novel therapeutic strategies for diabetes, obesity, and other conditions.

Systematic attempts to identify PPARγ target genes have been made in several rodent models of obesity and diabetes (Suzuki et al. (2000) Jpn. J. Pharmacol. 84:113-123; Way et al. (2001) Endocrinology 142:1269-1277). However, a serious drawback of the rodent gene expression studies is that significant differences exist between human and rodent models of adipogenesis, diabetes, and obesity (Taylor (1999) Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews 78:783-809). Therefore, an unbiased approach to identifying TZD regulated genes in primary cultures of human tissues is necessary to fully elucidate the molecular basis for diseases associated with PPARγ activity.

Tangier Disease

Tangier disease (TD) is a genetic disorder characterized by near absence of circulating high density lipoprotein (HDL) and the accumulation of cholesterol esters in many tissues, including tonsils, lymph nodes, liver, spleen, thymus, and intestine. Low levels of HDL represent a clear predictor of premature coronary artery disease and homozygous TD correlates with a four- to six-fold increase in cardiovascular disease compared to controls. HDL plays a cardio-protective role in reverse cholesterol transport, the flux of cholesterol from peripheral cells such as tissue macrophages through plasma lipoproteins to the liver. The HDL protein, apolipoprotein A-I, plays a major role in this process, interacting with the cell surface to remove excess cholesterol and phospholipids. This pathway is severely impaired in TD and the defect lies in a specific gene, the ABC1 transporter. This gene is a member of the family of ATP-binding cassette transporters, which utilize ATP hydrolysis to transport a variety of substrates across membranes.

Colon Cancer

The potential application of gene expression profiling is particularly relevant to improving the diagnosis, prognosis, and treatment of cancers, such as colon cancer and breast cancer. While soft tissue sarcomas are relatively rare, more than 50% of new patients diagnosed with the disease will die from it. The molecular pathways leading to the development of sarcomas are relatively unknown, due to the rarity of the disease and variation in pathology. Colon cancer evolves through a multi-step process whereby pre-malignant colonocytes undergo a relatively defined sequence of events leading to tumor formation. Several factors participate in the process of tumor progression and malignant transformation including genetic factors, mutations, and selection.

To understand the nature of gene alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. The first, Familial Adenomatous Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. The second known inherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in mismatch repair genes.

Although hereditary colon cancer syndromes occur in a small percentage of the population, and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be applied broadly. For instance, somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in disease progression. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer. Less is understood about downstream targets of these mutations and the role they may play in cancer development and progression.

Breast Cancer

More than 180,000 new cases of breast cancer are diagnosed each year, and the mortality rate for breast cancer approaches 10% of all deaths in females between the ages of 45-54 (Gish, K. (1999) AWIS Magazine 28:7-10). However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou, C. M. et al. (2000) Nature 406:747-752).

Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra). However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to non-inherited mutations that occur in breast epithelial cells.

The relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied. (See Khazaie, K. et al. (1993) Cancer and Metastasis Rev. 12:255-274, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR is one, have also been implicated in breast cancer. The abundance of erbB receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S. S. et al. (1994) Am. J. Clin. Pathol. 102:S13-S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix G1a protein which is overexpressed is human breast carcinoma cells; Drg1 or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen, L. et al. (1990) Oncogene 5:1391-1395; Ulrix, W. et al (1999) FEBS Lett 455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Immunol. 213:51-64; and Lee, S. W. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2504-2508).

Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell 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 epithelial cells at various stages of malignant transformation.

Lung Cancer

Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologically distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). The fourth group of cancers is referred to as small cell lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K-ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease.

PBMCs

Human peripheral blood mononuclear cells (PBMC) can be classified into discrete cellular populations representing the major cellular components of the immune system. PBMCs contain about 52% lymphocytes (12% B lymphocytes, 40% T lymphocytes {25% CD4+ and 15% CD8+}), 20% NK cells, 25% monocytes, and 3% various other cells including dendritic cells and progenitor cells. Glucocorticoids are naturally occurring hormones that prevent or suppress inflammation and immune responses when administered at pharmacological doses. At the molecular level, unbound glucocorticoids readily cross cell membranes and bind with high affinity to specific cytoplasmic receptors. Subsequent to binding, transcription and, ultimately, protein synthesis are affected. The result can include inhibition of leukocyte infiltration at the site of inflammation, interference in the function of mediators of inflammatory response, and suppression of humoral immune responses. The antiinflammatory actions of corticosteroids are thought to involve phospholipase A2 inhibitory proteins, collectively called lipocortins. Lipocortins, in turn, control the biosynthesis of potent mediators of inflammation such as prostaglandins and leukotrienes by inhibiting the release of the precursor molecule arachidonic acid. Beclomethasone is a synthetic glucocorticoid that is used for treating steroid-dependent asthma, relieving symptoms associated with allergic or nonallergic (vasomotor) rhinitis, or preventing recurrent nasal polyps following surgical removal. The anti-inflammatory and vasoconstrictive effects of intranasal beclomethasone are 5000 times greater than those produced by hydrocortisone.

Prostate Cancer

Prostate cancer is a common malignancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year. Once cancer cells arise in the prostate, they are stimulated by testosterone to a more rapid growth. Thus, removal of the testes can indirectly reduce both rapid growth and metastasis of the cancer. Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous cell and transitional cell carcinomas, both of which arise in the prostatic ducts or other parts of the prostate gland.

As with most tumors, prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population. These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung. A variety of genes may be differentially expressed during tumor progression. For example, loss of heterozygosity (LOH) is frequently observed on chromosome 8p in prostate cancer. Fluorescence in situ hybridization (FISH) revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with a significantly higher frequency of the deletion on 8p21.2-p21.1 in advanced prostate cancer than in localized prostate cancer, implying that deletions on 8p22-p21.3 play an important role in tumor differentiation, while 8p21.2-p21.1 deletion plays a role in progression of prostate cancer (Oba, K. et al. (2001) Cancer Genet. Cytogenet. 124: 20-26).

A primary diagnostic marker for prostate cancer is prostate specific antigen (PSA). PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithelial cells. The quantity of PSA correlates with the number and volume of the prostatic epithelial cells, and consequently, the levels of PSA are an excellent indicator of abnormal prostate growth. Men with prostate cancer exhibit an early linear increase in PSA levels followed by an exponential increase prior to diagnosis. However, since PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer.

Current areas of cancer research provide additional prospects for markers as well as potential therapeutic targets for prostate cancer. Several growth factors have been shown to play a critical role in tumor development, growth, and progression. The growth factors Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFα) are important in the growth of normal as well as hyperproliferative prostate epithelial cells, particularly at early stages of tumor development and progression, and affect signaling pathways in these cells in various ways (Lin, J. et al. (1999) Cancer Res. 59:2891-2897; Putz, T. et al. (1999) Cancer Res. 59:227-233). The TGF-β family of growth factors are generally expressed at increased levels in human cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold, L. I. (1999) Crit. Rev. Oncog. 10:303-360). Finally, there are human cell lines representing both the androgen-dependent stage of prostate cancer (LNCap) as well as the androgen-independent, hormone refractory stage of the disease (PC3 and DU-145) that have proved useful in studying gene expression patterns associated with the progression of prostate cancer, and the effects of cell treatments on these expressed genes (Chung, T. D. (1999) Prostate 15:199-207).

Obesity

Adipose tissue stores and releases fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of excess energy use. Its primary purpose is mobilization during energy deprivation. Understanding how various molecules regulate adiposity and energy balance in physiological and pathophysiological situations may lead to the development of novel therapeutics for human obesity. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked and present intriguing relations. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance.

The majority of research in adipocyte biology to date has been done using transformed mouse preadipocyte cell lines. The culture condition which stimulates mouse preadipocyte differentiation is different from that for inducing human primary preadipocyte differentiation. In addition, primary cells are diploid and may therefore reflect the in vivo context better than aneuploid cell lines. Understanding the gene expression profile during adipogenesis in humans will lead to an understanding of the fundamental mechanism of adiposity regulation. Furthermore, through comparing the gene expression profiles of adipogenesis between donor with normal weight and donor with obesity, identification of crucial genes, potential drug targets for obesity and type II diabetes, will be possible.

Immune Response

The immune system is responsible for coping with trauma caused by a variety of agents. Different specialized immune cells detect and repair damage originating from physical trauma versus foreign agents. For instance, lymphocytes, including T- and B-cells, specifically recognize and respond to foreign pathogens. T-cells fight viral infections and activate other immune cell types, while B-cells secrete antibodies that neutralie bacteria and other microbes. Granulocytes and monocytes are primarily migratory, phagocytic cells that exit the bloodstream to fight infection in tissues. Monocytes, derived from immature pro-monocytes, are capable of differentiating into macrophages that engulf and digest microorganisms as well as damaged or dead cells. Monocytes and macrophages also modulate the immune response by secreting signaling molecules such as growth factors and cytokines, and are recruited to sites of infection and inflammation by signaling proteins secreted by other immune cells. The differentiation of the monocyte blood cell lineage, as well as responses to inflammatory stimuli, can be studied in vitro using cultured cell lines. For example, THP-1 is a human promonocyte cell line, derived from a 1-year-old male with acute monocytic leukemia, that can be induced to differentiate into macrophage-like cells by treatment with both phorbol ester such as phorbol myristate acetate (PMA). Further treatment of these cells with oxidized low density lipids (oxLDL) causes them to acquire a particular “foam” cell morphology that is typically associated with atherosclerosis and the presence of vascular lesions.

Monocytes are involved in the initiation and maintenance of inflammatory immune responses. The outer membrane of gram-negative bacteria expresses lipopolysaccharide (LPS) complexes called endotoxins. Toxicity is associated with the lipid component (Lipid A) of LPS, and immunogenicity is associated with the polysaccharide components of LPS. LPS elicits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections. For the most part, endotoxins remain associated with the cell wall until the bacteria disintegrate. LPS released into the bloodstream by lysing gram-negative bacteria is first bound by certain plasma proteins identified as LPS-binding proteins. The LPS-binding protein complex interacts with CD14 receptors on monocytes, macrophages, B cells, and other types of receptors on endothelial cells. Activation of human B cells with LPS results in mitogenesis as well as immunoglobulin synthesis. In monocytes and macrophages three types of events are triggered during their interaction with LPS: 1) production of cytokines, including IL-1, IL-6, IL-8, TNF-α, and platelet-activating factor, which stimulate production of prostaglandins and leukotrienes that mediate inflammation and septic shock; 2) activation of the complement cascade; and 3) activation of the coagulation cascade.

There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and cell proliferative disorders, including cancer.

SUMMARY OF THE INVENTION

Various embodiments of the invention provide purified polypeptides, cell adhesion and extracellular matrix proteins, referred to collectively as ‘CADECM’ and individually 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,’ ‘CADECM-26,’ ‘CADECM-27,’ ‘CADECM-28,’ ‘CADECM-29,’ ‘CADECM-30,’ and ‘CADECM-31,’ and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified cell adhesion and extracellular 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 cell adhesion and extracellular 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:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-31.

Still 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:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-31. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:32-62.

Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.

Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.

Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31.

Still 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:32-62, b) 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:32-62, 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). In other embodiments, 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:32-62, b) 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:32-62, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.

Still 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:32-62, b) 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:32-62, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. Other embodiments 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:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. The method comprises a) exposing a sample comprising the polypeptide to 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.

Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. The method comprises a) exposing a sample comprising the polypeptide to 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 pharmaceutically 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 specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. 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:1-31, b) a polypeptide comprising a naturally 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:1-31, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-31, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-31. 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.

Still 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:32-62, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.

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:32-62, 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:32-62, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:32-62, 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:32-62, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, 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.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.

Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotide embodiments.

Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.

Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, materials, and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with various embodiments of the invention. Nothing 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 substantially purified CADECM obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics the biological activity of CADECM. Agonists may include proteins, nucleic acids, carbohydrates, small 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.

An “allelic variant” is an alternative form of the gene encoding CADECM. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally 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 readily detectable using a particular oligonucleotide probe of the polynucleotide encoding CADECM, and improper or unexpected hybridization to allelic 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 silent change and result in a functionally equivalent CADECM. Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of CADECM is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.

The terms “amino acid” and “amino acid sequence” can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

“Amplification” relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.

The term “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, small 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.

The term “antibody” refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind CADECM polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, 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. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by EXponential Enrichment), described in U.S. Pat. No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2′-OH group of a ribonucleotide may be replaced by 2′-F or 2′-NH₂), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).

The term “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 naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.

The term “antisense” refers to any composition capable of base-pairing with the “sense” (coding) strand of a polynucleotide having a specific nucleic acid sequence. Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell 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.

The term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” or “immunogenic” refers to the capability of the natural, recombinant, or synthetic CADECM, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells 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′.

A “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. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.) 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 Mass.) or Phrap (University of Washington, Seattle Wash.). Some sequences have been both extended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially 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.

Original ResidueConservative SubstitutionAlaGly, SerArgHis, LysAsnAsp, Gln, HisAspAsn, GluCysAla, SerGlnAsn, Glu, HisGluAsp, Gln, HisGlyAlaHisAsn, Arg, Gln, GluIleLeu, ValLeuIle, ValLysArg, Gln, GluMetLeu, IlePheHis, Met, Leu, Trp, TyrSerCys, ThrThrSer, ValTrpPhe, TyrTyrHis, Phe, TrpValIle, Leu, Thr

Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.

“Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing 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, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, 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. Clearly 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:32-62 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:32-62, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:32-62 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:32-62 from related polynucleotides. The precise length of a fragment of SEQ ID NO:32-62 and the region of SEQ ID NO:32-62 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

A fragment of SEQ ID NO:1-31 is encoded by a fragment of SEQ ID NO:32-62. A fragment of SEQ ID NO:1-31 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-31. For example, a fragment of SEQ ID NO:1-31 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-31. The precise length of a fragment of SEQ ID NO:1-31 and the region of SEQ ID NO:1-31 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 “full length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wis.). 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). For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The “weighted” residue weight table is selected as the default.

Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, Md., and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Reward for match: 1

Penalty for mismatch: -2

Open Gap: 5 and Extension Gap: 2 penalties

Gap×drop-off: 50

Expect: 10

Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases “percent similarity” and “% similarity,” as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.

Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix is selected as the default residue weight table.

Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set at default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62

Open Gap: 11 and Extension Gap: 1 penalties

Gap×drop-off 50

Expect: 10

Word Size: 3

Filter: on

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” (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.

The term “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 ability.

“Hybridization” refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the “washing” step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing 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×SSC, about 1% (w/v) SDS, and about 100 μg/ml sheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_mis 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_mand conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D. W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor N.Y., 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×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×SSC, with SDS being present at about 0.1%. Typically, 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 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.

The term “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_ot or R_ot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

The words “insertion” and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment of CADECM which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of CADECM which is useful in any of the antibody production methods disclosed herein or known in the art.

The term “microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.

The terms “element” and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.

The term “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.

The phrases “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, 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.

“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. For instance, 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.

“Peptide nucleic acid” (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.

“Post-translational modification” of an CADECM may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of CADECM.

“Probe” refers to nucleic acids encoding CADECM, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. “Primers” are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).

Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30,40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.

Methods for preparing and using probes and primers are described in, for example, Sambrook, J. and D. W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor N.Y.), Ausubel, F. M. et al. (1999; Short Protocols in Molecular Biology, 4^thed., John Wiley & Sons, New York N.Y.), and Innis, M. et al. (1990; PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego Calif.). 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 Mass.).

Oligonucleotides 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 oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas Tex.) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge Mass.) allows the user to input a “mispriming library,” in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides 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 fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.

A “recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished 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 Russell (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 cell.

Alternatively, 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 usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.

An “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 all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

The term “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 cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small 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 will reduce the amount of labeled A that binds to the antibody.

The term “substantially 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 naturally associated.

A “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, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

A “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.

“Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well 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 cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells 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 deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, 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 Russell (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 7, 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 “allelic” (as defined above), “splice,” “species,” or “polymorphic” variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing 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 will generally 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.

A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May 07, 1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.

The Invention

Various embodiments of the invention include new human cell adhesion and extracellular 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 cell proliferative disorders, including cancer.

Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown. Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.

Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows the 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 applicable, all 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 potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accelrys, Burlington Mass.). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are cell adhesion and extracellular matrix proteins. For example, SEQ ID NO:1 is 91% identical, from residue L15 to residue V989, to human NrCAM protein (GenBank ID g2511666) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.0e-301, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:1 also has homology to neuronal cell adhesion molecule, a member of the immunoglobulin superfamily predicted to have a role in neuronal cell adhesion, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:1 also contains a fibronectin type III domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMARTs databases and an Ig superfamily from SCOP domain as determined by a search of the HMM-based INCY database. (See Table 3.) Data from MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:1 is a cell adhesion molecule. In another example, SEQ ID NO:9 is 96% identical, from residue M1 to residue P673, and 99% identical from residue G643 to residue S752 to human LI-adherin (GenBank ID g854175) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:9 also has homology to proteins that are localized to the plasma membrane, mediate calcium-dependent cell to cell adhesion, are expressed in gastric intestinal metaplasia and andenocarcinomas, and are liver-intestine cadherins, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:9 also contains cadherin domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:9 is a cadherin. In another example, SEQ ID NO:16 is 99% identical, from residue M1 to residue Q947, to human platelet glycoprotein IIb, a member of the integrin family that functions as a receptor for fibrinogen (GenBank ID g386753) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:16 also has homology to integrin alpha 2b, a subunit of the fibrinogen receptor that is involved in hemostasis, blood coagulation, platelet aggregation, cell adhesion and actin reoganization, and is associated with immune thrombocytopenic purpura and Glanzmann thrombasthenia, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:16 also contains an integrin alpha domain as well as FG-GAP repeats as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:16 is an integrin alpha subunit. In another example, SEQ ID NO:24 is 92% identical from residue M1 to residue V4560, to Rattus norvegicus fat3 (GenBank ID g19773543) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:24 also has homology to FAT tumor suppressor homolog, a member of the cadherin superfamily, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:24 also contains epidermal growth factor (EGF)-like domains, cadherin repeats, calcium-binding EGF domains, and a laminin G domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based SMART database of conserved protein families/domains, as well as an epidermal growth factor (EGF)-like domain and a laminin G domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:24 is a cadherin. SEQ ID NO:2-8, SEQ ID NO:10-15, SEQ ID NO:17-23, and SEQ ID NO:25-31 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-31 are described in Table 7.

As shown in Table 4, 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 lists 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 in basepairs. 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:32-62 or that distinguish between SEQ ID NO:32-62 and related polynucleotides.

The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, 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”). Alternatively, 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”). Alternatively, 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_XXXXXX_N_1—N_2—YYYYY_N_3—N₄represents a “stitched” sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N_{1,2,3 . . .}, if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an “exon-stretching” algorithm. For example, a polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_—1_N is a “stretched” sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the “exon-stretching” algorithm was applied, 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). In instances where a RefSeq sequence was used as a protein homolog for the “exon-stretching” algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, 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 lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).

PrefixType of analysis and/or examples of programsGNN, GFG,Exon prediction from genomic sequences using,ENSTfor example, GENSCAN (Stanford University,CA, USA) or FGENES (Computer Genomics Group,The Sanger Centre, Cambridge, UK).GBIHand-edited analysis of genomic sequences.FLStitched or stretched genomic sequences (see Example V).INCYFull length transcript and exon prediction from mappingof EST sequences to the genome. Genomic locationand EST composition data are combined to predictthe exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.

Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.

Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with allele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full-length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in the EST sequence. Columns 8 and 9 show the two alleles 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 allele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.

The invention also encompasses CADECM variants. Various embodiments of CADECM variants can have at least about 80%, at least about 90%, or at least about 95% 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. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:32-62, which encodes CADECM. The polynucleotide sequences of SEQ ID NO:32-62, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

The invention also encompasses variants of a polynucleotide encoding CADECM. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% 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:32-62 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:32-62. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CADECM.

In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding CADECM. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding CADECM, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding CADECM over its entire length; however, portions of the splice variant will 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. For example, a polynucleotide comprising a sequence of SEQ ID NO:33 and a polynucleotide comprising a sequence of SEQ ID NO:34 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:35 and a polynucleotide comprising a sequence of SEQ ID NO:36 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:39 and a polynucleotide comprising a sequence of SEQ ID NO:46 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:51 and a polynucleotide comprising a sequence of SEQ ID NO:57 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:59 and a polynucleotide comprising a sequence of SEQ ID NO:60 are splice variants of each other. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CADECM.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding CADECM, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring CADECM, and all such variations are to be considered as being specifically disclosed.

Although polynucleotides which encode CADECM and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring CADECM under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding CADECM or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally 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. Other reasons for substantially altering the nucleotide sequence encoding CADECM and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

The invention also encompasses production of polynucleotides which encode CADECM and CADECM derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many available expression vectors and cell, systems using reagents well known in the art. Moreover, 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:32-62 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 well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway N. J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad Calif.). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied 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 well known in the art (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., 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. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Applic. 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 (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth Minn.) 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.

When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5′ regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.

In another embodiment of the invention, 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 cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express CADECM.

The polynucleotides of the invention can be engineered using methods generally 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 oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of CADECM, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through “artificial” breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding CADECM may be synthesized, in whole or in part, using one or more chemical methods well 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). Alternatively, CADECM itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York N.Y., pp. 55-60; Roberge, J. Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, 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 naturally occurring polypeptide.

The peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).

In order to express a biologically active CADECM, the polynucleotides encoding CADECM or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ untranslated regions in the vector and in polynucleotides encoding 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. In cases where a polynucleotide sequence encoding CADECM and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, 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 cell system used (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

Methods which are well 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 Russell, 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 cell systems infected with viral expression vectors (e.g., baculovirus); plant cell 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 cell systems (Sambrook and Russell, supra; Ausubel et al., supra; Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355). Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Buller, R. M. et al. (1985) Nature 317:813-815; McGregor, D. P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I. M. and N. Sornia (1997) Nature 389:239-242). The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding CADECM. For example, routine cloning, subcloning, and propagation of polynucleotides encoding CADECM can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding CADECM into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, 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). When large quantities of CADECM are needed, e.g. for the production of antibodies, vectors which direct high level expression of CADECM may be used. For example, 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 pastoris. In addition, such vectors direct either the secretion or intracellular 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, C. A. et al. (1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of CADECM. Transcription of polynucleotides encoding CADECM may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191-196).

In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, polynucleotides encoding CADECM may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus-which expresses CADECM in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliver 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 delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems, stable expression of CADECM in cell lines is preferred. For example, polynucleotides encoding CADECM can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk⁻and apr⁻cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites (Hartnan, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β-glucuronidase and its substrate β-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, C. A. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding CADECM is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding CADECM can be identified by the absence of marker gene function. Alternatively, 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 usually indicates expression of the tandem gene as well.

In general, host cells that contain the polynucleotide encoding CADECM and that express 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 amplification, 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), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on CADECM is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York N.Y.; Pound, J. D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CADECM include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, 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 commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Biosciences, Promega (Madison Wis.), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with polynucleotides encoding CADECM may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode CADECM may be designed to contain signal sequences which direct secretion of CADECM through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, 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 cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure the correct modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding CADECM may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric CADECM protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of CADECM activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin(HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically 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 may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.

In another embodiment, 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, ³⁵S-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, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.

In related embodiments, 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. In an embodiment, 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:1-31. 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.

In an embodiment, a compound identified in a screen for specific binding to CADECM can be closely related to the natural ligand of CADECM, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Coligan, J. E. et al. (1991) Current Protocols in Immunology 1(2):Chapter 5). In another embodiment, 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).

In other embodiments, 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. For example, 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. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336). The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Amgen Inc., Thousand Oaks Calif.), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgG₁(Taylor, P. C. et al. (2001) Curr. Opin. Immunol. 13:611-616).

In one embodiment, two or more antibodies having similar 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. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of CADECM. In another embodiment, 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.

In an embodiment, 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. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. 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.

In one embodiment, 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 cell membrane. Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing CADECM or cell 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. For example, 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. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, 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 solid 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. Pat. No. 5,914,236 and U.S. Pat. No. 6,372,724. In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1:25-30). In another related embodiment, 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).

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. In one embodiment, 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. Alternatively, a test compound is combined with an in vitro or cell-free system comprising CADECM under conditions suitable for CADECM activity, and the assay is performed. In either of these assays, 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 plurality of test compounds may be screened.

In another embodiment, polynucleotides encoding CADECM or their mammalian homologs may be “knocked out” in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells 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. Alternatively, 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 cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically 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 cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, 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) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding CADECM is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells 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. Alternatively, 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. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CADECM and cell adhesion and extracellular matrix proteins. In addition, examples of tissues expressing CADECM can be found in Table 6 and can also be found in Example XI. Therefore, CADECM appears to play a role in immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, and cell proliferative 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.

Therefore, in one embodiment, 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. Examples of such disorders include, but are not limited 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 Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a developmental disorder, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a connective tissue disorder, such as osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial aortic aneurysm, achondroplasia, mucopolysaccharidoses, osteoporosis, osteopetrosis, Paget's disease, rickets, osteomalacia, hyperparathyroidism, renal osteodystrophy, osteonecrosis, osteomyelitis, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defect, nonossifying fibroma, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, primitive neuroectodermal tumor, giant cell tumor, osteoarthritis, rheumatoid arthritis, ankylosing spondyloarthritis, Reiter's syndrome, psoriatic arthritis, enteropathic arthritis, infectious arthritis, gout, gouty arthritis, calcium pyrophosphate crystal deposition disease, ganglion, synovial cyst, villonodular synovitis, systemic sclerosis, Dupuytren's contracture, hepatic fibrosis, lupus erythematosus, mixed connective tissue disease, epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus; and a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

In another embodiment, a vector capable of expressing 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 including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantially purified CADECM in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADECM including, but not limited to, those provided above.

In still another embodiment, 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 listed above.

In a further embodiment, 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 cell proliferative disorders, including cancer described above. In one aspect, an antibody which specifically binds CADECM may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express CADECM.

In an additional embodiment, 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.

In other embodiments, 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 skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically 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 generally known in the art. In particular, purified CADECM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind CADECM. Antibodies to CADECM may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeutically. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).

For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with CADECM or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such 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, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to CADECM have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantially 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 cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell 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. Cell Biol. 62:109-120).

In addition, techniques developed for the production of “chimeric antibodies,” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, 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 immunoglobulin libraries (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 libraries or panels of highly specific binding reagents as disclosed in the literature (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. For example, such fragments include, but are not limited to, F(ab′)₂fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow 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 established specificities are well known in the art. Such immunoassays typically 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 generally used, but a competitive binding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for CADECM. Affinity is expressed as an association constant, K_a, which is defined as the molar concentration of CADECM-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K_adetermined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple CADECM epitopes, represents the average affinity, or avidity, of the antibodies for CADECM. The K_adetermined 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_aranging from about 10⁹to 10¹²L/mole are preferred for use in immunoassays in which the CADECM-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_aranging from about 10⁶to 10⁷L/mole are preferred for use in immunopurification and similar 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 D.C.; Liddell, J. E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of CADECM-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding CADECM, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding CADECM. Such technology is well known in the art, and antisense oligonucleotides 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 N.J.).

In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. 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 Clin. Immunol. 102:469-475; Scanlon, K. J. et al. (1995) FASEB J. 9:1288-1296). Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A. D. (1990) Blood 76:271-278; Ausubel et al., supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Boado, R. J. et al. (1998) J. Pharm. Sci. 87:1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res. 25:2730-2736).

In another embodiment of the invention, polynucleotides encoding CADECM may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 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) Cell 75:207-216; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410; Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular 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. USA 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in CADECM expression or regulation causes disease, the expression of CADECM from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders caused by deficiencies in CADECM are treated by constructing mammalian expression vectors encoding CADECM and introducing these vectors by mechanical means into CADECM-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson (1993) Annu. Rev. Biochen. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Récipon (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 Calif.), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). 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 β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V. and H. M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding CADECM from a normal individual.

Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, 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 cells requires modification of these standardized mammalian transfection protocols.

In another embodiment of the invention, 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, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol. 61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 to Rigg (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”) discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4⁺ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding CADECM to cells which have one or more genetic abnormalities with respect to the expression of CADECM. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication 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). Potentially useful adenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano (“Adenovirus vectors for gene therapy”), hereby incorporated by reference. For 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).

In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding CADECM to target cells which have one or more genetic abnormalities with respect to the expression of CADECM. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing CADECM to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver 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 detail in U.S. Pat. No. 5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”), which is hereby incorporated by reference. U.S. Pat. No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W. F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.

In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding CADECM to target cells. The biology of the prototypic alphavirus, Seinliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates 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). Similarly, inserting the coding sequence for CADECM into the alphavirus genome in place of the capsid-coding region results in the production of a large number of CADECM-coding RNAs and the synthesis of high levels of CADECM in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S. A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of CADECM into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.

Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., 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, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding CADECM.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, 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 cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art. RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T. (2000; Nature 404:804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.

RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA. siRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease. siRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3′ overhangs. The use of siRNA for inducing RNAi in mammalian cells 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 cell. Alternatively, siRNA can be synthesized directly and introduced into a cell by transfection methods and agents described herein or known in the art (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/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-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration. The selected siRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commercially available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin Tex.).

In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomplished 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). In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing.

In various embodiments, 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, oligonucleotides, 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. Thus, in the treatment of disorders associated with increased CADECM expression or activity, 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 specifically promotes expression of the polynucleotide encoding CADECM may be therapeutically useful.

In various embodiments, one or more 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 naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a 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 permeabilized 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. Typically, 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 may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. 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. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. et al. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C. K. et al. (1997) Nat. Biotechnol. 15:462-466).

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa.). Such compositions may consist of CADECM, antibodies to CADECM, and mimetics, agonists, antagonists, or inhibitors of CADECM.

In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No. 5,997,848). Pulmonary delivery allows administration without needle injection, and obviates the need for potentially toxic penetration enhancers.

Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising CADECM or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, 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 cells of all tissues, including the brain, in a mouse model system (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, 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 therapeutically 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 ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₅₀(the dose therapeutically effective in 50% of the population) or LD₅₀(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 LD₅₀/ED₅₀ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell 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 ED₅₀with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug 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-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically 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 cells 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.

A variety of protocols for measuring CADECM, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of CADECM expression. Normal or standard values for CADECM expression are established by combining body fluids or cell extracts taken from normal mammalian 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 from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding CADECM may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, 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.

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding CADECM or closely related molecules may be used to identify nucleic acid sequences which encode CADECM. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding CADECM, allelic 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:32-62 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 commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

Polynucleotides encoding CADECM may be used for the diagnosis of disorders associated with expression of CADECM. Examples of such disorders include, but are not limited 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 Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder, such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a developmental disorder, such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a connective tissue disorder, such as osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial aortic aneurysm, achondroplasia, mucopolysaccharidoses, osteoporosis, osteopetrosis, Paget's disease, rickets, osteomalacia, hyperparathyroidism, renal osteodystrophy, osteonecrosis, osteomyelitis, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondroma, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defect, nonossifying fibroma, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma, Ewing's sarcoma, primitive neuroectodermal tumor, giant cell tumor, osteoarthritis, rheumatoid arthritis, ankylosing spondyloarthritis, Reiter's syndrome, psoriatic arthritis, enteropathic arthritis, infectious arthritis, gout, gouty arthritis, calcium pyrophosphate crystal deposition disease, ganglion, synovial cyst, villonodular synovitis, systemic sclerosis, Dupuytren's contracture, hepatic fibrosis, lupus erythematosus, mixed connective tissue disease, epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus; and a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Polynucleotides encoding CADECM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered CADECM expression. Such qualitative or quantitative methods are well known in the art.

In a particular embodiment, 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.

In order to provide a basis for the diagnosis of a disorder associated with expression of CADECM, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CADECM, under conditions suitable for hybridization or amplification. 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 substantially 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 establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocol is initiated, 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.

With respect to cancer, 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 allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.

Additional diagnostic uses for oligonucleotides designed from the sequences encoding CADECM may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding CADECM, or a fragment of a polynucleotide complementary to the polynucleotide encoding CADECM, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide 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. In SSCP, oligonucleotide primers derived from polynucleotides encoding CADECM are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily 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. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego Calif.).

SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell 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 life-threatening toxicity. For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well 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. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11:637-641).

Methods which may also be used to quantify the expression of CADECM include radiolabeling or biotinylating nucleotides, coamplification 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 may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides 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. In particular, 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. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.

In another embodiment, CADECM, fragments of CADECM, or antibodies specific for CADECM may be used as elements on a microarray. 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 cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell 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. Pat. No. 5,840,484; hereby expressly incorporated by reference herein). Thus 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 cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.

Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell 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 naturally-occurring environmental compounds. All 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 similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released Feb. 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.

In an embodiment, 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 corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.

Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, 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 visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of 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. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.

In another embodiment, 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.

In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/25116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662). Various types of microarrays are well known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Aproach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encoding CADECM may be used to generate hybridization probes useful in mapping the naturally 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. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; Trask, B. J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate 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).

Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding 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.

In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R. A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, CADECM, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries 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 solid support, borne on a cell surface, or located intracellularly. 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 application WO84/03564). In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with CADECM, or fragments thereof, and washed. Bound CADECM is then detected by methods well known in the art. Purified CADECM can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding CADECM specifically compete with a test compound for binding CADECM. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with CADECM.

In additional embodiments, the 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.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The disclosures of all patents, applications, and publications mentioned above and below, including U.S. Ser. No. 60/379,840, U.S. Ser. No. 60/381,291, U.S. Ser. No. 60/383,183, and U.S. Ser. No. 60/394,146, are hereby expressly incorporated by reference.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs are derived from cDNA libraries described in the LIFESEQ database (Incyte, Palo Alto Calif.). Some tissues are homogenized and lysed in guanidinium isothiocyanate, while others are homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates are centrifuged over CsCl cushions or extracted with chloroform RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA are repeated as necessary to increase RNA purity. In some cases, RNA is treated with DNase. For most libraries, poly(A)+ RNA is isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA is isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).

In some cases, Stratagene is provided with RNA and constructs the corresponding cDNA libraries. Otherwise, cDNA is synthesized and cDNA libraries are constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription is initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters are ligated to double stranded cDNA, and the cDNA is digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA is size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs are ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad Calif.), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo Alto Calif.), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof. Recombinant plasmids are transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I are recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids are purified using at least one of the following: 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. Following precipitation, plasmids are resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.

Alternatively, plasmid DNA is amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V. B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps are carried out in a single reaction mixture. Samples are processed and stored in 384-well plates, and the concentration of amplified plasmid DNA is quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene Oreg.) 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 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions are prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied 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 (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences are identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences are selected for extension using the techniques disclosed in Example VIII.

Polynucleotide sequences derived from Incyte cDNAs are validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof are then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS; PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte, Palo Alto Calif.); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences are assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) are used to extend Incyte cDNA assemblages to full length. Assembly is performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages are screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences are translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide. Full 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. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda Calif.) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

Table 7 summarizes tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).

The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences are also used to identify polynucleotide sequence fragments from SEQ ID NO:32-62. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.

IV. Identification and Editing of Coding Sequences from Genomic DNA

Putative cell adhesion and extracellular matrix proteins are initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (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. The output of 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. To determine which of these Genscan predicted cDNA sequences encode cell adhesion and extracellular matrix proteins, the encoded polypeptides are analyzed by querying against PFAM models for cell adhesion and extracellular matrix proteins. Potential cell adhesion and extracellular matrix proteins are also identified by homology to Incyte cDNA sequences that have been annotated as cell adhesion and extracellular matrix proteins. These selected Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri public 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 public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage is available, this information is used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences are obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences.

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 splice variants that are subsequently confirmed, edited, or extended to create a full 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., For example, if an interval is present on a cDNA and two genomic sequences, then all three intervals are considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified are then “stitched” together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) are given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences are translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan are corrected by comparison to the top BLAST hit from genpept. Sequences are further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.

“Stretched” Sequences

Partial DNA sequences are extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example III are queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog is then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein is generated by using the resultant high-scoring segment 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. The GenBank protein homolog, the chimeric protein, or both are used as probes to search for homologous genomic sequences from the public 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.

VI. Chromosomal Mapping of CADECM Encoding Polynucleotides

The sequences used to assemble SEQ ID NO:32-62 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID NO:32-62 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Généthon 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 all sequences of that cluster, including its particular SEQ ID NO:, to that map location.

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. (The centiMorgan (cM) 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 Généthon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI “(GeneMap'99” World Wide Web site (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.

VII. Analysis of Polynucleotide Expression

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 cell type or tissue have been bound (Sambrook and Russell, 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 LIEESEQ (Incyte). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
$\frac{BLAST Score \times Percent Identity}{5 \times minimum {length (Seq . 1), length (Seq . 2)}}$

The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied 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 quality in a BLAST aligmnent. 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.

Alternatively, 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 III). Each cDNA sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system, embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding CADECM. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ database (Incyte, Palo Alto Calif.).

VIII. Extension of CADECM Encoding Polynucleotides

Full length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide 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 libraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed.

High fidelity amplification is obtained by PCR using methods well known in the art. PCR is performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, the parameters for primer pair T7 and SK+ are as follows: 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 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C.

The concentration of DNA in each well is determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1X TE and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent. The plate is scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot 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-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis., and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, 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 religated using T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37° C. in 384-well plates in LB/2× carb liquid media.

The cells are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following 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 reamplified 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 (Applied Biosystems).

In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5′ regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in CADECM Encoding Polynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) are identified in SEQ ID NO:32-62 using the 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 errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants. An automated procedure of advanced chromosome analysis is applied to the original chromatogram files in the vicinity of the putative SNP. Clone error filters use statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters use statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removes duplicates and SNPs found in immunoglobulins or T-cell receptors.

Certain SNPs are selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprises 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprises 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprises 324 individuals (162 male, 162 female), all Mexican Hispanic. The Asian population comprises 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies are first analyzed in the Caucasian population; in some cases those SNPs which show no allelic variance in this population are not further tested in the other three populations.

X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:32-62 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston Mass.). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 10⁷counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.). Hybridization is carried out for 16 hours at 40° C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1× saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.

XI. Microarrays

The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill 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; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well 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; After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element. Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)⁺RNA is purified using the oligo-(dT) cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1× first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM 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 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto Calif.) 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 N.Y.) and resuspended in 14 μl 5× SSC/0.2% SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Amplified array elements are then purified using SEPHACRYL400 (Amersham Biosciences).

Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (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 slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522, incorporated herein by reference. 1 μl of the array element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.

Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5× SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65° C. for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm²coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5× SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C. in a first wash buffer (1× SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in a second wash buffer (0.1× 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 Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20× microscope objective (Nikon, Inc., Melville N.Y.). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm×1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) 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 array 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 typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, 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 Mass.) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). 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 differentially expressed.

Expression

For example, SEQ ID NO:33 and SEQ ID NO:34 showed increased expression in ovarian cancer, as determined by microarray analysis. A normal ovary from a 79 year-old female donor was compared to an ovarian tumor from the same donor (Huntsman Cancer Institute, Salt Lake City, Utah).

Expression of SEQ ID NO:33 and SEQ ID NO:34 increased by at least twofold in ovarian tumor tissue versus normal ovary tissue. Therefore, SEQ ID NO:33 and SEQ ID NO:34 are useful in monitoring treatment of, and diagnostic assays for, ovarian cancer and other cell proliferative disorders.

As another example, SEQ ID NO:33 and SEQ ID NO:34 showed decreased expression in preadipocytes treated with differentiation medium. Primary subcutaneous preadipocytes were isolated from adipose tissue of a 40-year-old healthy female with a body mass index (BMI) of 32.47. The preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in the differentiation medium containing active components PPAR-γ agonist and human insulin (Zen-Bio). Thiazolidinediones or PPAR-γ agonists can bind and activate an orphan nuclear receptor, PPAR-γ, and some of them have been proven to be able to induce human adipocyte differentiation. The preadipocytes were treated with human insulin and PPAR-γ agonist for 3 days and subsequently were switched to medium containing insulin alone for a variety of time periods ranging from one to 20 days before the cells were collected for analysis. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. Between 80% and 90% of the preadipocytes finally differentiated to adipocytes observed under phase contrast microscope. Expression of SEQ ID NO:33 and SEQ ID NO:34 decreased by at least twofold in differentiated adipocytes versus untreated preadipocytes. Therefore, SEQ ID NO:33 and SEQ ID NO:34 are useful for the diagnosis, prognosis, or treatment of diabetes mellitus and other disorders, such as obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers including breast, prostate, and colon.

As another example, SEQ ID NO:36 showed decreased expression in fibroblasts associated with Tangier disease, as determined by microarray analysis. Normal and Tangier disease derived fibroblasts were compared. Human fibroblasts were obtained from skin explants from both normal subjects and two patients with homozygous Tangier disease. Cell lines were immortalized by transfection with human papillomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker. In addition, both types of cells were cultured in the presence of cholesterol and compared with the same cell type cultured in the absence of cholesterol. TD derived cells are shown to be deficient in an assay of apoA-I mediated tritiated cholesterol efflux. Expression of SEQ ID NO:36 decreased by at least twofold in fibroblasts associated with Tangier disease versus normal fibroblasts. Therefore, SEQ ID NO:36 is useful in diagnostic assays for Tangier disease.

SEQ ID NO:40 showed differential expression in association with colon cancer from three separate donors, as determined by microarray analysis. Tissue samples were provided by the Huntsman Cancer Institute, Salt Lake City, Utah. The gene expression profile of messenger RNA isolated from grossly uninvolved colon tissue was compared to that isolated from cancerous colon tissue from three separate donors in individual matched tissue experiments. The expression of SEQ ID NO:40 was decreased by three-fold in the tumorous colon tissue as compared to the normal colon tissue from one donor, while the expression of SEQ ID NO:40 was decreased by two-fold in the tumorous colon tissue as compared to the normal colon tissue in two other donors. Thus, in an embodiment, SEQ ID NO:40 can be used in diagnostic assays for colon cancer, as well as for monitoring the progression and treatment of colon cancer, and related diseases and conditions.

SEQ ID NO:42 showed differential expression in association with breast cancer, as determined by microarray analysis. Gene expression profiles of nonmalignant mammary epithelial cells were compared to gene expression profiles of various breast carcinoma lines at different stages of tumor progression. The cells were grown in defined serum-free H14 medium to 70-80% confluence prior to RNA harvest. Cell lines compared included: a) MCF-10A, a breast mammary gland (luminal ductal characteristics) cell line isolated from a 36-year-old woman with fibrocystic breast disease, b) MCF7, a nonmalignant breast adenocarcinoma cell line isolated from the pleural effusion of a 69-year-old female, c) T-47D, a breast carcinoma cell line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast, d) Sk-BR-3, a breast adenocarcinoma cell line isolated from a malignant pleural effusion of a 43-year-old female, e) BT-20, a breast carcinoma cell line derived in vitro from the cells emigrating out of thin slices of the tumor mass isolated from a 74-year-old female, and f) MDA-mb-231, a breast tumor cell line isolated from the pleural effusion of a 51-year old female. The expression of SEQ ID NO:42 was decreased by at least two-fold in all five (MCF7, T-47D, Sk-BR-3, BT-20, MDA-mb-231) of the breast carcinoma cells lines assayed as compared to the nonmalignant breast epithelial cell line (MCF-10A). Therefore, in an embodiment, SEQ ID NO:42 can be used in diagnostic assays for breast cancer, as well as for monitoring the progression and treatment of breast cancer, and related diseases and conditions.

In addition, SEQ ID NO:39 and SEQ ID NO:42 demonstrated tissue-specific expression. 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%), small 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 individually hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another. The expression of SEQ ID NO:39 was increased by at least two-fold in small intestine and blood leukocytes as compared to the reference sample. Therefore, SEQ ID NO:39 can be used as a marker for small intestine and blood leukocytes. In addition, the expression of SEQ ID NO:42 was increased by at least two-fold in aortic tissue as compared to the reference sample. Therefore, SEQ ID NO:42 can be used as a marker for aortic tissue.

In another example, SEQ ID NO:44 showed differential expression in breast cancer cell lines as compared to non-cancerous breast epithelial cell lines as determined by microarray analysis. Gene expression profiles of nonmalignant mammary epithelial cells were compared to gene expression profiles of various breast carcinoma lines at different stages of tumor progression. Cell lines compared included: a) HMEC, a primary breast epithelial cell line isolated from a normal donor, b) MCF-10A, a breast mammary gland cell line isolated from a 36-year-old woman with fibrocystic breast disease, c) MCF7, a nonmalignant breast adenocarcinoma cell line isolated from the pleural effusion of a 69-year-old female, d) T-47D, a breast carcinoma cell line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast, e) Sk-BR-3, a breast adenocarcinoma cell line isolated from a malignant pleural effusion of a 43-year-old female, f) BT-20, a breast carcinoma cell line derived in vitro from cells emigrating out of thin slices of the tumor mass isolated from a 74-year-old female, g) MDA-mb-231, a breast tumor cell line isolated from the pleural effusion of a 51-year-old female, and h) MDA-mb-435S, a spindle-shaped strain that evolved from the parent line (435) isolated by R. Cailieau from pleural effusion of a 31-year-old female with metastatic, ductal adenocarcinoma of the breast. Expression of SEQ ID NO:44 was decreased by at least two-fold in the MCF7 breast cancer cell lines as compared to the non-malignant HMEC and MCF-10A cells. Therefore, in an embodiment, SEQ ID NO:44 can be used in diagnostic assays for, and/or monitoring treatment of, early stages of breast cancer and related diseases and conditions.

In a further example, SEQ ID NO:44 and SEQ ID NO:48 showed differential expression in colon tumor tissues compared to normal colon tissue from the same donor as determined by microarray analysis. Samples of normal colon were compared to colon tumor from the same donor (Huntsman Cancer Institute, Salt Lake City, Utah). The expression of SEQ ID NO:44, was decreased at least two-fold in tumor tissue as compared to matched normal colon tissue for one donor and increased by at least twofold in another, while the expression of SEQ ID NO:48 was increased at least two-fold in tumor tissue as compared to matched normal colon tissue for one donor. Therefore, in an embodiment, SEQ ID NO:44 and SEQ ID NO:48 can be used in diagnostic assays for, and/or monitoring treatment of, colon cancer and related diseases and conditions.

In a further example, SEQ ID NO:44 and SEQ ID NO:47 showed differential expression in lung tumor tissues compared to normal lung tissue from the same donor as determined by microarray analysis. Samples of normal lung were compared to lung tumor from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). The expression of SEQ ID NO:44 was increased by at least two-fold in tumor tissue as compared to the matched normal lung for one donor, while the expression of SEQ ID NO:47 was decreased by at least two-fold in tumor tissue as compared to the matched normal lung for two different donors. Therefore, in an embodiment, SEQ ID NO:44 and SEQ ID NO:47 can be used in diagnostic assays for, and/or monitoring treatment of, lung cancer and related diseases and conditions.

In a further example, SEQ ID NO:46 showed differential expression associated with the immune response. PBMCs from the blood of 6 healthy volunteer donors were incubated for 24 hours in the presence of graded doses of beclomethasone dissolved in DMSO. In addition, matching PBMCs were treated for the same duration with matching doses of DMSO in order to monitor the possible effects of the vehicle alone. The treated PBMC were compared to matching untreated PBMCs maintained in culture for the same duration. The expression of SEQ ID NO:46 was decreased by at least two-fold in the PBMCs treated with beclomethasone, but not in PBMCs treated with DMSO alone, as compared to untreated PBMCs. Therefore, in an embodiment, SEQ ID NO:46 can be used in diagnostic assays for, and/or monitoring treatment of autoimmune and inflammatory disorders.

In a further example, SEQ ID NO:46 and SEQ ID NO:47 showed tissue-specific expression. RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues in the reference sample were selected for their ability to provide a complete representation of all RNA expressed in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), small 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 individually hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another. The expression of both SEQ ID NO:46 and SEQ ID NO:47 was increased by at least two-fold in blood as compared to the reference sample. Therefore, SEQ ID NO:46 and SEQ ID NO:47 can be used as blood markers.

In another example, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:61 showed differential expression associated with prostate cancer, as determined by microarray analysis. Primary prostate epithelial cells were compared with prostate carcinomas representative of the different stages of tumor progression. In two experiments, cell lines compared included: a) PrEC, a primary prostate epithelial cell line isolated from a normal donor, b) DU 145, a prostate carcinoma cell line isolated from a metastatic site in the brain of 69-year old male with widespread metastatic prostate carcinoma, c) LNCaP, a prostate carcinoma cell line isolated from a lymph node biopsy of a 50-year-old male with metastatic prostate carcinoma, and d) PC-3, a prostate adenocarcinoma cell line isolated from a metastatic site in the bone of a 62-year-old male with grade IV prostate adenocarcinoma. In the first experiment, cells were grown in basal medium in the absence of growth factors and hormones. In the second experiment, cells were cultured, under optimal growth conditions, in medium supplemented with growth factors and nutrients. The expression of SEQ ID NO:61 was downregulated by at least two-fold in all three carcinoma cell lines, as compared to the primary prostate epithelial cell line, whether cells were grown in basal or supplemented medium. The expression of SEQ ID NO:56 was downregulated by at least two-fold in DU145 cells and LNCaP cells, whether cells were grown in basal or supplemented medium. The expression of SEQ ID NO:52 was decreased by at least two-fold in DU145 cells and LNCaP cells, as compared to PrEC cells, when grown in basal medium. The expression of SEQ ID NO:51 and SEQ ID NO:57 was downregulated by at least two-fold in DU145 cells grown in supplemented medium. Therefore, in various embodiments, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:57 and SEQ ID NO:61 can each be used for one or more of the following: i) monitoring treatment of prostate cancer, ii) diagnostic assays for prostate cancer, and iii) developing therapeutics and/or other treatments for prostate cancer.

SEQ ID NO:61 showed differential expression associated with lung cancer, as determined by microarray analysis. Grossly uninvolved tissue from a donor was compared to lung tumor tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). The expression of SEQ ID NO:61 was upregulated by at least 10-fold in the lung tumor tissue as compared to uninvolved lung tissue. Therefore, in various embodiments, SEQ ID NO:61 can be used for one or more of the following: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii) developing therapeutics and/or other treatments for lung cancer.

SEQ ID NO:61 showed differential expression associated with obesity, as determined by microarray analysis. In two parallel experiments, pre-adipocytes from a healthy 28-year-old female donor, with a body mass index (BMI) of 23.59 and pre-adipocytes from an obese 40-year-old female donor with a BMI of 32.47 were induced to differentiate into adipocytes by treatment with human insulin and peroxisome proliferation-activated receptor gamma agonists for three days followed by growth in medium containing only insulin for 24 hours, 48 hours, 4 days, 1.1 weeks or 2.1 weeks. The expression of SEQ ID NO:61 was decreased by at least two-fold in differentiating pre-adipocytes from the donor with a BMI of 32.47 at the 1.1 and 2.1 weeks timepoints, as compared to the pre-adipocytes from the donor with a BMI of 23.59. Therefore, in various embodiments, SEQ ID NO:61 can be used for one or more of the following: i) monitoring treatment of, and ii) developing therapeutics and/or other treatments for obesity.

SEQ ID NO:56 showed differential expression associated with atherosclerosis and inflammatory immune responses, as determined by microarray analysis. Promonocyte THP-1 cells were incubated first with phorbol myristate acetate (PMA), inducing differentiation into macrophage-like cells, and then with oxidized low density lipid (oxLDL) leading to the development of a “foam” cell morphology, typically observed in macrophages that localize to vascular lesions. Macrophage and foam cells thus derived were activated by treatment with lipopolysaccharide (LPS) and examined for changes in gene expression. The expression of SEQ ID NO:56 was upregulated by at least two-fold in both activated macrophages and activated foam cells as compared to cells that were not treated with LPS, and therefore not activated. Therefore, in various embodiments, SEQ ID NO:56 can be used for one or more of the following: i) monitoring treatment of, ii) diagnostic assays for, and iii) developing therapeutics and/or other treatments for atherosclerosis.

SEQ ID NO:53 and SEQ ID NO:61 showed tissue-specific expression, as determined by microarray analysis. RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), small 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 individually hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another. The expression of SEQ ID NO:53 was increased by at least two-fold in brain tissue, specifically occipital and temporal cortex, as compared to the reference sample. The expression of SEQ ID NO:61 was increased by at least two-fold in esophageal tissue as compared to the reference sample. Therefore, in an embodiment, SEQ ID NO:53 can be used as a tissue marker for cortical tissue of the brain, and, in another embodiment, SEQ ID NO:61 can be used as a tissue marker for the esophagus.

XII. Complementary Polynucleotides

Sequences complementary to the CADECM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring CADECM. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CADECM. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the CADECM-encoding transcript.

XIII. Expression of CADECM

Expression and purification of CADECM is achieved using bacterial or virus-based expression systems. For expression of CADECM in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory 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). Expression of CADECM in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. 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 Spodoptera frugiperda (Sf9) insect cells in most 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, V. et al. (1996) Hum. Gene Ther. 7:1937-1945).

In most expression systems, 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 cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). Following purification, the GST moiety can be proteolytically cleaved from CADECM at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 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 directly in the assays shown in Examples XVII and XVIII, where applicable.

XIV. Functional Assays

CADECM function is assessed by expressing the sequences encoding CADECM at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad Calif.) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994; Flow Cytometry, Oxford, New York N.Y.).

The influence of CADECM on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CADECM and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding CADECM and other genes of interest can be analyzed by northern analysis or microarray techniques.

XV. Production of CADECM Specific Antibodies

CADECM substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.

Alternatively, the CADECM amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel et al., supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-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.

XVI. Purification of Naturally Occurring CADECM Using Specific Antibodies

Naturally occurring or recombinant CADECM is substantially purified by immunoaffinity chromatography using antibodies specific for CADECM. An immunoaffinity column is constructed by covalently coupling anti-CADECM antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing CADECM are passed over the immunoaffinity column, and the column is washed under conditions that allow 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 chaotrope, such as urea or thiocyanate ion), and CADECM is collected.

XVII. Identification of Molecules Which Interact with CADECM

CADECM, or biologically active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled CADECM, washed, and any wells 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.

Alternatively, 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 available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).

CADECM may also be used in the PATHCALLING process (CuraGen Corp., New Haven Conn.) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Pat. No. 6,057,101).

XVIII. Demonstration of CADECM Activity

An assay for CADECM activity measures the expression of CADECM on the cell surface. cDNA encoding CADECM is transfected into a non4eukocytic cell line. Cell surface proteins are labeled with biotin (de la Fuente, M. A. et al. (1997) Blood 90:2398-2405). Immunoprecipitations are performed using 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 cell surface.

Alternatively, an assay for CADECM activity measures the amount of cell aggregation induced by overexpression of CADECM. In this assay, cultured cells such as NIH3T3 are transfected with cDNA encoding CADECM contained within a suitable mammalian expression vector under control of a strong promoter. Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH), is useful for identifying stable transfectants. The amount of cell agglutination, or clumping, associated with transfected cells is compared with that associated with untransfected cells. The amount of cell agglutination is a direct measure of CADECM activity.

Alternatively, an assay for CADECM activity measures the disruption of cytoskeletal filament networks upon overexpression of CADECM in cultured cell lines (Rezniczek, G. A. et al. (1998) J. Cell Biol. 141:209-225). cDNA encoding CADECM is subcloned into a mammalian expression vector that drives high levels of cDNA expression. This construct is transfected into cultured cells, such as rat kangaroo PtK2 or rat bladder carcinoma 804G cells. Actin filaments and intermediate filaments such as keratin and vimentin are visualized by immunofluorescence microscopy using antibodies and techniques well known in the art. The configuration and abundance of cyoskeletal filaments can be assessed and quantified using confocal imaging techniques. In particular, the bundling and collapse of cytoskeletal filament networks is indicative of CADECM activity.

Alternatively, cell adhesion activity in CADECM is measured in a 96-well plate in which wells are first coated with CADECM by adding solutions of CADECM of varying concentrations to the wells. Excess CADECM is washed off with saline, and the wells incubated with a solution of 1% bovine serum albumin to block non-specific cell binding. Aliquots of a cell suspension of a suitable cell type are then added to the wells and incubated for a period of time at 37 ° C. Non-adherent cells are washed off with saline and the cells stained with a suitable cell stain such as Coomassie blue. The intensity of staining is measured using a variable wavelength multi-well plate reader and compared to a standard curve to determine the number of cells adhering to the CADECM coated plates. The degree of cell staining is proportional to the cell adhesion activity of CADECM in the sample.

Various modifications and variations of the described compositions, methods, and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

TABLE 1IncytePolypeptideIncytePolynucleotidePolynucleotideIncyte Project IDSEQ ID NO:Polypeptide IDSEQ ID NO:IDIncyte Full Length Clones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

TABLE 2

Poly-

GenBank

peptide
Incyte
ID NO: or

SEQ
Polypeptide
PROTEOME
Probability

ID NO:
ID
ID NO:
Score
Annotation

1
7506690CD1
g2511666
2.0e−301
[Homo sapiens] NrCAM protein

Wang, B.et al.

Alternative splicing of human NrCAM in neural and nonneural tissues

Mol. Cell. Neurosci. 10 (5-6), 287-295 (1998)

341732|
2.3E−296
[Homo sapiens][Adhesin/agglutinin][Plasma membrane] Neuronal cell adhesion molecule, a

NRCAM

member of the immunoglobulin superfamily, predicted to have a role in neuronal cell

adhesion

Sehgal, A. et al.

Cell adhesion molecule Nr-CAM is over-expressed in human brain tumors.

Int J Cancer 76, 451-8 (1998).

330958|
1.1E−287
[Rattus norvegicus][Adhesin/agglutinin][Plasma membrane] Neuronal cell adhesion

Nrcam

molecule, a member of the immunoglobulin superfamily, predicted to be cell adhesion

molecule involved in the guidance of axonal growth cones during neuronal development

Lambert, S. et al.

Morphogenesis of the node of Ranvier: co-clusters of ankyrin and ankyrin-binding integral

proteins define early developmental intermediates.

J Neurosci 17, 7025-36. (1997).

2
7506536CD1
g16191616
3.0E−218
[Homo sapiens] putative emu1 protein

578546|
5.7E−48
[Mus musculus][Structural protein][Extracellular matrix (cuticle and basement membrane);

Collal

Extracellular (excluding cell wall)] Alpha 1 subunit of type I collagen, a structural protein;

mutations in human COL1A1 cause Ehlers-Danlos syndrome type VII, osteoporosis, and

osteogenesis imperfecta

Francki, A. et al.

SPARC regulates the expression of collagen type I and transforming growth factor-beta1 in

mesangial cells.

J Biol Chem 274, 32145-52 (1999).

762505|
7.5E−47
[Homo sapiens][Structural protein][Extracellular matrix (cuticle and basement membrane);

COL9A3

Extracellular (excluding cell wall)] Collagen (type IX, alpha 3), a structural component of

cartilage extracellular matrix which may be involved in cartilage and muscle development;

mutation of the corresponding gene causes epiphyseal dysplasia

Paassilta, P. et al.

COL9A3: A third locus for multiple epiphyseal dysplasia.

Am J Hum Genet 64, 1036-44. (1999).

3
7506537CD1
g16191616
1.5E−58
[Homo sapiens] putative emu1 protein

428756|
9.7E−20
[Homo sapiens][Structural protein][Extracellular matrix (cuticle and basement membrane);

EMILIN

Extracellular (excluding cell wall)] Elastin microfibril interface located protein, an

extracellular matrix protein found between amorphous elastin and microfibrils that may play

a role in elastin deposition

Doliana, R. et al.

EMILIN, a component of the elastic fiber and a new member of the C1q/tumor necrosis

factor superfamily of proteins.

J Biol Chem 274, 16773-81 (1999).

716229|
1.6E−16
[Homo sapiens] Elastin microfibril interface located 2, a secreted glycoprotein with a

EMILIN-2

globular C1q domain, a short collagenous stalk, a potential coiled-coil region, a proline-rich

region and a cysteine-rich domain (EMI domain)

4
7506655CD1
g31419
6.8E−180
[Homo sapiens] fibulin-1 C

Korenberg, J. R. et al.

Localization of the human gene for fibulin-1 (FBLN1) to chromosome band 22q13.3

Cytogenet. Cell Genet. 68, 192-193 (1995)

568154|
5.8E−181
[Homo sapiens][Structural protein][Extracellular matrix (cuticle and basement membrane);

FBLN1

Extracellular (excluding cell wall)] Fibulin 1, an extracellular matrix and plasma

glycoprotein that may connect elements of the extracellular matrix, may play a role in

hemostasis, altered expression may play a role in tumor invasion, thrombosis, and

connective tissue and blood diseases

Hayashido, Y. et al.

Estradiol and fibulin-1 inhibit motility of human ovarian- and breast-cancer cells induced by

fibronectin.

Int J Cancer 75, 654-8. (1998).

584763|Fbln1
2.8E−142
[Mus musculus][Structural protein][Extracellular matrix (cuticle and basement membrane);

Extracellular (excluding cell wall)] Fibulin 1, an extracellular matrix glycoprotein that may

connect elements of the extracellular matrix, may play roles in heart, lung, and kidney

development, epithelial mesenchymal transitions, tensile strength of cardiac valves, and

tumor invasion

Olin, A. I. et al.

The Proteoglycans Aggrecan and Versican Form Networks with Fibulin-2 through Their

Lectin Domain Binding.

J Biol Chem 276, 1253-1261. (2001).

5
7506656CD1
g31419
2.7E−146
[Homo sapiens] fibulin-1 C

Korenberg, J. R. et al. (supra)

568154|
2.3E−147
[Homo sapiens][Structural protein][Extracellular matrix (cuticle and basement membrane);

FBLN1

Extracellular (excluding cell wall)] Fibulin 1, an extracellular matrix and plasma

glycoprotein that may connect elements of the extracellular matrix, may play a role in

hemostasis, altered expression may play a role in tumor invasion, thrombosis, and

connective tissue and blood diseases

Hayashido, Y. et al. (supra)

584763|Fbln1
4.5E−110
[Mus musculus][Structural protein][Extracellular matrix (cuticle and basement membrane);

Extracellular (excluding cell wall)] Fibulin 1, an extracellular matrix glycoprotein that may

connect elements of the extracellular matrix, may play roles in heart, lung, and kidney

development, epithelial mesenchymal transitions, tensile strength of cardiac valves, and

tumor invasion

Olin, A. I. et al. (supra)

6
7510567CD1
g2842786
2.9E−34
[Homo sapiens] INTEGRIN BETA-8 SUBUNIT PRECURSOR

Tin-Wollam, A. et al.

336074|
2.4E−35
[Homo sapiens][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] Integrin beta

ITGB8

8 subunit, a member of the integrin family of adhesion receptors, forms a heterodimer with

alpha v integrin (ITGAV), cytoplasmic domain apparently does not promote adhesion

Neufert, C. et al.

Mycobacterium tuberculosis 19-kDa lipoprotein promotes neutrophil activation.

J Immunol 167, 1542-9. (2001).

7
7506072CD1
g930343
1.7E−101
[Homo sapiens] LAR-interacting protein 1b

Serra-Pages, C. et al.

The LAR transmembrane protein tyrosine phosphtase and a coiled-coil LAR-interacting

protein co-localize at focal adhesions

EMBO J. 14, 2827-2838 (1995)

337114|
1.5E−102
[Homo sapiens][Cytoplasmic; Plasma membrane; Cell junction] Protein tyrosine

PPFIA1

phosphatase receptor type f polypeptide (PTPRF) interacting protein (liprin) alpha 1,

localizes the PTPRF transmembrane protein tyrosine phosphatase to focal adhesions; also

interacts with PTPRS and PTPRD

Pulido, R. et al.

The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-

phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a

tissue-specific manner and associate with the LAR-interacting protein LIP.1.

Proc Natl Acad Sci USA 92, 11686-90 (1995).

748465|
7.6E−68
[Homo sapiens][Cytoplasmic; Plasma membrane; Cell junction] Protein phosphatase

PPFIA3

receptor f interacting protein alpha 3, member of the alpha liprin family, interacts with

transmembrane protein tyrosine phosphatase PTPRF

Serra-Pages, C. et al.

Liprins, a family of LAR transmembrane protein-tyrosine phosphatase-interacting proteins.

J Biol Chem 273, 15611-20 (1998).

8
7511354CD1
g16198471
0.0
[Homo sapiens] integrin, beta 7

336072|
0.0
[Homo sapiens][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] Beta 7

ITGB7

subunit of integrin, a member of a family of cell-surface proteins involved in cell-cell and

cell-matrix interactions, involved in cell migration, cell clustering, and inflammatory

responses; may play a role in Sjogren's Syndrome.

Jakubowski, A., Ehrenfels, B. N., Pepinsky,

R. B., and Burkly, L. C. Vascular cell adhesion

molecule-Ig fusion protein selectively targets activated alpha 4-integrin receptors in vivo.

Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.

J Immunol 155, 938-46 (1995).

587101|
0.0
[Mus musculus][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] Beta 7

Itgb7

subunit of integrin, a member of a family of cell-surface proteins involved in cell-cell and

cell-matrix interactions; may be involved in inflammatory responses.

Wagner, N., Lohler, J., Kunkel, E. J., Ley, K.,

Leung, E., Krissansen, G., Rajewsky, K., and

Muller, W. Critical role for beta7 integrins in formation of the gut-associated lymphoid

tissue. Nature 382, 366-70 (1996).

331094|
1.0E−229
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Beta 7 subunit of integrin, a

Itgb7

member of the family of cell-surface proteins involved in cell-cell and cell-matrix

interactions; may be involved in cell migration and inflammatory responses.

Feng, C. G., Britton, W. J., Palendira, U., Groat, N. L., Briscoe, H., and Bean, A. G. Up-

regulation of VCAM-1 and differential expansion of beta integrin-expressing T

lymphocytes are associated with immunity to pulmonary Mycobacterium tuberculosis

infection. J Immunol 164, 4853-60 (2000).

9
7511643CD1
g854175
0.0
[Homo sapiens] LI-cadherin

Grotzinger C. et al. LI-cadherin: a marker of gastric metaplasia and neoplasia. Gut. 2001

Jul; 49(1): 73-81.

752595|
0.0
[Homo sapiens][Adhesin/agglutinin; Transporter][Plasma membrane] Cadherin 17 (liver-

CDH17

intestine cadherin), which mediates calcium-dependent cell to cell adhesion; is expressed in

gastric intestinal metaplasia and adenocarcinomas.

Meli, M. L. et al. Anti-neuroblastoma antibody chCE7 binds to an isoform of L1-CAM

present in renal carcinoma cells. Int J Cancer 83, 401-8 (1999).

608812|
0.0
[Mus musculus][Adhesin/agglutinin; Ligand; Receptor (signalling)][Plasma membrane]

Cdh17

Cadherin 17 (liver-intestine cadherin), which mediates calcium-dependent cell to cell

adhesion; the human protein is expressed in gastric intestinal metaplasia and

adenocarcinomas.

Angres, B., Kim, L., Jung, R., Gessner, R., and Tauber, R. LI-cadherin gene expression

during mouse intestinal development. Dev Dyn 221, 182-93. (2001).

757578|
0.0
[Rattus norvegicus][Adhesin/agglutinin][Basolateral plasma membrane; Plasma membrane]

Cdh17

Cadherin 17 (liver-intestine cadherin), which mediates calcium-dependent cell to cell

adhesion; the human protein is expressed in gastric intestinal metaplasia and

adenocarcinomas.

Hou, D. X. et al. Expression of cell adhesion molecule and albumin genes in

primary culture of rat hepatocytes. Cell Biol Int 25, 239-44. (2001).

10
7511400CD1
g18490857
2.1E−31
[Homo sapiens] thrombospondin

731643|
1.7E−32
[Homo sapiens] Protein containing a type 1 thrombospondin domain, has high similarity to

FLJ14440

uncharacterized mouse 2810459H04Rik.

368602|
1.6E−29
[Mus musculus] Protein containing a type 1 thrombospondin domain, has high similarity to

Mm.42202

uncharacterized human FLJ14440.

11
7511507CD1
g4490530
4.9E−165
[Homo sapiens] fibulin-5

567846|
4.0E−166
[Homo sapiens][Ligand][Extracellular matrix (cuticle and basement membrane);

FBLN5

Extracellular (excluding cell wall)] Fibulin 5, a member of the EGF-like family,

serves as an integrin ligand during cell adhesion.

Nakamura, T. et al. DANCE, a novel secreted RGD protein expressed in developing,

atherosclerotic, and balloon-injured arteries. J Biol Chem 274, 22476-83 (1999).

Nakamura, T. et al. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature 415,

171-5. (2002).

429808|
4.5E−158
[Mus musculus][Ligand][Extracellular (excluding cell wall)] Fibulin 5, a member of the

Fbln5

EGF-like family, a putative ligand that functions in the response to wounding and may

contribute to cell adhesion, angiogenesis, and epithelial-mesenchymal transitions during

development.

Yanagisawa, H. et al. Fibulin-5 is an elastin-binding protein essential for elastic fibre

development in vivo. Nature 415, 168-71. (2002).

Nakamura, T. et al. (1999) (supra)

Nakamura, T. et al. (2002) (supra)

609665|
9.4E−158
[Rattus norvegicus][Ligand][Extracellular (excluding cell wall)] Fibulin 5, a member of the

Fbln5

EGF-like family, an integrin ligand that is secreted, functions in cell adhesion, and may be

involved in vascular repair after injury.

Kowal, R. C. et al. EVEC, a novel epidermal growth factor-like repeat-containing protein

upregulated in embryonic and diseased adult vasculature. Circ Res 84, 1166-76 (1999).

Nakamura, T. et al. (1999) (supra)

Nakamura, T. et al. (2002) (supra)

12
7511819CD1
g2695574
0.0
[Homo sapiens] leukocyte function-associated molecule-1 alpha subunit

Loftus, B. J. et al., Genome duplications and other features in 12 Mb of DNA sequence

from human chromosome 16p and 16q, Genomics 60, 295-308 (1999)

336054|
0.0
[Homo sapiens][Adhesin/agglutinin; Receptor (signaling)][Plasma membrane] Integrin

ITGAL

alpha L subunit, a heterophilic cell adhesion molecule that forms a heterodimer with

ITGB2, function in cell-cell adhesion via an interaction with intercellular adhesion molecule

1 (ICAM-1), signals through a protein kinase C pathway

Weber, K. S. et al., Specific activation of leukocyte beta2 integrins lymphocyte function-

associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha

subunit cytoplasmic domains., Mol Biol Cell 10, 861-73 (1999).

Peled, A. et al., The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5

on immature human CD34(+) cells: role in transendothelial/stromal migration and

engraftment of NOD/SCID mice., Blood 95, 3289-96 (2000).

319762|Itgal
1.6E−265
[Mus musculus][Adhesin/agglutinin; Receptor (signaling)][Plasma membrane] Integrin

alpha L subunit, a heterophilic cell adhesion molecule forms a heterodimer with ITGB2,

interacts with intercellular adhesion molecule 1 and 2 (Icam1)(Icam2), involved in cell

migration, neutrophil rolling and in allograft rejection

Stein, J. V. et al., The CC chemokine thymus-derived chemotactic agent 4 (TCA-4,

secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-

associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node

high endothelial venules., J Exp Med 191, 61-76 (2000).

13
7511338CD1
g17939541
1.9E−14
[Homo sapiens] integrin beta 4 binding protein

336066|
1.5E−15
[Homo sapiens][Anchor Protein][Nuclear; Cytoskeletal; Plasma membrane] Integrin beta 4

ITGB4BP

binding protein (translation initiation factor eIF6), prevents formation of the 80S ribosome

by inhibiting the 60S ribosomal subunit, may play a role in the allergic response,

upregulated during progression of colorectal cancer

Sanvito, F. et al., The beta 4 integrin interactor p27(BBP/eIF6) is an essential nuclear

matrix protein involved in 60S ribosomal subunit assembly., J Cell Biol 144, 823-837

(1999).

Sanvito, F. et al., Expression of a highly conserved protein, p27BBP, during the progression

of human colorectal cancer., Cancer Res 60, 510-6 (2000).

676861|
6.6E−15
[Mus musculus][Anchor Protein][Nuclear; Cytoplasmic; Unspecified membrane] Integrin

Itgb4bp

beta 4 binding protein (translation initiation factor eIF6), may prevent formation of the 80S

ribosome by inhibiting the 60S ribosomal subunit, may play a role in the allergic response,

induced in inflamed asthmatic lung tissues

Sanvito, F. et al. (supra)

Oh, C. K. et al., Eukaryotic Translation Initiation Factor-6 Enhances Histamine and IL-2

Production in Mast Cells., J Immunol 166, 3606-3611. (2001).

14
7511425CD1
g3930583
0.0
[Homo sapiens] integrin alpha-7

Vizirianakis, I. S. et al., Transfection of MCF-7 carcinoma cells with human integrin alpha7

cDNA promotes adhesion to laminin, Arch. Biochem. Biophys. 385, 108-116 (2001)

336050|
0.0
[Homo sapiens][Adhesin/agglutinin; Receptor (signaling)][Plasma membrane] Integrin

ITGA7

alpha 7, alpha subunit of an integrin laminin receptor, involved in cell adhesion and muscle

development; gene mutation is associated with congenital myopathy

Song, W. K. et al., Expression of alpha 7 integrin cytoplasmic domains during skeletal

muscle development: alternate forms, conformational change, and homologies with

serine/threonine kinases and tyrosine phosphatases., J Cell Sci, 1139-52 (1993).

Hayashi, Y. K. et al., Mutations in the integrin alpha7 gene cause congenital

myopathy., Nat Genet 19, 94-7 (1998).

Burkin, D. J. et al., The alpha7beta1 integrin in muscle development and disease., Cell

Tissue Res 296, 183-90 (1999).

583421|Itga7
0.0
[Mus musculus][Receptor (signaling)][Plasma membrane] Integrin alpha 7, alpha subunit of

an integrin laminin receptor, involved in cell adhesion, motility, and muscle development;

human ITGA7 gene mutation is associated with congenital myopathy

Ziober, B. L. et al., Alternative extracellular and cytoplasmic domains of the integrin alpha

7 subunit are differentially expressed during development., J Biol Chem 268, 26773-83

(1993).

Mayer, U. et al., Absence of integrin alpha 7 causes a novel form of muscular dystrophy.,

Nat Genet 17, 318-23 (1997). (supra)

15
7511534CD1
g244681
0.0
[Homo sapiens] integrin beta 7 subunit

Yuan, Q. A. et al., Cloning and sequence analysis of a novel beta 2-related integrin

transcript from T lymphocytes: homology of integrin cysteine-rich repeats to domain III of

laminin B chains, Int. Immunol. 3, 1373-1374 (1991)

336072|
0.0
[Homo sapiens][Adhesin/agglutinin; Receptor (signaling)][Plasma membrane] Beta 7

ITGB7

subunit of integrin, a member of a family of cell-surface proteins involved in cell-cell and

cell-matrix interactions, involved in cell migration, cell clustering, and inflammatory

responses; may play a role in Sjogren's Syndrome

Higgins, J. M. et al., The role of alpha and beta chains in ligand recognition by beta 7

integrins., J Biol Chem 275, 25652-64 (2000).

587101|Itgb7
6.7E−283
[Mus musculus][Adhesin/agglutinin; Receptor (signaling)][Plasma membrane] Beta 7

subunit of integrin, a member of a family of cell-surface proteins involved in cell-cell and

cell-matrix interactions; may be involved in inflammatory responses

Gurish, M. F. et al., Expression of murine beta 7, alpha 4, and beta 1 integrin genes by

rodent mast cells., J Immunol 149, 1964-72 (1992).

16
7511648CD1
g386753
0.0
[Homo sapiens] platelet Glycoprotein IIb (GPIIb)

Heidenreich, R. et al., Organization of the gene for platelet glycoprotein IIb, Biochemistry

29, 1232-1244 (1990)

336042|
0.0
[Homo sapiens][Adhesin/agglutinin; Isomerase; Chaperones; Receptor

ITGA2B

(signaling)][Cytoskeletal; Plasma membrane] Integrin alpha 2b, subunit of the fibrinogen

receptor, involved in hemostasis, blood coagulation, platelet aggregation, cell adhesion and

actin reoganization, associated with immune thrombocytopenic purpura; mutation causes

Glanzmann thrombasthenia

Basani, R. B. et al., Glanzmann thrombasthenia due to a two amino acid deletion in the

fourth calcium-binding domain of alpha IIb: demonstration of the importance of calcium-

binding domains in the conformation of alpha IIb beta 3., Blood 88, 167-73 (1996).

Furman, M. I. et al., The cleaved peptide of the thrombin receptor is a strong platelet

agonist., Proc Natl Acad Sci USA 95, 3082-7 (1998).

429948|
0.0
[Mus musculus][Receptor (signaling)][Plasma membrane] Integrin alpha 2b, subunit of the

Itga2b

fibrinogen receptor, involved in blood coagulation, platelet aggregation, cell adhesion and

actin reoganization, human ITGA2B is associated with immune thrombocytopenic purpura

and Glanzmann thrombasthenia

Chen, Y. Q. et al., Ectopic expression of platelet integrin alphaIIb beta3 in tumor cells from

various species and histological origin., Int J Cancer 72, 642-8 (1997).

Nieswandt, B. et al., Acute systemic reaction and lung alterations induced by an antiplatelet

integrin gpIIb/IIIa antibody in mice., Blood 94, 684-93 (1999).

17
7511600CD1
g6960195
2.6E−147
[Mus musculus] adhesion regulating molecule ARM-1

711654|
5.6E−148
[Rattus norvegicus] Integral plasma membrane glycoprotein, type I transmembrane

Gp110

glycoprotein that mediates Ca2+ - independent cell-cell adhesion, functions as a carrier in

the transport of bile salts from hepatocytes into bile

Lucka, L. et al., A short isoform of carcinoembryonic-antigen-related rat liver cell-cell

adhesion molecule (C-CAM/gp110) mediates intercellular adhesion. Sequencing and

recombinant functional analysis., Eur J Biochem 234, 527-35 (1995).

428282|
1.2E−116
[Homo sapiens][Plasma membrane] Cell membrane glycoprotein 110 kDa, putative integral

ADRM1

plasma membrane glycoprotein, putative tumor antigen and is expressed on human gastric

carcinoma cells; upregulated in response to IFNgamma (IFNG)

Shimada, S. et al., Molecular cloning and characterization of the complementary DNA of an

M(r) 110,000 antigen expressed by human gastric carcinoma cells and upregulated by

gamma-interferon., Cancer Res 54, 3831-6 (1994).

18
7511783CD1
g13111859
8.2E−85
[Homo sapiens] intercellular adhesion molecule 2

335916|
6.6E−86
[Homo sapiens][Adhesin/agglutinin; Ligand][Plasma membrane] Intercellular adhesion

ICAM2

molecule 2, a surface glycoprotein and member of the immunoglobulin superfamily, binds

the integrin LFA-1 (ITGB2) and promotes cell adhesion during immunological and

inflammatory reactions

Thomson, A. J. et al., Expression of intercellular adhesion molecules ICAM-1 and ICAM-2

in human endometrium: regulation by interferon-gamma., Mol Hum Reprod 5, 64-70

(1999).

Shimaoka, M. et al., Reversibly locking a protein fold in an active conformation with a

disulfide bond: integrin alphaL I domains with high affinity and antagonist activity in vivo.,

Proc Natl Acad Sci USA 98, 6009-14. (2001).

585029|
3.4E−50
[Mus musculus][Adhesin/agglutinin; Ligand][Plasma membrane] Intercellular adhesion

Icam2

molecule 2, a surface glycoprotein and member of the immunoglobulin superfamily, binds

the integrin LFA-1 (Itgb2) and promotes cell adhesion during immunological and

inflammatory reactions

Reiss, Y. et al., T cell interaction with ICAM-1-deficient endothelium in vitro:

transendothelial migration of different T cell populations is mediated by endothelial ICAM-

1 and ICAM-2., Int. Immunol 11, 1527-39 (1999).

Gerwin, N. et al., Prolonged eosinophil accumulation in allergic lung interstitium of ICAM-

2 deficient mice results in extended hyperresponsiveness., Immunity 10, 9-19 (1999).

19
7512383CD1
g14318638
0.0
[Homo sapiens] chondroitin sulfate proteoglycan BEHAB/brevican

625835|
0.0
[Homo sapiens][Structural protein][Extracellular matrix (cuticle and basement membrane)]

BCAN

Brevican (brain enriched hyaluronan binding protein), member of the lectican family of

chondroitin sulfate proteoglycans, a brain extracellular matrix protein, may function in brain

development; overexpression may enhance glioma invasiveness

Zhang, H. et al., Expression of a cleaved brain-specific extracellular matrix protein

mediates glioma cell invasion in vivo., J Neurosci 18, 2370-6 (1998).

Gary, S. C. et al., cDNA cloning, chromosomal localization, and expression analysis of

human BEHAB/brevican, a brain specific proteoglycan regulated during cortical

development and in glioma, Gene 256, 139-47 (2000).

589875|Bcan
1.7E−229
[Rattus norvegicus][Structural protein; Small molecule-binding protein][Extracellular

matrix (cuticle and basement membrane); Extracellular (excluding cell wall); Plasma

membrane] Brevican (brain enriched hyaluronan binding protein), member of the lectican

family of chondroitin sulfate proteoglycans, a brain extracellular matrix protein, promotes

neuronal cell adhesion and migration and may function in brain development

Milev, P. et al., Differential regulation of expression of hyaluronan-binding proteoglycans

in developing brain: aggrecan, versican, neurocan, and brevican., Biochem Biophys Res

Commun 247, 207-12 (1998).

Thon, N. et al., The chondroitin sulphate proteoglycan brevican is upregulated by astrocytes

after entorhinal cortex lesions in adult rats., Eur J Neurosci 12, 2547-58 (2000).

21
7512842CD1
g3169830
3.4E−65
[Homo sapiens] epithelial V-like antigen 1

Guttinger, M. et al. Epithelial V-like antigen (EVA), a novel member of the

immunoglobulin superfamily, expressed in embryonic epithelia with a potential role as

homotypic adhesion molecule in thymus histogenesis. J Cell Biol 141: 1061-71 (1998).

342924|EVA1
2.6E−66
[Homo sapiens][Cytoplasmic; Cytoskeletal] Epithelial V-like antigen, a member of the

immunoglobulin superfamily, may mediate cell adhesion, may play a role in trophoblast

invasion, placental development, and thymus organogenesis.

Guttinger, M. et al. (supra)

586553|Eva
9.4E−55
[Mus musculus][Cytoplasmic; Cytoskeletal] Epithelial V-like antigen, a member of the

immunoglobulin superfamily, may mediate cell adhesion, may play a role in thymus

organogenesis

Teesalu, T. et al. Expression pattern of the epithelial v-like antigen (Eva) transcript suggests

a possible role in placental morphogenesis. Dev Genet 23, 317-23 (1998).

22
90190613CD1
g7407146
0.0
[Homo sapiens] protocadherin Flamingo 1

Wu, Q. et al. A striking organization of a large family of human neural cadherin-like cell

adhesion genes. Cell 97: 779-790 (1999)

Wu, Q. et al. Large exons encoding multiple ectodomains are a characteristic feature of

protocadherin genes. Proc. Natl. Acad. Sci. U.S.A. 97: 3124-3129 (2000)

705126|
0.0
[Rattus norvegicus][Ligand; Receptor (signalling)][Plasma membrane] Protein with high

Celsr3

similarity to murine Celsr1, member of the secretin family of G protein-coupled receptors

(GPCR), contains nine cadherin domains, six EGF-like domains, two extracellular laminin

G domains, and a Latrophilin/CL-1-like GPS domain

Hill, E. et al. Cadherin Superfamily Proteins in Caenorhabditis elegans and Drosophila

melanogaster. J Mol Biol 305: 1011-1024 (2001).

Bockaert, J. et al. Molecular tinkering of G protein-coupled receptors: an evolutionary

success. Embo Journal 18: 1723-9 (1999).

690794|
0.0
[Homo sapiens] Cadherin-EGF-LAG seven-pass G-type receptor 2, may be involved in

CELSR2

transduction of extracellular signals by interacting with SH3 domain-containging proteins

Vincent, J. B. et al. The human homologue of flamingo, EGFL2, encodes a brain-expressed

large cadherin-like protein with epidermal growth factor-like domains, and maps to

chromosome 1p13.3-p21.1. DNA Res 7: 233-5 (2000).

Formstone, C. J. et al. Chromosomal localization of Celsr2 and Celsr3 in the mouse; Celsr3

is a candidate for the tippy (tip) lethal mutant on chromosome 9. Mamm Genome 11: 392-4

(2000).

23
7511894CD1
g9622236
4.8E−119
[Homo sapiens] cadherin-like protein VR20

743150|
6.0E−92
[Homo sapiens][Adhesin/agglutinin][Plasma membrane] Cadherin 2 type 1 (N-cadherin), a

CDH2

calcium-dependent glycoprotein that mediates cell-cell interactions, inhibits apoptosis, has a

role in development and is likely involved in tumor metastasis

Williams, C. L. et al. Regulation of E-cadherin-mediated adhesion by muscarinic

acetylcholine receptors in small cell lung carcinoma. J Cell Biol 121, 643-54 (1993).

Husmark, J. et al. N-cadherin-mediated adhesion and aberrant catenin expression in

anaplastic thyroid-carcinoma cell lines. Int J Cancer 83, 692-9 (1999).

583765|Cdh2
2.6E−91
[Mus musculus][Adhesin/agglutinin][Plasma membrane; Cell junction] Cadherin 2 (N-

cadherin), a calcium-dependent glycoprotein that mediates cell-cell interactions, inhibits

apoptosis, has a role in embryogenesis and brain development, and facilitates tumor

metastasis

Miyatani, S. et al. Neural cadherin: role in selective cell-cell adhesion. Science 245, 631-5

(1989).

Hermiston, M. L. et al. Inflammatory bowel disease and adenomas in mice expressing a

dominant negative N-cadherin. Science 270, 1203-7 (1995).

24
3604804CD1
g19773543
0.0
[Rattus norvegicus] (AB076401) fat3

Mitsui, K. et al. Mammalian fat3: a large protein that contains multiple cadherin and EGF-

like motifs. Biochem. Biophys. Res. Commun. 290: 1260-1266 (2002)

342032|FAT
0.0
[Homo sapiens][Adhesin/agglutinin][Plasma membrane] FAT tumor suppressor precursor,

member of the cadherin superfamily, possibly functions in developmental processes and cell

communication

Dunne, J. et al. Molecular cloning and tissue expression of FAT, the human homologue of

the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative

adhesion molecule. Genomics 30, 207-23 (1995).

Matsuyoshi, N. et al. Identification of novel cadherins expressed in human melanoma cells.

J Invest Dermatol 108, 908-13 (1997).

711832|Fat
0.0
[Rattus norvegicus] FAT tumor suppressor (Drosophila) homolog, member of the cadherin

superfamily, has 34 cadherin repeats, five EGF-like repeats, a laminin A-G domain, a

putative transmembrane domain, and a PDZ domain-binding motif, a component of

intercellular junctions

Ponassi, M. et al. Expression of the rat homologue of the Drosophila fat tumour suppressor

gene. Mech Dev 80, 207-12. (1999).

Inoue, T. et al. FAT is a component of glomerular slit diaphragms. Kidney Int 59, 1003-12.

(2001).

25
7512568CD1
g31191
8.5E−179
[Homo sapiens] epican

Kugelman, L. C. et al. The core protein of epican, a heparan sulfate proteoglycan on

keratinocytes, is an alternative form of CD44. J. Invest. Dermatol. 99: 381-385 (1992)

309453|
6.7E−180
[Homo sapiens] Protein with high similarity to mouse Cd44, which functions as a receptor

Hs.169322

for both soluble and membrane-bound hyaluronic acid, contains an extracellular link

(hyaluronan-binding) domain

575773|Cd44
4.5E−144
[Mus musculus][Adhesin/agglutinin; Receptor (signalling); Small molecule-binding

protein][Plasma membrane] Cd44 antigen, type I transmembrane protein that functions as a

receptor for soluble and membrane-bound hyaluronic acid, mediates cell adhesion and

migration, may contribute to angiogenesis, mammary gland development, tumor metastasis

and atherosclerosis

Wolffe, E. J. et al. The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell

surface antigen and proteoglycan core/link proteins. J Biol Chem 265, 341-7 (1990).

Yu, Q. et al. A new alternatively spliced exon between v9 and v10 provides a molecular

basis for synthesis of soluble CD44. J Biol Chem 271, 20603-7 (1996).

Screaton, G. R. et al. The identification of a new alternative exon with highly restricted

tissue expression in transcripts encoding the mouse Pgp-1 (CD44) homing receptor.

Comparison of all 10 variable exons between mouse, human, and rat. J Biol Chem 268,

12235-8 (1993).

Tolg, C. et al. Splicing choice from ten variant exons establishes CD44 variability. Nucleic

Acids Res 21, 1225-9 (1993).

26
7512812CD1
g9446402
0.0
[Homo sapiens] integrin beta-subunit

Sheppard, D. et al. Complete amino acid sequence of a novel integrin beta subunit (beta 6)

identified in epithelial cells using the polymerase chain reaction. J. Biol.

Chem. 265: 11502-11507 (1990)

606202|
0.0
[Homo sapiens][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] Integrin beta

ITGB6

6, member of a family of cell-surface proteins, binds fibronectin, mediates epithelial cell-

matrix interactions in development, wound repair, and neoplasia, regulates lung

inflammatory response, receptor for foot and mouth disease virus

Jackson, T. et al. The epithelial integrin alpha v beta 6 is a receptor for foot-and-mouth

disease virus. J Virol 74, 4949-56 (2000).

Agrez, M. et al. The alpha v beta 6 integrin induces gelatinase B secretion in colon cancer

cells. Int J Cancer 81, 90-7 (1999).

Agrez, M. et al. The alpha v beta 6 integrin promotes proliferation of colon carcinoma cells

through a unique region of the beta 6 cytoplasmic domain. J Cell Biol 127, 547-56 (1994).

Thorne, R. F. et al. The integrins alpha 3beta 1 and alpha 6beta 1 physically and

functionally associate with CD36 in human melanoma cells. REQUIREMENT FOR THE

EXTRACELLULAR DOMAIN OF CD36. J Biol Chem 275: 35264-75 (2000).

585099|Itgb5
1.3E−158
[Mus musculus][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] Integrin

beta 5, forms a heterodimer with alpha V integrin (Itgav) and acts as a vitronectin receptor,

involved in cell adhesion and motility.

Lai, C. F. et al. Transforming Growth Factor-beta Up-regulates the beta 5 Integrin Subunit

Expression via Sp1 and Smad Signaling. J Biol Chem 275, 36400-36406 (2000).

Huang, X. et al. Normal development, wound healing, and adenovirus susceptibility in

beta5-deficient mice. Mol Cell Biol 20, 755-9. (2000).

27
7512826CD1
g529724
0.0
[Homo sapiens] MUC18 glycoprotein

Lehmann, J. M. et al. MUC18, a marker of tumor progression in human melanoma, shows

sequence similarity to the neural cell adhesion molecules of the immunoglobulin

superfamily. Proc. Natl. Acad. Sci. U.S.A. 86, 9891-9895 (1989)

432384|
0.0
[Homo sapiens][Adhesin/agglutinin][Plasma membrane] Melanoma cell adhesion molecule,

MCAM

an adhesion molecule and tumor antigen that may play roles in cytoskeletal rearrangement,

embryo implantation, development, angiogenesis, and inflammation, expression correlates

with tumor progression and metastasis

Pickl, W. F. et al. MUC18/MCAM (CD146), an activation antigen of human T

lymphocytes. J Immunol 158, 2107-15 (1997).

Mintz-Weber, C. S. et al. Identification of the elements regulating the expression of the cell

adhesion molecule MCAM/MUC18. LOSS OF AP-2 IS NOT REQUIRED FOR MCAM

EXPRESSION IN MELANOMA CELL LINES. J. Biol. Chem. 275: 34672-80 (2000).

662705|
2.8E−260
[Mus musculus] Protein with high similarity to melanoma cell adhesion molecule (human

Mcam

MCAM), which is an adhesion molecule and tumor antigen that may play roles in

cytoskeletal rearrangement, development, and angiogenesis, contains five immunoglobulin

(Ig) domains

Vainio, O. et al. HEMCAM, an adhesion molecule expressed by c-kit+ hemopoietic

progenitors. J Cell Biol 135, 1655-68 (1996).

Yang, H. et al. Isolation and characterization of mouse MUC18 cDNA gene, and correlation

of MUC18 expression in mouse melanoma cell lines with metastatic ability.

Gene 265, 133-45. (2001).

28
7512908CD1
g5764665
1.2E−154
[Homo sapiens] cerebral cell adhesion molecule

Starzyk, R. et al. Cerebral Cell Adhesion Molecule: a novel leukocyte adhesion determinant

on blood brain barrier capillary endothelium. J. Infect. Dis. 181: 181-187 (2000).

475793|
9.7E−156
[Homo sapiens][Adhesin/agglutinin][Plasma membrane] Cerebral cell adhesion molecule,

LOC51148

an adhesion molecule that is predicted to be involved in leukocyte transmigration across the

blood-brain barrier

Starzyk, R. M. et al. (supra)

752557|C1orf17
1.3E−105
[Homo sapiens] mRNA KIAA0584, isolated from human brain cDNA library

Sood, R. et al. Cloning and characterization of 13 novel transcripts and the human rgs8

gene from the 1q25 region encompassing the hereditary prostate cancer

(hpc1) locus. Genomics 73, 211-22. (2001).

29
7512909CD1
g5764665
9.7E−246
[Homo sapiens] cerebral cell adhesion molecule

Starzyk, R. et al. (supra)

475793|
7.6E−247
[Homo sapiens][Adhesin/agglutinin][Plasma membrane] Cerebral cell adhesion molecule,

LOC51148

an adhesion molecule that is predicted to be involved in leukocyte transmigration across the

blood-brain barrier

Starzyk, R. M. et al. (supra)

752557|C1orf17
1.7E−148
[Homo sapiens] mRNA KIAA0584, isolated from human brain cDNA library

Sood, R. et al. (supra)

30
7512769CD1
g2654433
2.6E−35
[Homo sapiens] extracellular matrix protein 1

Smits, P. et al. The human extracellular matrix gene 1 (ECM1): genomic structure, cDNA

cloning, expression pattern, and chromosomal localization. Genomics 45: 487-495 (1997)

662414|
2.0E−36
[Homo sapiens][Extracellular matrix (cuticle and basement membrane); Extracellular

ECM1

(excluding cell wall)] Extracellular matrix protein 1, may possess angiogenic properties that

promote tumor progression

Deckers, M. M. et al. Recombinant human extracellular matrix protein 1 inhibits alkaline

phosphatase activity and mineralization of mouse embryonic metatarsals in vitro. Bone 28,

14-20. (2001).

Han, Z. et al. Extracellular matrix protein 1 (ECM1) has angiogenic properties and is

expressed by breast tumor cells. FASEB J 15: 988-94. (2001).

584057|Ecm1
1.6E−16
[Mus musculus][Structural protein][Extracellular matrix (cuticle and basement membrane);

Extracellular (excluding cell wall)] Extracellular matrix protein 1, functions as a negative

regulator of bone formation and may contribute to angiogenesis; human ECM1 may

promote tumor progression

Bhalerao, J. et al. Molecular cloning, characterization, and genetic mapping of the cDNA

coding for a novel secretory protein of mouse. Demonstration of alternative splicing in skin

and cartilage. J Biol Chem 270, 16385-94 (1995).

31
7512871CD1
g1911530
2.6E−168
[Homo sapiens] ficolin

Lu, J. et al. Human ficolin: cDNA cloning, demonstration of peripheral blood leucocytes as

the major site of synthesis and assignment of the gene to chromosome 9. Biochem. J.

313: 473-478 (1996)

571024|FCN1
1.8E−170
[Homo sapiens][Structural protein; Small molecule-binding protein] Ficolin-1, a peripheral

leukocyte GlcNAc-binding lectin containing collagen- and fibrinogen-like domains, forms a

complex with mannose-binding lectin-associated serine proteases (MASPs) that activates

complement, may play a role in innate immunity

Matsushita, M. et al. A novel human serum lectin with collagen- and fibrinogen-like

domains that functions as an opsonin. J Biol Chem 271, 2448-54 (1996).

Matsushita, M. et al. Cutting edge: complement-activating complex of ficolin and mannose-

binding lectin-associated serine protease. J Immunol 164, 2281-4. (2000).

418853|Fcnb
9.0E−121
[Mus musculus][Extracellular (excluding cell wall)] Ficolin B, a member of the ficolin

family of proteins that bind carbohydrates, elastin, and corticosteroids, expressed in liver

and forms 12-mers in plasma

Ohashi, T. et al. Oligomeric structure and tissue distribution of ficolins from mouse, pig and

human. Arch Biochem Biophys 360, 223-32 (1998).

TABLE 3

SEQ
Incyte

Potential

ID
Polypeptide
Amino Acid
Phosphorylation
Potential

Analytical Methods

NO:
ID
Residues
Sites
Glycosylation Sites
Signature Sequences, Domains and Motifs
and Databases

1
7506690CD1
1252
S47 S91 S96 S129
N240 N322 N426
Signal_cleavage: M1-A24
SPSCAN

S178 S190 S243
N463 N500 N769

S252 S306 S324
N795 N883 N898

S336 S341 S347
N1027 N1038

S418 S441 S451
N1059 N1236

S488 S567 S660

S734 S837 S918

S956 S1018 S1176

S1202 T230 T427

T465 T478 T554

T613 T634 T756

T789 T876 T1001

T1040 T1061

T1086 T1188

T1193 T1225 Y516

Y582

Signal Peptide: P6-A24
HMMER

Signal Peptide: M1-G22
HMMER

Signal Peptide: M1-A24
HMMER

Signal Peptide: M1-I27
HMMER

Fibronectin type III domain: P645-S731, P842-V929, E1022-S1098, P744-P830
HMMER_PFAM

Immunoglobulin domain: G278-A335, G553-A611, Y462-A520, G368-T427, G155-A215, R56-A120
HMMER_PFAM

Fibronectin type 3 domain: P645-S728, P842-G926, E745-G827, E1022-S1098
HMMER_SMART

Immunoglobulin: A270-K351, P545-L627, L147-L234, N454-K536, P360-L443, A48-S137
HMMER_SMART

Immunoglobulin C-2 Type: L276-G340, I460-G525, A366-G432, R551-D616, D54-G125, Q153-T220
HMMER_SMART

I type Ig domains from SCOP: K536-P633, T260-L357, V445-V535
HMMER_INCY

Ig superfamily from SCOP: W356-R448, L450-T539, P266-P354, I541-Q630, T44-P140, P143-H218
HMMER_INCY

Cytosolic domain: K1144-A1252;
TMHMMER

Transmembrane domain: G1121-I1143;

Non-cytosolic domain: M1-Q1120

CELL ADHESION PRECURSOR SIGNAL MOLECULE
BLAST_PRODOM

IMMUNOGLOBULIN GLYCOPROTEIN

TRANSMEMBRANE REPEAT FOLD PD003273: I1139-S1243

PRECURSOR SIGNAL ADHESION CELL
BLAST_PRODOM

GLYCOPROTEIN IMMUNOGLOBULIN FOLD REPEAT MOLECULE

NEURAL PD003129: N122-L231

NEUROFASCIN PRECURSOR SIGNAL PD065767:
BLAST_PRODOM

E1017-T1108

PRECURSOR SIGNAL CONTACTIN CELL ADHESION
BLAST_PRODOM

NEUROFASCIN GLYCOPROTEIN GP135

IMMUNOGLOBULIN FOLD PD001890: L732-A844

NEURAL CELL ADHESION MOLECULE L1 DM02463|
BLAST_DOMO

S26180|1027-1247: G1012-K1228

P35331|1009-1259: V923-A969, I1025-K1228

S26180|1027-1247: Q922-S1011

IMMUNOGLOBULIN DM00001
BLAST_DOMO

|S26180|352-436: K351-A436

|S26180|45-129: T44-S129

2
7506536CD1
421
S31 S72 S103 S188
N51 N138 N374
Signal_cleavage: M1-A21
SPSCAN

T80 T89 T122

T146 T160 T172

T242 T282

Signal Peptide: P4-A22
HMMER

Signal Peptide: M1-A21
HMMER

Signal Peptide: M1-A22
HMMER

Signal Peptide: M1-S24
HMMER

Signal Peptide: M1-A28
HMMER

Collagen triple helix repeat (20 copies): G299-R358, G208-P267
HMMER_PFAM

Cytosolic domain: M1-A6; Transmembrane
TMHMMER

domain: W7-P29; Non-cytosolic

domain: F30-P421

COLLAGEN ALPHA PRECURSOR CHAIN REPEAT
BLAST_PRODOM

SIGNAL CONNECTIVE TISSUE EXTRACELLULAR

MATRIX PD000007: G223-G332, P291-E369, G293-E369,

G199-P277, P165-G256, G293-G368, Q164-G238

SIMILAR TO CUTICULAR COLLAGEN PD067228:
BLAST_PRODOM

G217-G314, D191-G305, P224-G317, E163-R270, P291-K370,

P291-G368, P306-G409, P297-D398, P257-Q357,

G235-G332, P165-G259

PRECOLLAGEN P PRECURSOR SIGNAL PD072959:
BLAST_PRODOM

G293-H372, G223-G329, G293-H372, G181-P277 G247-P349,

G229-G341G256-P367, G299-P401

ZK265.2 PROTEIN PD068401: G226-P337, P294-P367,
BLAST_PRODOM

P173-G259, G190-P310

FIBRILLAR COLLAGEN CARBOXYL-TERMINAL
BLAST_DOMO

DM00019

|S18803|1304-1528: P176-K370

|P20908|1300-1524: P176-K370

|P20908|1300-1524: P167-G368

|P02457|915-1139: G181-G396

|P20908|1300-1524: G202-G368

S18803|1304-1528: G181-G368

|P05997|957-1181: G181-G409

FIBRILLAR COLLAGEN CARBOXYL-TERMINAL
BLAST_DOMO

DM00019

|P20908|1300-1524: P168-G368

|S18803|1304-1528: P168-G368

|S18803|1304-1528: G205-G368

|P02457|915-1139: G202-G420

|P05997|957-1181: P167-G368

|P05997|957-1181: P167-G368

FIBRILLAR COLLAGEN CARBOXYL-TERMINAL
BLAST_DOMO

DM00019

|P02457|915-1139: P168-G338

|P05997|957-1181: V166-G332

|P20908|1300-1524: G293-P421

S18803|1304-1528: G293-P421

|P02457|915-1139: G296-P421

|S18803|1304-1528: P165-G314

FIBRILLAR COLLAGEN CARBOXYL-TERMINAL
BLAST_DOMO

DM00019

|P20908|1300-1524: P167-G308

|P20908|1300-1524: G296-G420

|S18803|1304-1528: G296-G420

|P02457|915-1139: P167-G265

|P02457|915-1139: P167-G238

3
7506537CD1
124
S31 S72 S103 T80
N51 N120
Signal_cleavage: M1-A21
SPSCAN

T89

Signal Peptide: P4-A22
HMMER

Signal Peptide: M1-A21
HMMER

Signal Peptide: M1-A22
HMMER

Signal Peptide: M1-S24
HMMER

Signal Peptide: M1-A28
HMMER

4
7506655CD1
398
S7 S71 S213 S226
N98
Signal_cleavage: M1-A29
SPSCAN

S239 S259 S285

T161 T193 T250

T333 T369 T391

Signal Peptide: P11-A29
HMMER

Signal Peptide: R9-A29
HMMER

Signal Peptide: R8-A29
HMMER

Signal Peptide: M1-A25
HMMER

Signal Peptide: M1-A29
HMMER

Signal Peptide: L12-A29
HMMER

Signal Peptide: P6-A29
HMMER

Signal Peptide: V10-A29
HMMER

Anaphylotoxin-like domain: C36-C69, C112-C144, H77-C110
HMMER_PFAM

Epidermal growth factor-like domain.: R179-E215, E265-I307,
HMMER_SMART

E219-K261

Calcium-binding EGF-like domain: D262-I307, D216-K261
HMMER_SMART

Anaphylatoxin domain proteins BL01177: G18-A29, S200-E215,
BLIMPS_BLOCKS

F277-L303

GLYCOPROTEIN EGFLIKE DOMAIN PRECURSOR
BLAST_PRODOM

FIBULIN1 ISOFORM SIGNAL EXTRACELLULAR

MATRIX PLASMA PD006208: V27-N177

FIBULIN2 PRECURSOR SIGNAL GLYCOPROTEIN
BLAST_PRODOM

EXTRACELLULAR MATRIX PLASMA EGFLIKE

DOMAIN CALCIUMBINDING ALTERNATIVE

SPLICING REPEAT PD166194: C186-C227

EGF DM00003|P98095|720-782: V217-N281
BLAST_DOMO

EGF-LIKE DOMAIN DM00864|I55476|159-241: R181-E267
BLAST_DOMO

Anaphylatoxin domain signature: C36-C69, H77-C110,
MOTIFS

C112-C144

EGF-like domain signature 2: C199-C214
MOTIFS

Calcium-binding EGF-like domain pattern signature: D216-C242,
MOTIFS

D262-C288

5
7506656CD1
288
S7 S71 S213 S226
N98
Signal_cleavage: M1-A29
SPSCAN

S239 S259 T161

T193 T250 T274

Signal Peptide: P11-A29
HMMER

Signal Peptide: R9-A29
HMMER

Signal Peptide: R8-A29
HMMER

Signal Peptide: M1-A25
HMMER

Signal Peptide: M1-A29
HMMER

Signal Peptide: L12-A29
HMMER

Signal Peptide: P6-A29
HMMER

Signal Peptide: V10-A29
HMMER

Anaphylotoxin-like domain: C36-C69, C112-C144, H77-C110
HMMER_PFAM

EGF-like domain: C180-C214
HMMER_PFAM

Anaphylatoxin homologous domain: C36-C69, C112-C144,
HMMER_SMART

H77-C110

Calcium-binding EGF-like domain: D216-K261
HMMER_SMART

Anaphylatoxin domain proteins BL01177: G18-A29, S200-E215,
BLIMPS_BLOCKS

G184-F202, S209-S226

GLYCOPROTEIN EGFLIKE DOMAIN PRECURSOR
BLAST_PRODOM

FIBULIN1 ISOFORM SIGNAL EXTRACELLULAR

MATRIX PLASMA PD006208: V27-N177

FIBULIN2 PRECURSOR SIGNAL GLYCOPROTEIN
BLAST_PRODOM

EXTRACELLULAR MATRIX PLASMA EGFLIKE

DOMAIN CALCIUMBINDING ALTERNATIVE

SPLICING REPEAT PD166194: C186-C227

Anaphylatoxin domain signature: C36-C69, H77-C110,
MOTIFS

C112-C144

EGF-like domain signature 2: C199-C214
MOTIFS

Calcium-binding EGF-like domain pattern signature: D216-C242
MOTIFS

6
7510567CD1
78

Signal_cleavage: M1-G42
SPSCAN

Signal Peptide: R21-G42
HMMER

Signal Peptide: P23-G42
HMMER

Signal Peptide: R20-G42
HMMER

INTEGRIN BETA8 SUBUNIT PRECURSOR CELL
BLAST_PRODOM

ADHESION TRANSMEMBRANE GLYCOPROTEIN

REPEAT EXTRACELLULAR PD136783: M1-A52

INTEGRINS BETA CHAIN CYSTEINE-RICH DOMAIN
BLAST_DOMO

DM00846|P26012|24-457: A24-E72

7
7506072CD1
203
S10 S28 S34 S42
N119 N186
PROTEIN LAR-INTERACTING LIPRINALPHA2 1A 1B
BLAST_PRODOM

S133 S138 S156

F59F5.6 PD023244: Q29-T94, P87-K180, E89-Q147, Q88-Q147

S166 T8 T53 T190

T197

8
7511354CD1
650
S15 S19 S27 S163
N68 N279 N434
signal_cleavage: M1-S19
SPSCAN

S188 S195 S410
N477 N517 N526
Signal Peptide: M1-G17, M1-S19, M1-A23
HMMER

S611 T131 T218

Cytosolic domain: R599-L650
TMHMMER

T287 T294 T323

Transmembrane domain: T576-Y598

T472 Y327

Non-cytosolic domain: M1-H575

Integrins, beta chain: S50-C476
HMMER_PFAM

Integrin beta subunits (N-terminal): S50-C476
HMMER_SMART

Domain found in Plexins, Semaphorins and Integrins: S44-P92
HMMER_SMART

Integrin beta, C-terminus IPB001169: R79-L96, L140-V180,
BLIMPS_BLOCKS

R181-C216, V232-L283, L284-L313, F325-V368, G369-S410,

C478-C497

Integrin beta subunit signature PR01186: A48-C64, C80-P99,
BLIMPS_PRINTS

Q122-G135, Y150-L168, R190-P209, C223-F242,

D256-R278, R282-D297, D326-T349, L362-Y386, S489-D502,

D574-G591, G591-E609, S626-T636

INTEGRIN GLYCOPROTEIN CELL ADHESION
BLAST_PRODOM

TRANSMEMBRANE REPEAT PRECURSOR

EXTRACELLULAR SUBUNIT SIGNAL

PD001811: S50-C476

PD001794: C497-A645

INTEGRIN BETA-7 SUBUNIT PRECURSOR CELL
BLAST_PRODOM

ADHESION TRANSMEMBRANE GLYCOPROTEIN

REPEAT EXTRACELLULAR PD041877: M1-P49

INTEGRINS BETA CHAIN CYSTEINE-RICH DOMAIN
BLAST_DOMO

DM00846:

|P26010|17-465: G17-L466

|S32659|2-437: S50-E464

|P32592|3-435: S50-E464

|P12607|2-453: A48-L466

9
7511643CD1
752
S37 S129 S134
N149 N184 N250
signal_cleavage: M1-G22
SPSCAN

S212 S223 S272
N419 N456 N546
Signal Peptide: M1-G22
HMMER

S409 S425 S484
N587
Cadherin domain: F571-C685, T345-I440, Y131-T235,
HMMER_PFAM

S601 S649 S686

G455-T558, K243-K331, M34-K117

T113 T151 T228

Cadherin repeats.: I259-P338, L362-P447, V148-P242, Y39-P124,
HMMER_SMART

T345 T363 T385

I472-Q565, V588-P672

T458 T475 T589

Cytosolic domain: R729-S752
TMHMMER

T653 Y418

Transmembrane domain: G706-I728

Non-cytosolic domain: M1-V705

Cadherin domain IPB002126: D605-E628, E657-V705
BLIMPS_BLOCKS

Cadherins extracellular repeated domain signature: L94-F145
PROFILESCAN

Cadherin signature PR00205: D67-R86, F126-D155, S206-G218,
BLIMPS_PRINTS

P319-P338, P564-E577, Q288-D314

CELL ADHESION GLYCOPROTEIN
BLAST_PRODOM

TRANSMEMBRANE CALCIUM BINDING REPEAT LI

CADHERIN LIVER INTESTINE CADHERIN

PRECURSOR CADHERIN 17

PD024554: I2-G64

PD024555: H698-S752

PD151760: E236-G268

CADHERIN REPEAT DM00030
BLAST_DOMO

|S55396|481-593: F481-E594

|S55396|265-369: N265-E370

|S55396|371-479: N371-P480

|S55396|54-154: N54-D155

Cell attachment sequence: R603-D605
MOTIFS

Cadherins extracellular repeated domain signature: I114-P124,
MOTIFS

V328-P338

10
7511400CD1
180
S29 S130 T126
N17 N97
signal_cleavage: M1-C21
SPSCAN

T136 T146 T175

Signal Peptide: M1-G23
HMMER

Signal Peptide: M1-C21
HMMER

Furin-like repeats: Y18-P71
HMMER_SMART

Thrombospondin type 1 repeats: V84-P141
HMMER_SMART

11
7511507CD1
407
S163 S205 S211
N242 N255
signal_cleavage: M1-A23
SPSCAN

S244 S316 S344

Signal Peptide: M1-A23, M1-A25, I4-A23
HMMER

T48 T156 T330

EGF-like domain: C170-C204, C131-C164,
HMMER_PFAM

C210-C245, C251-C291

Epidermal growth factor-like domain.: E209-Q246, E169-S205,
HMMER_SMART

E130-Q165, E250-M292

Calcium-binding EGF-like domain: D206-Q246, D166-S205
HMMER_SMART

D127-Q165, D247-M292, D42-T86

EGF-like domain IPB000561: C149-L157
BLIMPS_BLOCKS

Calcium-binding EGF-like domain IPB001881: C149-E159,
BLIMPS_BLOCKS

C180-C191

Laminin-type EGF-like (LE) domain IPB002049: R290-C300
BLIMPS_BLOCKS

Type II EGF-like signature PR00010: D127-Q138, G201-D208,
BLIMPS_PRINTS

G226-Y236

LAMININ CHAIN EGF-LIKE DOMAIN PD00320: G226-L239
BLIMPS_PRODOM

GLYCOPROTEIN EGF-LIKE DOMAIN T16 H411
BLAST_PRODOM

PRECURSOR SIGNAL UPH1 UP50 PD030337: Q26-E130

EGF-LIKE DOMAIN GLYCOPROTEIN PRECURSOR
BLAST_PRODOM

SIGNAL EXTRACELLULAR MATRIX PLASMA

CALCIUMBINDING REPEAT PD008104: R290-V402

EGF-LIKE DOMAIN DM00864|I55476|159-241: G134-C210,
BLAST_DOMO

C176-E252

EGF DM00003|P35556|2219-2292: N174-D247
BLAST_DOMO

EGF DM00003|P35556|2219-2292: C140-D206
BLAST_DOMO

EGF DM00003|A57278|2213-2286: C140-D206
BLAST_DOMO

EGF DM00003|A57278|2213-2286: N174-D247
BLAST_DOMO

EGF DM00003|P98163|1373-1460: C136-M207
BLAST_DOMO

Cell attachment sequence: R54-D56
MOTIFS

Aspartic acid and asparagine hydroxylation site: C140-C151,
MOTIFS

C180-C191, C221-C232

EGF-like domain signature 2: C149-C164, C189-C204,
MOTIFS

C230-C245

Calcium-binding EGF-like domain pattern signature: D42-C68,
MOTIFS

E121-C149, D127-C149, D166-C189, D206-C230,

D247-C273

12
7511819CD1
681
S187 S201 S338
N65 N89 N188
signal_cleavage: M1-S25
SPSCAN

S566 S611 S625
N649

S641 T84 T91

T204 T263 T337

T681 Y124 Y211

Signal Peptide: M1-A23, M1-S25, M9-S25
HMMER

Integrin alpha (beta-propellor repeats: P517-E570, I456-Q511,
HMMER_SMART

R400-H452, A41-G87, G577-E628

von Willebrand factor (vWF) type A domain: N154-K329
HMMER_SMART

FG-GAP repeat: L518-L575, G457-Y516, A401-Q455, I578-P630,
HMMER_PFAM

G42-T91

von Willebrand factor type A domain: D156-L327
HMMER_PFAM

Cytosolic domain: M1-S4
TMHMMER

Transmembrane domain: C5-N27

Non-cytosolic domain: L28-T681

Integrins alpha chain IPB000413: S66-T77, G366-G375,
BLIMPS_BLOCKS

G453-A482, R520-P544

Von Willebrand factor type A domain signature PR00453:
BLIMPS_PRINTS

V155-F172, Q192-F206, V258-E266

Integrin alpha subunit signature PR01185: A401-S413, L419-M430,
BLIMPS_PRINTS

G453-G473, R520-P544, F581-G602, Q606-S625

von Willebrand factor type A domain proteins. PF00092:
BLIMPS_PFAM

G481-G491

INTEGRIN PRECURSOR SIGNAL GLYCOPROTEIN
BLAST_PRODOM

ALPHA CELL ADHESION TRANSMEMBRANE

EXTRACELLULAR MATRIX PD001221: K381-G503,

L484-S658, S35-Y71, V56-G106

GLYCOPROTEIN PRECURSOR CELL LEUKOCYTE
BLAST_PRODOM

ADHESION LFA1 ALPHA CHAIN FUNCTION

ASSOCIATED PD152352: G114-V155

VON WILLEBRAND FACTOR TYPE A REPEAT
BLAST_DOMO

DM00219|P20701|148-316: Q148-F317

DM00219|P24063|145-314: Q148-F317

INTEGRINS ALPHA CHAIN DM00458|P20701|35-147:
BLAST_DOMO

S35-F147

CELL SURFACE GLYCOPROTEIN CD11B
BLAST_DOMO

DM02945|P20701|560-1036: G560-Q664

13
7511338CD1
59
S51

signal_cleavage: M1-S31
SPSCAN

14
7511425CD1
1113
S97 S127 S234
N86 N742 N921
signal_cleavage: M1-A33
SPSCAN

S269 S301 S461
N957 N977

S659 S744 S788

S796 S832 S852

S914 S983 S1082

T229 T532 T599

T625 T776 T786

T959 T979

Integrin alpha (beta-propellor repeats: P377-E430, L316-R372,
HMMER_SMART

P48-Q110, L259-S312, G435-E491

FG-GAP repeat: D378-G435, S318-P377, G49-M114, I436-D489,
HMMER_PFAM

N260-T317

Cytosolic domains: M1-G11, K1037-A1113
TMHMMER

Transmembrane domains: A12-F34, W1014-W1036

Non-cytosolic domain: N35-P1013

Integrins alpha chain IPB000413: R87-L98, G215-G224,
BLIMPS_BLOCKS

G313-P342, M380-P404, W1015-R1043

Integrins alpha chain signature: V1016-T1074
PROFILESCAN

Integrin alpha subunit signature PR01185: N260-G272, S280-K291,
BLIMPS_PRINTS

G313-G333, M380-P404, F439-G460, T465-A484,

D584-L597, G1024-R1043

INTEGRIN PRECURSOR SIGNAL GLYCOPROTEIN
BLAST_PRODOM

ALPHA CELL ADHESION TRANSMEMBRANE

EXTRACELLULAR MATRIX PD001221: G46-G132,

Q193-Q361, V339-Q880, V882-R1043

INTEGRIN ALPHA PRECURSOR SIGNAL ALPHA7
BLAST_PRODOM

PD146538: W1036-A1113

INTEGRIN PRECURSOR CELL GLYCOPROTEIN
BLAST_PRODOM

SIGNAL ADHESION TRANSMEMBRANE

EXTRACELLULAR MATRIX CYTOSKELETON

PD001587: A33-R147

INTEGRIN PRECURSOR SIGNAL ALPHA ALPHA6
BLAST_PRODOM

VLA6 CELL ADHESION GLYCOPROTEIN

TRANSMEMBRANE PD001486: E155-E192

INTEGRINS ALPHA CHAIN
BLAST_DOMO

DM01028|I61186|420-951: G179-Q238, P307-A341, F381-D406,

G420-Q880, D881-R928

DM01028|P23229|416-932: P307-A341, F381-D406, G420-Q897

DM01028|P26007|410-931: P307-A341, S379-D406, G420-Q897

DM00458|I61186|42-233: L42-S233

Integrins alpha chain signature: W1036-R1043
MOTIFS

15
7511534CD1
601
S15 S19 S27 S163
N68 N279 N434
signal_cleavage: M1-S19
SPSCAN

S188 S195 S410
N477 N531

S514 S519 S538

T131 T218 T287

T294 T323 T472

Y327

Signal Peptide: M1-G17, M1-S19, M1-A23
HMMER

Integrin beta subunits (N-terminal portion of extracellular
HMMER_SMART

region): S50-C476

Domain found in plexins, semaphorins and integrins: S44-P92
HMMER_SMART

Integrins, beta chain: S50-C476
HMMER_PFAM

Integrin beta, C-terminus IPB001169: R79-L96, L140-V180,
BLIMPS_BLOCKS

R181-C216, V232-L283, L284-L313, F325-V368, G369-S410,

G491-C513, A529-C550

Integrins beta chain cysteine-rich domain signature: R506-G571
PROFILESCAN

Integrin beta subunit signature PR01186: A48-C64, C80-P99,
BLIMPS_PRINTS

Q122-G135, Y150-L168, R190-P209, C223-F242,

D256-R278, R282-D297, D326-T349, L362-Y386, C537-C550

INTEGRIN GLYCOPROTEIN CELL ADHESION
BLAST_PRODOM

TRANSMEMBRANE REPEAT PRECURSOR

EXTRACELLULAR SUBUNIT SIGNAL PD001811: S50-C476

INTEGRIN GLYCOPROTEIN CELL ADHESION
BLAST_PRODOM

TRANSMEMBRANE REPEAT SUBUNIT PRECURSOR

EXTRACELLULAR MATRIX PD149771: P483-L558

INTEGRIN BETA7 SUBUNIT PRECURSOR CELL
BLAST_PRODOM

ADHESION TRANSMEMBRANE GLYCOPROTEIN

REPEAT EXTRACELLULAR PD041877: M1-P49

INTEGRIN CELL ADHESION TRANSMEMBRANE
BLAST_PRODOM

GLYCOPROTEIN REPEAT PRECURSOR SIGNAL BETA

GPD154651: A485-S551

INTEGRINS BETA CHAIN CYSTEINE-RICH DOMAIN
BLAST_DOMO

DM00846|P26010|17-465: G17-L466

DM00846|P32592|3-435: S50-E464

DM00846|S32659|2-437: S50-E464

DM00846|P12607|2-453: A48-L466

EGF-like domain signature 1: C500-C511, C548-C559
MOTIFS

Integrins beta chain cysteine-rich domain signature: C537-C550
MOTIFS

16
7511648CD1
1005
S77 S130 S168
N46 N280 N601
signal_cleavage: M1-A31
SPSCAN

S237 S257 S347
N711

S415 S451 S695

T117

Signal Peptide: M1-A26, M1-A31, Q9-A26, L11-A26
HMMER

Integrin alpha (beta-propellor repeats): L383-D439, M316-L377,
HMMER_SMART

N46-L103, T444-D495, F262-R312

FG-GAP repeat: A317-Y384, G47-T107, G445-L497, G385-T444,
HMMER_PFAM

D263-M316

Integrin alpha cytoplasmic region: K986-D1000
HMMER_PFAM

Cytosolic domain: K986-E1005
TMHMMER

Transmembrane domain: I963-W985

Non-cytosolic domain: M1-P962

Integrins alpha chain IPB000413: E80-A91, G215-G224,
BLIMPS_BLOCKS

G313-P342, R386-P410, W964-R992

Integrins alpha chain signature: V966-E1005
PROFILESCAN

Integrin alpha subunit signature PR01185: D263-F275, E283-T294,
BLIMPS_PRINTS

G313-G333, R386-P410, F448-G469, Q475-Q494,

D591-L604, G973-R992

INTEGRIN PRECURSOR SIGNAL GLYCOPROTEIN
BLAST_PRODOM

ALPHA CELL ADHESION TRANSMEMBRANE

EXTRACELLULAR MATRIX PD001221: S192-V420,

E355-E608, Q707-L952, E957-R992

PLATELET MEMBRANE GLYCOPROTEIN IIB
BLAST_PRODOM

PRECURSOR GPIIB INTEGRIN ALPHA CD41 CELL

ADHESION TRANSMEMBRANE EXTRACELLULAR

MATRIX CYTOSKELETON SIGNAL POLYMORPHISM

DISEASE MUTATION PD175312: N145-F191

INTEGRIN PRECURSOR CELL GLYCOPROTEIN
BLAST_PRODOM

SIGNAL ADHESION TRANSMEMBRANE

EXTRACELLULAR MATRIX CYTOSKELETON

PD001587: A31-E148

INTEGRINS ALPHA CHAIN
BLAST_DOMO

DM01028|P08514|429-913: P36-A70, S318-A341, G429-A914

DM01028|A60163|428-911: P36-A70, 318-A341, G429-A914

DM00458|P08514|40-239: T40-P240

DM01028|P53708|390-888: G429-P877

Integrins alpha chain signature: W985-R992
MOTIFS

17
7511600CD1
330
S19 S32 S104 S169
N123
signal_cleavage: M1-G17
SPSCAN

S185 S315 T75

T284 T320 T323

T326 T330

Dopamine D4 receptor signature PR00569: G140-G157,
BLIMPS_PRINTS

T182-A196, L304-R325

PROTEIN CELL MEMBRANE GLYCOPROTEIN
BLAST_PRODOM

C56G2.7 CHROMOSOME III T9J22.26 PD022277: S15-V85,

E72-I133

PROTEIN CELL MEMBRANE GLYCOPROTEIN
BLAST_PRODOM

C56G2.7 CHROMOSOME III T9J22.26 PD021964: S214-Q299

18
7511783CD1
186
S88 Y69
N64 N87 N98
C2 type Ig domains from SCOP: M1-Y127
HMMER_INCY

Cytosolic domain: F159-P186
TMHMMER

Transmembrane domain: V136-I158

Non-cytosolic domain: M1-M135

Intercellular adhesion molecule/vascular cell adhesion
BLIMPS_PRINTS

molecule PR01472: P20-T33, V37-P52, P52-E70

Intercellular adhesion molecule (ICAM) family sign
BLIMPS_PRINTS

PR01473: P32-C45, G111-Y127

PRECURSOR SIGNAL ADHESION TRANSMEMBRANE
BLAST_PRODOM

INTERCELLULAR IMMUNOGLOBULIN FOLD CELL

GLYCOPROTEIN REPEAT PD005863: E21-C156

INTERCELLULAR ADHESION ICAM2 MOLECULE2
BLAST_PRODOM

PRECURSOR CD102 IMMUNOGLOBULIN FOLD CELL

GLYCOPROTEIN PD024043: F157-P186

IMMUNOGLOBULIN
BLAST_DOMO

DM00001|P13598|115-200: V26-G112

DM00001|P35330|115-199: V26-G112

19
7512383CD1
806
S29 S67 S74 S116
N130 N337
signal_cleavage: M1-A22
SPSCAN

S165 S310 S319

S419 S446 S615

S704 S708 S727

T212 T219 T269

T382 T386 T397

T420 T430 T545

T558 T660 T705

T728 Y135 Y459

Signal Peptide: M1-A17, M1-A19, M1-A20, M1-A22, M1-V24
HMMER

C-type lectin (CTL) or carbohydrate-recognition domain:
HMMER_SMART

C688-R806

Epidermal growth factor-like domain.: D649-D682
HMMER_SMART

Immunoglobulin: D42-K155
HMMER_SMART

Immunoglobulin V-Type: A52-V139
HMMER_SMART

Link (Hyaluronan-binding): N256-R354, K155-A252
HMMER_SMART

EGF-like domain: C650-C681
HMMER_PFAM

Extracellular link domain: G257-F353, G156-Y251
HMMER_PFAM

Immunoglobulin domain: G50-V139
HMMER_PFAM

Lectin C-type domain: T705-L764, E266-T286, Y168-E190
HMMER_PFAM

Link domain IPB000538: G174-G226
BLIMPS_BLOCKS

C-type lectin domain IPB001304: W692-C716, W744-F756
BLIMPS_BLOCKS

Link module signature PR01265: R167-C179, A195-L208,
BLIMPS_PRINTS

V213-C224, Y246-L255

BREVICAN CORE PROTEIN PRECURSOR
BLAST_PRODOM

GLYCOPROTEIN HYALURONIC ACID

PROTEOGLYCAN LECTIN SIGNAL PD022317: L479-V651

BREVICAN CORE PROTEIN PRECURSOR
BLAST_PRODOM

GLYCOPROTEIN HYALURONIC ACID

PROTEOGLYCAN LECTIN SIGNAL PD021260: R354-E455

CORE PROTEIN PRECURSOR GLYCOPROTEIN
BLAST_PRODOM

HYALURONIC ACID PROTEOGLYCAN EGFLIKE

DOMAIN REPEAT PD150847: D28-V154

GLYCOPROTEIN PRECURSOR PROTEIN
BLAST_PRODOM

PROTEOGLYCAN SIGNAL REPEAT CORE EGFLIKE

DOMAIN IMMUNOGLOBULIN PD000918: G156-Y251,

K267-F353

COMPLEMENT FACTOR H REPEAT
BLAST_DOMO

DM00260|A54423|142-252: G142-E253, G238-D355

DM00260|P41725|142-252: G142-E253, G238-D355

DM00260|A53908|141-251: G142-E253, G238-D355

DM00260|A54423|254-355: K153-E253, D254-S356

EGF-like domain signature 1: C670-C681
MOTIFS

EGF-like domain signature 2: C670-C681
MOTIFS

Immunoglobulins and major histocompatibility complex
MOTIFS

proteins signature: Y135-H141

Link domain signature: C179-C224, C277-C322
MOTIFS

20
7512813CD1
32
S28

signal_cleavage: M1-H18
SPSCAN

Cadherins extracellular repeated domain signature: E4-S28
PROFILESCAN

21
7512842CD1
142
S6 S88 S127 T31
N39 N118
signal_cleavage: M1-L21
SPSCAN

Signal Peptide: M1-A20, M1-W22, M1-A26
HMMER

Immunoglobulin: S32-L137
HMMER_SMART

Immunoglobulin V-Type: D42-Y125
HMMER_SMART

Immunoglobulin domain: G40-Y125
HMMER_PFAM

V type Ig domains: V27-H142
HMMER_INCY

Ig superfamily: E28-L128
HMMER_INCY

Cytosolic domain: M1-R8
TMHMMER

Transmembrane domain: A9-T31

Non-cytosolic domain: S32-H142

EPITHELIAL V-LIKE ANTIGEN PRECURSOR SIGNAL
BLAST_PRODOM

PD178398: M1-A26 (P = 1.1E−08)

PRECURSOR GLYCOPROTEIN SIGNAL CHANNEL
BLAST_PRODOM

TRANSMEMBRANE IMMUNOGLOBULIN FOLD

PROTEIN MYELIN SODIUM PD013099: I29-C123

IMMUNOGLOBULIN
BLAST_DOMO

DM00001|P10522|3-108: I29-C123

DM00001|P37301|32-137: I29-C123

DM00001|P20938|30-135: I29-C123

DM00001|A57843|32-137: L35-C123

22
90190613CD1
3317
S30 S94 S187 S195
N632 N847 N1182
signal_cleavage: M1-Q31
SPSCAN

S223 S241 S410
N1222 N1317

S498 S546 S659
N1327 N1649

S834 S866 S881
N1713 N1770

S883 S984 S1031
N2053 N2182

S1181 S1231 S1348
N2201 N2391

S1367 S1579 S1631
N2479 N2511

S1660 S1661 S1686
N2702 N2751

S1738 S1830 S1904
N2813 N3259

S1935 S1995 S2132

S2341 S2374 S2378

S2387 S2532 S2568

S2571 S2682 S2725

S2838 S2841 S2849

S2866 S2897 S2899

S2904 S2916 S2938

S2975 S2999 S3072

S3300

T201 T251 T267

Signal Peptide: M2-E32
HMMER

T308 T404 T422

T441 T458 T528

T534 T568 T570

T745 T757 T779

T832 T853 T932

T945 T986 T1021

T1090 T1224

T1247 T1276

T1305 T1434

T1466 T1489

T1842 T2085

T2203 T2235

T2501 T2728

T2882 T2983

T2998 T3292 Y400

Y450 Y843 Y2318

Cadherin repeats: I673-P754, L455-P543, I983-P1065, I880-P959,
HMMER_SMART

V347-P431, V567-P649, I1089-P1167, V778-P856,

I1195-L1271

Epidermal growth factor-like domain: L1438-E1471, F1725-Q1758,
HMMER_SMART

A1949-V1982, A1984-E2020, V1378-E1433,

R1478-E1514, P2039-N2108

Calcium-binding EGF-like domain: E1435-E1471, C1479-E1514,
HMMER_SMART

N1948-V1982, C1726-Q1758, D1983-E2020

G-protein-coupled receptor proteolytic site: S2481-R2534
HMMER_SMART

Domain present in hormone receptors: L2125-S2192
HMMER_SMART

Laminin G domain: F1535-R1702, V1785-T1924
HMMER_SMART

7 transmembrane receptor (Secretin family): L2541-A2783
HMMER_PFAM

EGF-like domain: C1726-C1757, C1479-C1513, C1985-C2019,
HMMER_PFAM

C1439-C1470, C1950-C1981, R1420-C1432, C1379-V1393

Latrophilin/CL-1-like GPS domain: S2481-R2534
HMMER_PFAM

Hormone receptor domain: Y2126-F2188
HMMER_PFAM

Cadherin domain: Y550-V642, F656-L747, Y966-Q1058,
HMMER_PFAM

Y438-L536, Y330-A424, F1072-V1160, Y863-N952, Y761-T849,

P1188-I1266

Laminin G domain: F1543-D1705, F1793-G1927
HMMER_PFAM

Cytosolic domains: S2568-G2578, R2634-G2645, R2711-R2730
TMHMMER

Transmembrane domains: A2545-L2567, I2579-T2601,

A2611-Y2633, A2646-L2668, I2688-A2710, S2731-V2750

Non-cytosolic domains: M1-L2544, H2602-V2610, D2669-L2687,

N2751-S3317

G-protein coupled receptors family 2 (secretin-like)
BLIMPS_BLOCKS

IPB000832: C2129-A2156, V2551-L2596, C2607-L2632,

G2654-F2678, W2689-Q2718, S2725-G2746, G2766-R2794

Laminin-type EGF-like (LE) domain IPB002049: R1420-D1430,
BLIMPS_BLOCKS

S2034-V2044, S2086-C2096, C2098-F2114

Cadherin domain IPB002126: D1005-E1028
BLIMPS_BLOCKS

Cadherins extracellular repeated domain signature: V403-I452,
PROFILESCAN

V512-V564, L618-V670, G725-V775, A930-V980,

V1032-V1086

Type II EGF-like signature PR00010: E1435-N1446, G1487-P1494,
BLIMPS_PRINTS

G1742-F1752

Cadherin signature PR00205: E374-R393, F545-K574, E615-R627,
BLIMPS_PRINTS

P735-P754, P856-E869, D909-D935, S1151-V1168

MEGF2 CELL ADHESION GLYCOPROTEIN
BLAST_PRODOM

TRANSMEMBRANE CALCIUM-BINDING REPEAT

PD185010: S260-F282, W2787-S3317

PD182204: Q31-L333

TRANSMEMBRANE SEVEN PASS RECEPTOR
BLAST_PRODOM

PRECURSOR SIGNAL MEGF2 CELL ADHESION

GLYCOPROTEIN CALCIUM-BINDING

PD155621: M1761-V1946

PD155993: E2246-E2407

HORMONE; EMR1; LEUCOCYTE; ANTIGEN;
BLAST_DOMO

DM05221|A57172|465-886: M2444-R2784

DM05221|I37225|347-738: R2480-L2792

DM05221|P48960|347-738: R2480-L2792

G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO

DM00378|P47866|1-405: Y2126-W2175 V2554-A2786

Aspartic acid and asparagine hydroxylation site: C1961-C1972
MOTIFS

Cadherins extracellular repeated domain signature: V421-P431,
MOTIFS

I533-P543, I639-P649, V744-P754, V949-P959,

V1055-P1065, V1157-P1167

EGF-like domain signature 1: C1421-C1432, C1459-C1470,
MOTIFS

C1746-C1757, C1970-C1981, C2008-C2019, C2096-C2107

EGF-like domain signature 2: C1421-C1432, C1746-C1757,
MOTIFS

C1970-C1981, C2008-C2019

Laminin-type EGF-like (LE) domain signature: C2096-C2122
MOTIFS

23
7511894CD1
638
S3 S23 S56 S111
N85 N89 N175
signal_cleavage: M1-I24
SPSCAN

S589 T63 T145
N181 N466

T177 T332 T503

T552 T567 T606

Y50

Signal Peptide: M1-Q21, M1-V22, M1-I24, M1-S27, M1-I25,
HMMER

M1-Q30

Cadherin repeats: M191-P277, F422-P502, L300-A399, P76-P167
HMMER_SMART

Cadherin domain: F174-Q270, F405-S495, Y284-Q390,
HMMER_PFAM

R509-C599, E116-S160

Cytosolic domain: M1-H6
TMHMMER

Transmembrane domain: P7-I25

Non-cytosolic domain: D26-C638

Cadherin domain IPB002126: L53-N85, D438-E461, T485-L494
BLIMPS_BLOCKS

Cadherins extracellular repeated domain signature: T246-L298,
PROFILESCAN

F469-L522

Cadherin signature PR00205: L109-R128, F169-E198, Q244-E256,
BLIMPS_PRINTS

S258-P277, P277-E290, N337-N363, A486-T503

CADHERIN REPEAT
BLAST_DOMO

DM00030|P33150|392-505: T310-S429

DM00030|P55290|392-505: T310-S429

DM00030|P33147|413-526: T310-D428

DM00030|P55291|290-403: W313-S429

Cadherins extracellular repeated domain signature: I157-P167,
MOTIFS

L492-P502

24
3604804CD1
4560
S85 S132 S189
N49 N342 N482
Signal Peptide: M5-S30, M5-G32, V9-G32
HMMER

S216 S384 S412
N563 N668 N800

S461 S495 S497
N880 N899 N1007

S529 S538 S601
N1368 N1430

S617 S677 S682
N1755 N1948

S813 S862 S901
N1997 N2000

S942 S1025 S1051
N2212 N2296

S1072 S1081 S1092
N2335 N2471

S1130 S1161 S1178
N3004 N3205

S1209 S1232 S1294
N3333 N3453

S1314 S1315 S1374
N3622 N3745

S1404 S1499 S1505
N3930 N4186

S1574 S1643 S1780
N4191 N4205

S1808 S1868 S1909
N4472

S1924 S1993 S2153

S2197 S2247 S2422

S2427 S2438 S2473

S2611 S2649 S2728

S2820 S2860 S2914

S2950 S3021 S3047

S3055 S3172 S3207

S3280 S3297 S3491

S3529 S3616 S3624

S3660 S3687 S3747

S3819 S3857 S3911

S3935 S3939 S3972

S4007 S4250 S4273

S4300 S4304 S4331

S4335 S4349 S4369

S4389 S4516 S4534

S4538

T51 T71 T166

Cadherin repeats: V3156-P3237, V1065-P1146, V3261-P3342,
HMMER_SMART

T181 T183 T249

V2839-P2925, I1170-P1252, V494-P576, L3366-P3447,

T286 T431 T437

V853-P934, L958-H1039, I1801-P1884, V2312-P2395,

T448 T530 T565

V3054-P3132, I748-P829, I1696-P1770, I1482-P1564,

T654 T656 T764

L2949-P3030, V180-P264, V2521-P2601, I2211-P2288,

T802 T921 T1009

V2007-P2085, V1588-P1672, V394-P470, V2419-P2497,

T1066 T1101

I1380-P1458, L3471-P3552, V1908-L1983, A1278-P1354,

T1121 T1184

I70-P156, G2727-P2815, V600-H673, V2109-P2187,

T1208 T1225

V2625-P2709

T1328 T1342

T1361 T1370

T1468 T1485

T1589 T1665

T1728 T1804

T1822 T1911

T1943 T2110

Cadherin domain: Y3244-T3335, Y3349-S3440, Y3139-L3230,
HMMER_PFAM

T2173 T2214

V1048-E1139, Y1153-L1245, Y941-V1032, Y836-D927,

T2260 T2298

Y477-G569, Y2504-V2594, Y1465-E1557, Y2822-T2918,

T2351 T2364

Y1679-V1763, Y1777-E1875, Y163-E257, Y3037-E3125,

T2524 T2558

Y1571-T1665, Y2194-N2281, Y2295-S2388, S731-E822,

T2588 T2719

Y1259-I1350, Y2092-V2182, Y1990-E2078, Y2932-S3023,

T2732 T2767

Y2402-L2490, Y2608-L2702, Y3454-I3545, C583-L672,

T2880 T3006

Y1367-L1451, F1886-K1976, Y2716-L2808, F3560-E3641,

T3228 T3247

Y381-E463, Y48-L149

T3264 T3270

T3474 T3549

T3563 T3579

T3653 T3675

T3682 T3772

T4056 T4097

T4278 Y506 Y1000

Y1080 Y1276

Y1545 Y1966

Y2020 Y2468

Y2822 Y4397

Epidermal growth factor-like domain: A4027-E4061, E4104-G4137,
HMMER_SMART

A4066-E4099, P3801-S3836

Calcium-binding EGF-like domain: D4101-G4137, D4026-E4061,
HMMER_SMART

E4063-E4099

Laminin G domain: E3860-N3997
HMMER_SMART

EGF-like domain: C4105-C4136, C4067-C4098, C4028-C4060,
HMMER_PFAM

C3802-C3835

Laminin G domain: L3868-P4000
HMMER_PFAM

Cytosolic domain: R4179-V4560
TMHMMER

Transmembrane domain: I4159-F4178

Non-cytosolic domain: M1-L4158

Cadherin domain IPB002126: S3176-E3199, S3430-I3439,
BLIMPS_BLOCKS

E4315-M4363

Cadherins extracellular repeated domain signature: T1117-V1167,
PROFILESCAN

V1224-G1272, L1742-V1791, L2259-V2309,

V2367-V2416, F2784-L2836, F2892-V2946, I3206-V3258,

V3313-V3363, L3418-I3468

Cadherin signature PR00205: V418-T437, F472-K501,
BLIMPS_PRINTS

E1219-P1231, S3218-P3237, P3342-E3355, V3397-D3423,

S3431-V3448

CADHERIN-RELATED TUMOR SUPPRESSOR
BLAST_PRODOM

HOMOLOG PRECURSOR FAT PROTEIN HOMOLOG

CELL ADHESION SIGNAL GLYCOPROTEIN

TRANSMEMBRANE CALCIUM-BINDING REPEAT

EGF-LIKE DOMAIN

PD140312: V4153-V4560

PD131818: V2088-A2196

CELL ADHESION GLYCOPROTEIN
BLAST_PRODOM

TRANSMEMBRANE CALCIUM-BINDING REPEAT

HOMOLOG PROTEIN MEGF1 CADHERIN-RELATED

PD152384: F41-T166

CADHERIN REPEAT
BLAST_DOMO

DM00030|P33450|3576-3680: P3375-S3478, G3269-S3373,

G756-D859, N2322-S2426, R1074-D1176, G966-S1072,

N758-L860, G3164-D3267, N3271-D3372

DM00030|P08641|300-409: N3376-S3478, N2849-D2955

Aspartic acid and asparagine hydroxylation site: C4116-C4127
MOTIFS

Cadherins extracellular repeated domain signature: I460-P470,
MOTIFS

I819-P829, I1136-P1146, V1242-P1252, I1448-P1458,

V1554-P1564, I1760-P1770, I1874-P1884, V2075-P2085,

L2278-P2288, I2385-P2395, V2591-P2601, I2805-P2815,

V2915-P2925, V3020-P3030, L3122-P3132, I3227-P3237,

I3332-P3342, I3437-P3447

EGF-like domain signature 1: C4049-C4060, C4087-C4098,
MOTIFS

C4125-C4136

EGF-like domain signature 2: C4125-C4136
MOTIFS

Calcium-binding EGF-like domain pattern signature: D4101-C4125
MOTIFS

25
7512568CD1
330
S45 S112 S131
N25 N57 N100
signal_cleavage: M1-A20
SPSCAN

S182 S184 S190
N110 N120 N224

S209 S260 S285

T27 T76 T163

T174 T314 Y169

Signal Peptide: M1-A20
HMMER

Link (Hyaluronan-binding): F30-N120
HMMER_SMART

Extracellular link domain: A31-F119
HMMER_PFAM

Cytosolic domain: R261-V330
TMHMMER

Transmembrane domain: W238-S260

Non-cytosolic domain: M1-E237

Link domain IPB000538: T226-A278
BLIMPS_BLOCKS

CD44 antigen precursor signature PR00658: D23-Y42, G73-P93,
BLIMPS_PRINTS

Y114-D134, N149-I168, E220-L239, I240-R262, C264-K283,

D310-G329

Link module signature PR01265: R41-C53, A69-I82, V86-C97,
BLIMPS_PRINTS

Y114-A123

CD44 GLYCOPROTEIN ANTIGEN PRECURSOR
BLAST_PRODOM

PHAGOCYTIC I PGP1 HUTCHI EXTRACELLULAR

MATRIX

PD006761: V254-V330

PD004309: N120-S227, Y161-W238

GLYCOPROTEIN PRECURSOR PROTEIN
BLAST_PRODOM

PROTEOGLYCAN SIGNAL REPEAT CORE EGFLIKE

DOMAIN IMMUNOGLOBULIN PD000918: V33-F119

CD44; LONG; SURFACE; ADHESION;
BLAST_DOMO

DM03720|S24631|462-698: V178-V330

DM03720|A53286|153-365: Y161-V330

DM03720|B38745|157-502: T163-V330

DM03720|P20944|155-361: R90-P124, S187-V330

Link domain signature: C53-C97
MOTIFS

26
7512812CD1
681
S80 S144 S176
N48 N97 N260
Signal Peptide: M1-G21
HMMER

S270 S297 S366
N387 N396 N463

S373 S407 S464
N471 N541 N664

S501 S512 S589

S628 T30 T151

T188 T189 T305

T579 T661 T666

Integrin beta subunits (N-terminal portion): T30-C454
HMMER_SMART

Domain found in Plexins, Semaphorins and Integrins: G22-Q71
HMMER_SMART

Integrins, beta chain: T30-C454
HMMER_PFAM

Cytosolic domain: K624-C681
TMHMMER

Transmembrane domain: I601-W623

Non-cytosolic domain: M1-N600

Integrin beta, C-terminus IPB001169: R58-I75, L121-K161,
BLIMPS_BLOCKS

E162-C197, I213-H264, L266-L295, L307-V350, G351-A392,

G470-C492, K503-C524

Integrins beta chain cysteine-rich domain signature: G487-G549
PROFILESCAN

Type III EGF-like signature PR00011: G472-C490
BLIMPS_PRINTS

Integrin beta subunit signature PR01186: A28-C44, C59-P78,
BLIMPS_PRINTS

Q103-G116, Y131-L149, R171-P190, C204-F223,

D237-R259, H264-D279, E308-T331, L344-Y368, C511-C524,

P599-G616, G616-E634, T651-T661

INTEGRIN GLYCOPROTEIN CELL ADHESION
BLAST_PRODOM

TRANSMEMBRANE REPEAT PRECURSOR

EXTRACELLULAR SUBUNIT SIGNAL

PD001811: T30-C454

PD001794: S556-Y667

INTEGRIN GLYCOPROTEIN CELL ADHESION
BLAST_PRODOM

TRANSMEMBRANE REPEAT SUBUNIT PRECURSOR

EXTRACELLULAR MATRIX PD149771: D455-Y536

INTEGRIN CELL ADHESION TRANSMEMBRANE
BLAST_PRODOM

GLYCOPROTEIN REPEAT PRECURSOR SIGNAL BETA

G PD154651: N463-Y536

INTEGRINS BETA CHAIN CYSTEINE-RICH DOMAIN
BLAST_DOMO

DM00846|P18564|1-442: M1-A443

DM00846|P18084|7-451: C23-A443

DM00846|P05106|9-449: C23-A443

DM00846|P12607|2-453: Q20-D442

Cell attachment sequence: R514-D516
MOTIFS

Integrins beta chain cysteine-rich domain signature: C511-C524
MOTIFS

27
7512826CD1
614
S58 S93 S290 S315
N56 N418 N449
signal_cleavage: M1-G23
SPSCAN

S369 S471 S561
N467 N508

S571 S582 S595

T107 T249 T298

T469 T495 Y222

Signal Peptide: M1-C17, M1-C18, M1-R20, M1-G23, M1-V24,
HMMER

M1-A28,

Immunoglobulin: P33-Y133, P350-F428, E257-N341, E437-E517,
HMMER_SMART

I148-P244

Immunoglobulin C-2 Type: K263-T327, Q356-I414, K153-K209
HMMER_SMART

Immunoglobulin domain: G358-A409, G265-G322, N445-A501,
HMMER_PFAM

G41-G118, E156-L225

Ig superfamily from SCOP: V346-G416, W253-L325, E29-E138,
HMMER_INCY

M433-E517, I141-P248

b7/CD80/CD86 multiple Ig domain: E29-E255
HMMER_INCY

Cytosolic domain: Y551-H614
TMHMMER

Transmembrane domain: V528-L550

Non-cytosolic domain: M1-G527

GLYCOPROTEIN ANTIGEN PRECURSOR
BLIMPS_PRODOM

IMMUNOGLOBULIN. PD02327: L130-I141, T160-L181

PRECURSOR SIGNAL IMMUNOGLOBULIN FOLD
BLAST_PRODOM

GLYCOPROTEIN TRANSMEMBRANE CELL ANTIGEN

ADHESION RECEPTOR PD004088: F10-L229

PRECURSOR SIGNAL HEMCAM CELL ADHESION
BLAST_PRODOM

SURFACE GLYCOPROTEIN MUC18 MELANOMA-

ASSOCIATED ANTIGEN

PD012722: L323-T469

PD012723: Q478-T567

CELL SURFACE GLYCOPROTEIN MUC18
BLAST_PRODOM

PRECURSOR MELANOMA-ASSOCIATED ANTIGEN

A32 SENDO 1 ENDOTHELIAL ASSOCIATED CD146

MELANOMA ADHESION MOLECULE

IMMUNOGLOBULIN FOLD TRANSMEMBRANE

SIGNAL PD074165: A470-L516

IMMUNOGLOBULIN
BLAST_DOMO

DM00001|P43121|142-233: Q142-H234

DM00001|P43121|434-509: A434-S510

DM00001|P43121|254-330: L254-L331

DM00001|P43121|347-417: R347-N418

28
7512908CD1
385
S220 S311 S315
N75 N153 N237
signal_cleavage: M1-A22
SPSCAN

S340 S347 T155

Y105

Signal Peptide: M1-A22, M1-V25, M1-S28, M1-A26, M1-E27
HMMER

29
7512909CD1
527
S220 S311 S315
N75 N153 N237
signal_cleavage: M1-A22
SPSCAN

S340 S347 S380
N360

S397 S479 S485

S491 S500 S506

T155 T385 T503

Y105

Signal Peptide: M1-A22, M1-V25, M1-S28, M1-A26, M1-E27
HMMER

Glycosyltransferase family 25 (LPS biosynthesis protein):
HMMER_PFAM

F319-L478

30
7512769CD1
84
T4

signal_cleavage: M1-A19
SPSCAN

Signal Peptide: M1-A19, M1-S21, M1-G23, M1-F25, M1-G29,
HMMER

M1-A20

Cytosolic domain: M1-R6
TMHMMER

Transmembrane domain: A7-G29

Non-cytosolic domain: Q30-C84

MATRIX PROTEIN EXTRACELLULAR PRECURSOR
BLAST_PRODOM

SECRETORY COMPONENT P85 SIGNAL

GLYCOPROTEIN REPEAT PD014357: M1-P81

31
7512871CD1
311
S44 S222 S300 T32
N290
signal_cleavage: M1-A27
SPSCAN

Y126 Y232

Signal Peptide: M8-A27, M8-A29, M8-T32, M1-A29
HMMER

Fibrinogen-related domains (FReDs): T113-A311
HMMER_SMART

Collagen triple helix repeat (20 copies): L50-S108
HMMER_PFAM

Fibrinogen beta and gamma chains, C-terminal: G114-A311
HMMER_PFAM

Fibrinogen beta and gamma chains C-terminal globular
BLIMPS_BLOCKS

IPB002181: A59-I69, L122-L136, V144-G180, E185-T197,

L204-Y220, Y232-Q246, N254-E283, A286-P310

Fibrinogen beta and gamma chains C-terminal domain
PROFILESCAN

signature: D245-G296

PRECURSOR GLYCOPROTEIN SIGNAL FIBRINOGEN
BLAST_PRODOM

BLOOD COAGULATION CHAIN PLASMA PROTEIN

PLATELET PD001241: T113-A244, D245-P310

FICOLIN PD168236: G52-C111
BLAST_PRODOM

FIBRINOGEN BETA/GAMMA
BLAST_DOMO

DM00531|S61517|64-326: G64-A311

DM00531|B47172|64-326: G64-R309

DM00531|JN0596|27-305: E95-W264, D245-P310

DM00531|A45445|1099-1341: P115-P310

Fibrinogen beta and gamma chains C-terminal domain
MOTIFS

signature: W263-G275

Lipocalin signature: N254-A266
MOTIFS

TABLE 4

Polynucleotide

SEQ ID NO:/

Incyte ID/Sequence

Length
Sequence Fragments

32/7506690CB1/
1-652, 1-724, 1-779, 1-812, 1-3988, 9-571, 10-735, 26-733, 26-778,

4000
226-641, 226-781, 282-907, 308-554, 340-907, 341-820, 371-907, 391-731,

411-731, 421-934, 421-1091, 455-728, 783-1408, 787-1510, 965-1580,

965-1669, 965-1679, 965-1724, 985-1646, 994-1444, 999-1907,

1008-1664, 1032-1685, 1090-1701, 1115-1886, 1160-1907, 1184-1536,

1210-1907, 1257-1907, 1259-1906, 1278-1847, 1283-2114,

1284-2114, 1285-2114, 1326-1905, 1327-1584, 1327-1768,

1329-2114, 1337-1907, 1341-1907, 1344-2114, 1372-1907,

1379-2114, 1401-1926,

1450-2106, 1462-2054, 1493-2106, 1535-2060, 1589-2247,

1768-2171, 1926-2277, 1997-2454, 1997-2467, 1997-2505, 1997-2515,

1997-2541, 2052-2651, 2105-2386, 2162-2836, 2167-2836, 2238-2752,

2257-2524, 2257-2676, 2257-2758, 2257-2799, 2257-2919, 2267-2391,

2267-2517, 2267-2535, 2267-2795, 2267-2844, 2269-2795, 2286-2962,

2350-2516, 2373-2457, 2460-2659, 2465-3044, 2502-2876,

2505-2597, 2609-2917, 2614-3012, 2791-3004, 2893-3049, 3046-3239,

3065-3231, 3086-3239, 3191-3280, 3232-3670, 3468-3991,

3469-4000, 3549-3699, 3670-4000, 3768-4000

33/7506536CB1/
1-505, 1-711, 10-483, 10-1448, 18-651, 101-651, 217-372, 270-472,

1448
327-641, 327-893, 335-581, 417-1023, 492-697, 539-856, 552-863,

593-834, 650-1091, 763-1050, 763-1213, 774-1005, 774-1045, 774-1200,

774-1217, 778-1041, 787-1083, 914-1403, 914-1407, 967-1202,

968-1433, 1144-1382, 1243-1433

34/7506537CB1/
1-590, 1-699, 1-792, 1-1331, 60-759, 176-331, 229-376,

1430
374-669, 374-676, 375-677, 406-647, 463-904, 576-863, 576-1065, 587-818, 587-858,

587-1013, 587-1047, 587-1065, 587-1127, 587-1133, 587-1187, 587-1218,

587-1219, 587-1236, 587-1237, 587-1245, 587-1247, 587-1249,

587-1290, 587-1305, 587-1315, 587-1317, 591-854, 600-896, 682-1269,

682-1318, 690-1346, 715-1057, 769-1208, 780-1015, 794-1089,

795-1092, 824-1108, 830-1430, 970-1216, 974-1114, 1141-1369

35/7506655CB1/
1-616, 5-2437, 17-500, 17-608, 17-631, 23-471, 31-250, 32-324, 67-253,

2529
73-645, 76-299, 76-314, 96-209, 118-484, 126-845, 131-382, 132-720,

133-794, 138-249, 143-401, 145-434, 147-582, 153-835, 160-620, 163-408,

177-474, 180-386, 180-435, 180-439, 189-424, 191-447,

192-370, 192-509, 193-458, 199-744, 203-770, 211-506, 211-616, 221-378,

223-883, 225-331, 229-596, 247-694, 259-482, 261-918, 262-535,

262-715, 262-723, 262-745, 262-818, 262-846, 262-851, 262-878, 262-933,

262-947, 262-950, 262-967, 264-934, 267-961, 273-590,

295-884, 308-524, 308-582, 310-557, 312-451, 312-582, 317-563, 332-966,

335-966, 338-458, 346-595, 346-907, 346-1164, 349-1140, 354-588,

360-447, 364-653, 364-665, 366-628, 367-606, 376-672, 380-967, 386-581,

388-966, 391-914, 395-633, 398-619, 398-656, 407-636,

416-709, 429-687, 431-651, 431-706, 435-815, 435-839, 442-710, 448-967,

454-756, 456-706, 458-712, 475-753, 479-725, 479-740, 479-757,

480-943, 493-691, 497-781, 503-835, 503-883, 503-920, 503-957, 503-961,

503-967, 504-733, 506-742,

506-746, 509-902, 518-775, 519-743, 521-760, 522-863, 523-804, 527-754,

529-662, 530-967, 534-835, 538-835, 539-720, 544-967, 551-637,

555-831, 561-821, 562-861, 571-850, 581-967, 586-902, 590-789, 611-904,

616-896, 616-906, 622-907, 625-885, 636-908, 638-785,

645-963, 645-967, 648-941, 650-907, 655-848, 655-884, 663-757, 663-875,

663-907, 663-964, 666-934, 668-955, 672-823, 675-916, 677-936,

681-916, 687-954, 687-960, 689-967, 691-945, 698-967, 702-967, 707-963,

710-967, 716-967, 730-943, 735-939, 739-967, 743-962,

794-893, 796-967, 849-967, 862-967, 873-967, 963-1374, 963-1457, 963-1500, 963-1520,

963-1529, 963-1559, 965-1081, 967-1224, 972-1686,

973-1233, 974-1815, 986-1255, 989-1235, 1006-1175, 1018-1173, 1018-1250,

1022-1252, 1029-1303, 1035-1703, 1046-1520, 1056-1348,

1064-1285, 1066-1370, 1072-1336, 1074-1325, 1078-1539, 1086-1333, 1098-1669,

1099-1627, 1101-1259, 1101-1336, 1104-1391,

1106-1668, 1110-1333, 1114-1664, 1117-1416, 1117-1422, 1120-1755, 1121-1241,

1121-1409, 1134-1382, 1135-1712,

1139-1422, 1141-1341, 1145-1735, 1154-1398, 1164-1460, 1165-1412, 1171-1429,

1171-1740, 1175-1448, 1181-1621, 1184-1476, 1187-1429,

1187-1487, 1192-1449, 1192-1746, 1192-1760, 1193-1897, 1206-1385, 1206-1439,

1208-1475, 1208-1485, 1208-1532, 1208-1726,

1212-1486, 1212-1492, 1212-1550, 1213-1785, 1224-1809, 1229-1543, 1232-1818,

1233-1555, 1235-1529, 1237-1691, 1238-1540, 1242-1440,

1245-1518, 1246-1744, 1247-1557, 1248-1885, 1250-1498, 1253-1700, 1256-1880,

1257-1897, 1260-1529, 1260-1540, 1262-1555,

1262-1560, 1262-1726, 1263-1528, 1263-1831, 1266-1630, 1277-1402, 1277-1500,

1277-1791, 1283-1562, 1290-1575, 1300-1557, 1300-1595,

1308-1565, 1313-1606, 1314-1558, 1319-1571, 1323-1515, 1323-1587, 1324-1598,

1329-1601, 1329-1758, 1331-1915, 1333-1556,

1335-1433, 1335-1574, 1335-1649, 1335-1795, 1336-1938, 1337-2009, 1338-1564,

1338-1595, 1341-1536, 1341-1582, 1341-1585, 1341-1627,

1341-1979, 1342-1978, 1343-1559, 1343-1606, 1348-1945, 1354-1599, 1354-1625,

1355-1605, 1356-1820, 1356-2137, 1362-1563,

1363-1898,

1369-1628, 1370-1649, 1371-1733, 1373-1644, 1375-1713, 1375-1837, 1383-1663,

1383-1692, 1383-1868, 1387-1573, 1390-1662, 1392-1636,

1395-1985, 1406-1677, 1406-1994, 1408-1864, 1409-1921, 1414-2040, 1414-2069,

1415-1921, 1416-1656, 1418-1834, 1419-1884,

1423-1694, 1423-1712, 1425-1876, 1431-1697, 1432-2029, 1434-1684, 1435-2015,

1438-1722, 1439-2037, 1442-1902, 1443-1964, 1447-1759,

1449-1726, 1450-1833, 1452-2089, 1460-1776, 1467-2074, 1468-1729, 1468-1947,

1470-1936, 1472-1941, 1474-1726, 1474-1735,

1475-1749, 1476-1590, 1483-1734, 1485-1775, 1485-2127, 1494-1755, 1495-1752,

1499-1746, 1499-2019, 1501-1640, 1509-1935, 1514-1713,

1514-1809, 1519-1634, 1519-1771, 1519-1788, 1519-1802, 1519-1821, 1519-2044,

1519-2055, 1522-1718, 1522-1772, 1522-2133,

1530-1769, 1530-1771, 1530-1772, 1530-1980, 1531-1816, 1531-2130, 1532-1813,

1532-1826, 1533-1777, 1535-1823, 1535-1993, 1536-1772,

1536-1783, 1536-1810, 1537-1765, 1537-1773, 1537-1780, 1537-1792, 1538-1846,

1541-2071, 1543-1779, 1543-2002, 1543-2094,

1546-2120,

1546-2126, 1548-1825, 1548-1975, 1550-2049, 1551-1841, 1551-2101, 1555-1823,

1555-1971, 1556-1845, 1556-2094, 1562-1818, 1563-1932,

1565-2039, 1566-1853, 1567-1839, 1576-1822, 1576-1830, 1578-1822, 1578-1839,

1578-1841, 1579-1847, 1579-1905, 1579-2099,

1581-1863, 1581-2174, 1582-1800, 1582-1833, 1584-1829, 1585-1837, 1587-1829,

1597-1842, 1598-1866, 1599-1748, 1599-2418, 1600-2132,

1616-1914, 1619-1791, 1622-2118, 1624-1945, 1624-2118, 1626-1866, 1632-2102,

1634-1910, 1635-1887, 1641-2096, 1644-2309,

1646-1904, 1652-2194, 1653-1934, 1656-1931, 1664-1779, 1669-1938, 1670-2250,

1671-1907, 1671-1957, 1671-2256, 1673-2179, 1673-2256,

1675-1940, 1675-2179, 1679-1939, 1683-2182, 1684-1929, 1686-1932, 1686-2018,

1688-1987, 1694-1958, 1695-1937, 1700-1946,

1706-1971, 1709-1959, 1709-1975, 1711-1972, 1713-1994, 1723-1881, 1723-1954,

1723-1978, 1724-1989, 1724-2214, 1730-2074, 1734-1970,

1734-1993, 1734-2015, 1739-2030, 1741-2125, 1745-2238, 1748-2076, 1751-2032,

1753-2057, 1753-2074, 1770-2304, 1778-1984,

1781-2050,

1781-2052, 1784-2093, 1794-2035, 1794-2077, 1794-2092, 1803-2081, 1804-2011,

1804-2242, 1804-2293, 1805-2041, 1806-1984, 1806-2253,

1807-2067, 1812-2041, 1818-2410, 1820-2005, 1822-2055, 1822-2351, 1824-2069,

1824-2366, 1825-2123, 1826-1945, 1832-2089,

1843-2371, 1844-2147, 1844-2294, 1847-2090, 1848-2113, 1848-2150, 1852-2102,

1853-2071, 1853-2094, 1853-2108, 1853-2117, 1858-2128,

1859-2333, 1861-2339, 1862-2071, 1862-2074, 1862-2160, 1863-2027, 1863-2173,

1865-2448, 1867-2102, 1867-2118, 1867-2225,

1868-2169, 1875-2135, 1876-2119, 1877-2134, 1877-2146, 1877-2396, 1880-2057,

1880-2130, 1880-2185, 1881-2143, 1886-2126, 1886-2133,

1890-2422, 1897-2131, 1903-2208, 1904-2451, 1910-2437, 1913-2362, 1914-2067,

1915-2409, 1921-2109, 1922-2170, 1922-2182,

1925-2226, 1933-2425, 1942-2423, 1943-2441, 1944-2122, 1944-2129, 1944-2441,

1948-2094, 1950-2119, 1950-2196, 1950-2423, 1961-2437,

1961-2444, 1962-2425, 1965-2440, 1969-2133, 1969-2194, 1973-2440, 1974-2271,

1975-2425, 1976-2423, 1978-2431, 1981-2402,

1982-2420,

1982-2427, 1984-2239, 1985-2421, 1986-2455, 1999-2370, 2000-2161, 2000-2443,

2001-2133, 2002-2449, 2003-2420, 2004-2236, 2004-2440,

2005-2421, 2005-2422, 2005-2447, 2006-2364, 2006-2422, 2007-2420, 2007-2443,

2011-2427, 2011-2449, 2012-2237, 2012-2263,

2012-2269, 2012-2424, 2012-2426, 2014-2423, 2015-2422, 2018-2440, 2022-2117,

2024-2421, 2027-2376, 2028-2422, 2029-2315, 2030-2324,

2030-2420, 2031-2422, 2032-2423, 2033-2443, 2034-2422, 2035-2442, 2036-2171,

2036-2419, 2040-2420, 2044-2422, 2047-2419,

2048-2422, 2048-2429, 2055-2423, 2056-2333, 2056-2419, 2057-2422, 2065-2419,

2068-2437, 2070-2321, 2070-2422, 2080-2439, 2085-2422,

2086-2417, 2087-2422, 2090-2424, 2091-2420, 2091-2424, 2093-2370, 2094-2420,

2098-2347, 2098-2422, 2099-2422, 2100-2419,

2100-2440, 2123-2386, 2125-2360, 2130-2422, 2134-2418, 2134-2420, 2136-2415,

2136-2419, 2136-2422, 2137-2418, 2143-2404, 2145-2388,

2151-2419, 2154-2443, 2162-2392, 2168-2431, 2169-2434, 2172-2448, 2175-2434,

2183-2274, 2191-2422, 2191-2432, 2191-2442,

2193-2431,

2198-2395, 2198-2436, 2200-2373, 2200-2435, 2202-2422, 2205-2422, 2210-2402,

2216-2419, 2224-2432, 2230-2420, 2233-2419, 2235-2455,

2239-2419, 2247-2415, 2247-2432, 2250-2457, 2251-2420, 2253-2422, 2253-2426,

2257-2423, 2277-2529, 2308-2498, 2310-2419,

2325-2417, 2338-2514, 2348-2461, 2348-2477, 2359-2467

36/7506656CB1/
1-616, 5-2763, 17-500, 17-608, 17-631, 23-471, 31-250, 32-324, 67-253,

2763
73-645, 76-299, 76-314, 96-209, 118-484, 126-796, 131-382, 132-720,

133-794, 138-249, 143-401, 145-434, 147-582, 160-620, 163-408, 177-474,

180-386, 180-435, 180-439, 189-424, 191-447, 192-370,

192-509, 193-458, 199-744, 203-770, 211-506, 211-616, 221-378, 225-331,

229-596, 247-694, 259-482, 262-535, 262-715, 262-723, 262-745,

262-799, 273-590, 308-524, 308-582, 309-988, 310-557, 312-451, 312-582,

317-563, 338-458, 346-595, 354-588, 360-447, 364-653,

364-665, 366-628, 367-606, 376-672, 386-581, 395-633, 398-619, 398-656,

407-636, 416-709, 429-687, 431-651, 431-706, 435-799, 442-710,

454-756, 456-706, 458-712, 475-753, 479-725, 479-740, 479-757, 493-691,

497-781, 504-733, 506-742, 506-746, 518-775, 519-743,

521-760, 522-799, 523-799, 527-754, 529-662, 539-720, 551-637, 568-1028,

586-789, 590-789, 611-799, 638-785, 655-799, 663-757, 793-1277,

797-985, 797-997, 797-1011, 797-1013, 797-1028, 797-1037, 797-1043, 797-1045,

797-1053, 797-1059,

797-1070, 797-1082, 797-1092, 797-1137, 797-1157, 797-1164, 797-1298, 797-1452,

797-1518, 797-1603, 803-1061, 803-1079, 803-1445,

806-924, 809-1521, 812-1090, 813-1051, 813-1060, 813-1241, 813-1487, 814-1053,

814-1061, 814-1092, 814-1272, 816-1065, 816-1105,

817-920, 817-1095, 817-1352, 817-1440, 818-1436, 818-1503, 823-1282, 827-1352,

832-1481, 833-1081, 834-1315, 840-1150, 842-1257,

842-1475, 845-1103, 845-1127, 845-1433, 846-1097, 846-1416, 847-1115, 855-1139,

855-1150, 855-1166, 856-1109, 856-1150, 856-1431,

858-1300, 858-1542, 862-1446, 863-1278, 865-1072, 865-1204, 867-1342, 868-1076,

868-1139, 868-1143, 870-1125, 870-1149, 870-1160,

877-1118, 877-1155, 877-1285, 878-1151, 878-1440, 879-1141, 879-1169, 879-1606,

881-1129, 882-1134, 882-1339, 882-1377, 882-1508,

883-1135, 883-1144, 883-1145, 883-1149, 884-1541, 886-1510, 889-1178, 889-1652,

892-1164, 892-1448, 893-1015, 893-1119, 893-1147,

893-1175, 894-1297, 896-1150, 896-1175, 897-1143, 897-1158, 897-1184, 899-1105,

899-1132, 899-1192, 899-1410,

900-1067, 901-1145, 901-1167, 901-1359, 903-1055, 903-1158, 905-1136, 905-1173,

905-1176, 910-1161, 910-1486, 911-1176, 911-1472,

914-1151, 914-1491, 914-1509, 921-1157, 922-1005, 922-1140, 922-1414, 922-1488,

923-1369, 933-1048, 937-1390, 939-1184, 941-1163,

941-1165, 941-1173, 941-1174, 943-1198, 944-1366, 946-1476, 948-1224, 952-1484,

954-1618, 955-1101, 955-1221, 955-1443, 955-1498,

956-1262, 959-1273, 959-1547, 960-1579, 961-1136, 961-1270, 962-1146, 962-1212,

962-1262, 963-1066, 963-1211, 963-1627, 971-1579,

972-1253, 975-1242, 975-1243, 975-1440, 977-1398, 978-1427, 978-1430, 979-1248,

980-1583, 991-1580, 992-1506, 993-1650, 994-1635,

999-1233, 1002-1139, 1002-1214, 1003-1305, 1003-1401, 1005-1511, 1006-1244, 1006-1254,

1008-1482, 1008-1780, 1009-1204, 1009-1267,

1009-1294, 1009-1606, 1010-1274, 1010-1315, 1011-1303, 1012-1275, 1013-1267, 1013-1278,

1013-1485, 1014-1250, 1014-1281,

1015-1542, 1017-1672, 1021-1313, 1023-1522, 1025-1607, 1027-1235, 1027-1294, 1027-1301,

1027-1308, 1030-1279, 1030-1284,

1031-1621, 1039-1324, 1039-1584, 1040-1475, 1041-1546, 1043-1353, 1044-1636, 1047-1329,

1048-1295, 1055-1347, 1057-1456, 1059-1311,

1060-1305, 1065-1270, 1065-1371, 1065-1653, 1068-1655, 1072-1264, 1072-1360, 1072-1518,

1072-1549, 1073-1389, 1077-1229,

1077-1232, 1079-1522, 1079-1644, 1083-1623, 1085-1200, 1085-1355, 1088-1342, 1088-1365,

1088-1655, 1090-1300, 1094-1339, 1096-1395,

1096-1588, 1099-1388, 1103-1384, 1103-1627, 1109-1502, 1109-1561, 1110-1592, 1115-1640,

1117-1374, 1117-1699, 1122-1584,

1122-1595, 1124-1383, 1125-1582, 1126-1401, 1126-1644, 1129-1357, 1129-1512, 1132-1388,

1132-1680, 1133-1632, 1138-1404, 1139-1373,

1139-1414, 1140-1385, 1141-1477, 1141-1487, 1143-1630, 1144-1318, 1144-1602, 1144-1607,

1145-1652, 1147-1385, 1147-1573,

1149-1655, 1150-1416, 1150-1421, 1150-1432, 1150-1637, 1151-1831, 1152-1398, 1152-1434,

1152-1640, 1156-1405, 1156-1442, 1157-1808,

1158-1648, 1159-1405, 1161-1391, 1161-1494, 1162-1449, 1162-1463, 1162-1472, 1163-1420,

1164-1739, 1168-1697, 1169-1641,

1171-1390,

1171-1414, 1171-1434, 1178-1449, 1178-1466, 1179-1963, 1181-1442, 1183-1400, 1186-1336,

1186-1598, 1188-1448, 1190-1476, 1190-1485,

1190-1666, 1192-1430, 1192-1472, 1192-1651, 1195-1433, 1198-1479, 1205-1588, 1207-1436,

1210-1456, 1211-1442, 1211-1481,

1211-1490, 1211-1508, 1211-1551, 1211-1598, 1213-1396, 1213-1509, 1213-1818, 1225-1476,

1225-1518, 1229-1520, 1229-1655, 1233-1629,

1235-1335, 1235-1655, 1240-1499, 1242-1458, 1242-1896, 1248-1433, 1248-1440, 1248-1536,

1249-1540, 1251-1449, 1253-1509,

1253-1562, 1253-1636, 1253-1654, 1255-1461, 1256-1529, 1258-1549, 1258-1589, 1258-1653,

1258-1655, 1260-1502, 1262-1552, 1264-1543,

1269-1538, 1274-1604, 1275-1628, 1276-1545, 1276-1871, 1278-1604, 1278-1632, 1281-1536,

1282-1492, 1282-1511, 1285-1574,

1285-1788, 1290-1496, 1290-1709, 1290-1729, 1290-1795, 1290-1838, 1290-1858, 1290-1867,

1291-1501, 1292-1712, 1294-1558, 1294-1566,

1295-1582, 1295-1583, 1295-1593, 1296-1578, 1297-1530, 1297-1546, 1297-1747, 1297-1786,

1298-1559, 1298-1593, 1299-1540,

1299-1897,

1303-1419, 1305-1559, 1305-1562, 1310-2024, 1311-1571, 1312-1494, 1312-2153, 1321-1636,

1324-1593, 1327-1573, 1335-1589, 1338-1553,

1341-1636, 1344-1513, 1345-1595, 1352-1655, 1354-1616, 1355-1562, 1355-1620, 1355-1655,

1356-1511, 1356-1588, 1357-1596,

1358-1486, 1360-1524, 1360-1590, 1360-1596, 1360-1604, 1360-1606, 1360-1617, 1360-1618,

1360-1627, 1360-1655, 1360-1659, 1361-1664,

1362-1661, 1363-1624, 1367-1625, 1367-1641, 1368-1573, 1371-1520, 1371-1655, 1371-1659,

1373-1623, 1373-2041, 1377-1635,

1381-1628, 1383-1655, 1384-1606, 1384-1655, 1384-1858, 1387-1588, 1387-1606, 1387-1613,

1387-1632, 1387-1636, 1387-1646, 1387-1655,

1389-1607, 1389-1655, 1394-1686, 1398-1651, 1402-1615, 1402-1623, 1404-1625, 1404-1708,

1407-1640, 1410-1674, 1412-1655,

1412-1663, 1416-1877, 1423-1709, 1424-1671, 1429-1621, 1432-1655, 1436-1655, 1436-2007,

1437-1965, 1438-1607, 1439-1597, 1439-1674,

1442-1729, 1444-2006, 1448-1604, 1448-1671, 1452-2002, 1455-1754, 1455-1760, 1458-2093,

1459-1579, 1459-1747, 1461-1655,

1462-1529,

1462-1809, 1463-1728, 1465-1655, 1472-1720, 1473-2050, 1477-1760, 1479-1679, 1483-2073,

1491-1797, 1492-1736, 1493-1781, 1502-1772,

1502-1798, 1503-1750, 1504-1626, 1509-1758, 1509-1767, 1509-2078, 1513-1786, 1519-1959,

1522-1814, 1525-1767, 1525-1825,

1530-1787, 1530-1814, 1530-2084, 1530-2098, 1531-2235, 1533-1798, 1544-1723, 1544-1777,

1546-1812, 1546-1813, 1546-1823, 1546-1870,

1546-2064, 1550-1824, 1550-1830, 1550-1888, 1551-2123, 1552-1804, 1562-2147, 1567-1881,

1570-2156, 1571-1893, 1573-1867,

1574-1820, 1575-2029, 1576-1878, 1580-1778, 1583-1856, 1584-2082, 1585-1895, 1586-2223,

1588-1836, 1591-2038, 1594-2218, 1595-2235,

1598-1867, 1598-1878, 1600-1893, 1600-1898, 1600-2064, 1601-1866, 1601-2169, 1604-1968,

1615-1740, 1615-1838, 1615-2129,

1621-1900, 1628-1913, 1638-1895, 1638-1933, 1646-1903, 1651-1944, 1652-1896, 1657-1909,

1661-1853, 1661-1925, 1662-1936, 1667-1939,

1667-2096, 1669-2253, 1671-1894, 1673-1771, 1673-1912, 1673-1987, 1673-2133, 1674-2276,

1675-2347, 1676-1902, 1676-1933,

1679-1874,

1679-1920, 1679-1923, 1679-1965, 1679-2317, 1680-2316, 1681-1897, 1681-1944, 1686-2283,

1692-1937, 1692-1963, 1693-1943, 1694-2158,

1694-2475, 1700-1901, 1701-2236, 1707-1966, 1708-1987, 1709-2071, 1711-1982, 1713-2051,

1713-2175, 1721-2001, 1721-2030,

1721-2206, 1725-1911, 1728-2000, 1730-1974, 1733-2323, 1744-2015, 1744-2332, 1746-2202,

1747-2259, 1752-2378, 1752-2407, 1753-2259,

1754-1994, 1756-2172, 1757-2222, 1761-2032, 1761-2050, 1763-2214, 1769-2035, 1770-2367,

1772-2022, 1773-2353, 1776-2060,

1777-2375, 1780-2240, 1781-2302, 1785-2097, 1787-2064, 1788-2171, 1790-2427, 1798-2114,

1805-2412, 1806-2067, 1806-2285, 1808-2274,

1810-2279, 1812-2064, 1812-2073, 1813-2087, 1814-1928, 1821-2072, 1823-2113, 1823-2465,

1832-2093, 1833-2090, 1837-2084,

1837-2357, 1839-1978, 1847-2273, 1852-2051, 1852-2147, 1857-1972, 1857-2109, 1857-2126,

1857-2140, 1857-2159, 1857-2382, 1857-2393,

1860-2056, 1860-2110, 1860-2471, 1868-2107, 1868-2109, 1868-2110, 1868-2318, 1869-2154,

1869-2468, 1870-2151, 1870-2164,

1871-2115,

1873-2161, 1873-2331, 1874-2110, 1874-2121, 1874-2148, 1875-2103, 1875-2111, 1875-2118,

1875-2130, 1876-2184, 1879-2409, 1881-2117,

1881-2340, 1881-2432, 1884-2458, 1884-2464, 1886-2163, 1886-2313, 1888-2387, 1889-2179,

1889-2439, 1893-2161, 1893-2309,

1894-2183, 1894-2432, 1900-2156, 1901-2270, 1903-2377, 1904-2191, 1905-2177, 1914-2160,

1914-2168, 1916-2160, 1916-2177, 1916-2179,

1917-2185, 1917-2243, 1917-2437, 1919-2201, 1919-2512, 1920-2138, 1920-2171, 1922-2167,

1923-2175, 1925-2167, 1935-2180,

1936-2204, 1937-2086, 1937-2756, 1938-2470, 1954-2252, 1957-2129, 1960-2456, 1962-2283,

1962-2456, 1964-2204, 1970-2440, 1972-2248,

1973-2225, 1979-2434, 1982-2647, 1990-2532, 1991-2272, 1994-2269, 2002-2117, 2002-2252,

2007-2276, 2008-2588, 2009-2245,

2009-2295, 2009-2594, 2011-2517, 2011-2594, 2013-2278, 2013-2517, 2017-2277, 2021-2520,

2022-2267, 2024-2270, 2024-2356, 2026-2325,

2032-2296, 2033-2275, 2044-2309, 2047-2274, 2047-2297, 2047-2313, 2049-2310, 2051-2332,

2061-2219, 2061-2292, 2061-2316,

2062-2327,

2062-2552, 2068-2412, 2072-2308, 2072-2331, 2072-2353, 2077-2368, 2079-2463, 2083-2576,

2086-2414, 2089-2370, 2091-2395, 2091-2412,

2108-2642, 2116-2322, 2119-2388, 2119-2390, 2122-2431, 2132-2373, 2132-2415, 2132-2430,

2141-2419, 2142-2349, 2142-2580,

2142-2631, 2143-2379, 2144-2322, 2144-2591, 2145-2405, 2150-2379, 2156-2748, 2158-2343,

2160-2393, 2160-2689, 2162-2407, 2162-2704,

2163-2461, 2164-2283, 2170-2427, 2181-2709, 2182-2485, 2182-2632, 2185-2428, 2186-2451,

2186-2488, 2190-2440, 2191-2409,

2191-2432, 2191-2446, 2191-2455, 2196-2466, 2197-2671, 2199-2677, 2200-2409, 2200-2412,

2200-2498, 2201-2365, 2201-2511, 2203-2763,

2205-2440, 2205-2456, 2205-2563, 2206-2507, 2213-2473, 2214-2457, 2215-2472, 2215-2484,

2215-2734, 2218-2395, 2218-2468,

2218-2523, 2219-2481, 2224-2464, 2224-2471, 2228-2760, 2235-2469, 2241-2546, 2242-2757,

2248-2757, 2251-2700, 2252-2405, 2253-2747,

2259-2447, 2260-2508, 2260-2520, 2263-2564, 2271-2763, 2280-2761, 2281-2754, 2282-2460,

2282-2467, 2282-2763, 2286-2432,

2288-2457,

2288-2534, 2288-2761, 2299-2763, 2300-2763, 2303-2763, 2307-2471, 2307-2532, 2311-2757,

2312-2609, 2313-2763, 2314-2761, 2316-2763,

2319-2740, 2320-2758, 2322-2577, 2323-2759, 2324-2762, 2337-2708, 2338-2499, 2338-2763,

2339-2471, 2340-2763, 2341-2758,

2342-2574, 2342-2757, 2343-2759, 2343-2760, 2343-2763, 2344-2702, 2344-2760, 2345-2758,

2345-2763, 2349-2763, 2350-2575, 2350-2601,

2350-2607, 2350-2762, 2350-2763, 2352-2761, 2353-2760, 2356-2757, 2360-2455, 2362-2759,

2365-2714, 2366-2760, 2367-2653,

2368-2662, 2368-2758, 2369-2760, 2370-2761, 2371-2763, 2372-2760, 2373-2763, 2374-2509,

2374-2757, 2378-2758, 2382-2760, 2385-2757,

2386-2760, 2386-2763, 2393-2761, 2394-2671, 2394-2757, 2395-2760, 2403-2757, 2406-2763,

2408-2659, 2408-2760, 2418-2758,

2423-2760, 2424-2755, 2425-2760, 2428-2762, 2429-2758, 2429-2762, 2431-2708, 2432-2758,

2436-2685, 2436-2760, 2437-2760, 2438-2754,

2438-2757, 2461-2724, 2463-2698, 2468-2760, 2472-2756, 2472-2758, 2474-2753, 2474-2757,

2474-2760, 2475-2756, 2481-2742,

2483-2726,

2489-2757, 2492-2757, 2500-2730, 2506-2757, 2507-2757, 2510-2763, 2513-2763, 2521-2612,

2529-2757, 2529-2760, 2531-2763, 2536-2733,

2536-2763, 2538-2711, 2538-2757, 2540-2760, 2543-2760, 2548-2740, 2554-2757, 2562-2763,

2568-2758, 2571-2757, 2573-2757,

2577-2757, 2585-2753, 2585-2763, 2588-2758, 2589-2758, 2591-2760, 2591-2763, 2595-2761,

2615-2763, 2646-2763, 2648-2757, 2663-2755,

2676-2761, 2686-2756, 2686-2758, 2697-2763

37/7510567CB1/
1-2454, 20-256, 105-610, 1151-1829, 1241-1513, 1397-1719, 1442-1706, 1493-2422,

2454
1623-2419, 1625-1924, 1641-1869, 1641-1891, 1733-2422,

1760-2422, 1796-2325, 2012-2422

38/7506072CB1/
1-431, 1-624, 1-2250, 124-515, 124-842, 255-1214, 305-1214, 386-495,

2255
741-920, 764-969, 802-1281, 806-1409, 815-1085, 822-1078, 830-1149,

834-1094, 835-1086, 835-1286, 883-1253, 886-1120, 888-1492, 895-1401, 904-1354, 923-1171,

925-1419, 938-1196, 938-1444, 939-1151,

940-1268, 944-1535, 958-1480, 982-1253, 986-1369, 999-1207, 999-1398, 1001-1336,

1001-1506, 1001-1512, 1001-1663, 1002-1150,

1002-1556, 1003-1326, 1006-1326, 1012-1298, 1049-1734, 1054-1286, 1056-1355, 1069-1313,

1078-1530, 1087-1543, 1109-1690,

1110-1282, 1111-1690, 1115-1244, 1117-1676, 1130-1771, 1131-1771, 1156-1377, 1156-1632,

1158-1729, 1159-1406, 1167-1773, 1195-1457,

1196-1706, 1211-1721, 1221-1468, 1226-1479, 1232-1364, 1233-1690, 1234-1339, 1235-1498,

1235-1499, 1239-1485, 1241-1815,

1245-1525, 1248-1844, 1251-1513, 1260-1550, 1282-1471, 1282-1771, 1289-1567, 1295-1953,

1302-1802, 1318-1680, 1327-1987, 1331-1580,

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1356-1880, 1365-1854, 1378-1604,

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1505-2218, 1518-1832, 1518-1976,

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1583-2239, 1586-1844, 1598-2218, 1608-1812, 1611-1850, 1621-1979, 1628-1986, 1633-2167,

1634-2154, 1641-1873, 1641-2227,

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1694-2245, 1704-1939, 1710-2236, 1715-2224,

1717-1958, 1717-2097, 1725-1853, 1725-1993, 1725-2148, 1725-2216, 1725-2220, 1725-2241,

1729-1987, 1729-1996, 1731-2006,

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1774-2071, 1775-2227, 1775-2251, 1781-2229,

1782-2246, 1783-2233, 1792-2229, 1798-2252, 1810-2217, 1813-2220, 1813-2227, 1813-2232,

1814-2227, 1815-2218, 1822-2219,

1823-2218,

1827-2250, 1829-2229, 1831-2229, 1832-2195, 1832-2232, 1838-2121, 1840-2186, 1845-2227,

1852-2255, 1853-2228, 1855-2136, 1857-2224,

1858-2229, 1858-2247, 1858-2255, 1862-2229, 1864-2116, 1864-2123, 1864-2230, 1865-2227,

1872-2158, 1876-2227, 1909-2120,

1909-2179, 1909-2217, 1909-2250, 1916-2135, 1916-2233, 1920-2168, 1923-2227, 1927-2086,

1934-2095, 1934-2227, 1947-2227, 1949-2227,

1951-2227, 1952-2227, 1992-2250, 1996-2229, 2030-2227, 2042-2229, 2049-2227, 2055-2227,

2083-2250, 2097-2244, 2165-2229

39/7511354CB1/
1-251, 1-609, 12-305, 12-544, 13-511, 13-666, 14-613, 14-657, 14-674,

2285
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179-638, 186-578, 186-580, 186-593, 186-622, 186-635, 186-685, 186-701,

186-713, 186-719, 186-764, 190-666, 191-808, 211-807, 234-869,

250-891, 268-920, 305-853, 323-583, 324-594, 335-909, 349-942, 353-694,

374-898, 382-1054, 408-498, 416-1049, 427-571, 435-1061,

435-1068, 436-1051, 449-979, 460-634, 463-1149, 467-1038, 468-1074, 505-1094,

508-1072, 512-1071, 529-1176, 539-1215, 551-688,

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704-862, 717-1316, 723-1342, 724-1293, 771-1383,

817-1177, 849-1588, 855-1225, 861-1566, 876-1578, 893-1363, 908-1494, 913-1493,

933-1472, 956-1547, 960-1599, 1000-1599,

1018-1576, 1018-1591, 1028-1497, 1032-1219, 1125-1222, 1153-1670, 1175-1434,

1190-1419, 1291-1540, 1366-1594, 1380-1620, 1405-1651,

1473-1723, 1473-1939, 1502-1830, 1599-1691, 1690-1931, 1691-1847, 1691-1969,

1691-2256, 1691-2259, 1691-2285,

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1774-2036, 1776-2285, 1779-2285, 1801-2226, 1820-2285, 1828-2285, 1835-2285,

1867-2134, 1869-2082, 1912-2127, 1920-2285,

1925-2285, 1940-2285, 1941-2285, 1968-2238, 1985-2200, 1988-2285, 1989-2236,

1989-2256, 1989-2285, 1991-2285, 1993-2214, 1997-2285,

2006-2235, 2006-2247, 2033-2285, 2034-2285, 2040-2285, 2056-2285, 2059-2284,

2077-2285, 2080-2246, 2112-2285, 2113-2285,

2116-2285, 2142-2285, 2145-2285, 2164-2285, 2171-2285, 2189-2285, 2243-2285

40/7511643CB1/
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2755
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458-1110, 525-610, 525-893, 525-949, 527-780, 532-1141, 684-914, 685-914,

846-1141, 895-1163, 896-1315, 923-1196, 930-1163, 1045-1492,

1058-1351, 1073-1686, 1087-1503, 1094-1674, 1103-1732, 1104-1616, 1104-1689,

1104-1785, 1104-1794, 1104-1796, 1104-1838,

1104-1839, 1104-1873, 1105-1744, 1106-1499, 1107-1676, 1109-1690, 1110-1290,

1110-1498, 1194-1714, 1216-1695, 1218-1987, 1219-1601,

1219-1837, 1219-1914, 1219-1982, 1220-1744, 1220-1796, 1220-1874, 1220-1912,

1220-1958, 1220-1959, 1222-1738, 1222-1899,

1222-1938, 1225-1883, 1306-1876, 1307-1603, 1324-1914, 1326-1564, 1331-1631,

1351-1838, 1401-1669, 1528-1781, 1549-1865, 1635-1900,

1650-1987, 1658-1855, 1765-1987, 1821-1954, 1901-2163, 1902-2334, 1986-2225,

1986-2233, 1986-2237, 1986-2432, 1989-2584,

1995-2189, 1998-2192, 2018-2283, 2032-2294, 2032-2297, 2033-2646, 2040-2321,

2059-2274, 2070-2278, 2070-2422, 2105-2644,

2116-2727, 2119-2724, 2134-2755, 2138-2544, 2192-2709, 2193-2395, 2201-2755,

2235-2751, 2249-2515, 2250-2453, 2261-2546, 2262-2493,

2284-2542, 2298-2638, 2326-2442, 2377-2652, 2385-2641

41/7511400CB1/
1-936, 194-870, 195-870

936

42/7511507CB1/
1-230, 12-624, 18-328, 18-329, 30-250, 30-442, 30-2456, 33-620, 45-285,

2457
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176-790, 186-461, 191-432, 191-501, 191-715, 193-447, 197-509, 205-478,

208-753, 208-790, 213-789, 215-777, 216-459, 221-517,

222-504, 223-479, 223-789, 237-470, 239-772, 241-538, 241-540, 241-789,

245-439, 245-787, 251-789, 265-523, 323-561, 355-626, 387-562,

446-724, 465-699, 477-738, 592-770, 629-790, 762-1022, 789-1038, 789-1041,

789-1243, 789-1253, 789-1291, 789-1309, 789-1369,

789-1376, 789-1607, 791-1055, 791-1753, 793-1346, 800-1237, 803-1270,

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867-1122, 867-1272, 867-1470, 871-1414, 874-1699, 875-1451,

877-1432, 878-1424, 880-1258, 884-1120, 884-1411, 890-1086, 890-1192,

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1086-1624, 1090-1290, 1090-1579, 1092-1162, 1094-1276, 1094-1293, 1094-1377,

1094-1457, 1094-1521, 1094-1720, 1095-1381,

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1107-1666, 1108-1648, 1109-1735, 1110-1692, 1115-1700,

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1131-1862, 1132-1660, 1133-1680, 1134-1734,

1136-1791, 1142-1718, 1145-1650, 1145-1738, 1147-1633, 1147-1810, 1150-1329,

1154-1680, 1155-1787, 1157-1425, 1160-1680, 1163-1707,

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43/7511819CB1/
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3916
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44/7511338CB1/
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964
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45/7511425CB1/
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3971
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46/7511534CB1/
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2509
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47/7511648CB1/
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3231
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3034-3221, 3100-3226

48/7511600CB1/
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1192
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49/7511783CB1/
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1242
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50/7512383CB1/
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3293
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3257-3293

51/7512813CB1/
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2533
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2188-2483, 2190-2504

52/7512842CB1/
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1192
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935-1097, 1013-1183, 1026-1184

53/90190613CB1/
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11740
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54/7511894CB1/
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2466
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1713-1869, 2058-2258

55/3604804CB1/
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14106
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10949-11388, 11020-11360

56/7512568CB1/
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1874
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1021-1421, 1026-1269, 1027-1262, 1028-1262, 1029-1262, 1029-1266, 1029-1269,

1030-1541, 1032-1498, 1040-1198, 1040-1264, 1040-1286,

1042-1488, 1045-1153, 1046-1305, 1049-1300, 1054-1313, 1054-1343, 1055-1635,

1057-1262, 1065-1262, 1075-1262, 1077-1403,

1079-1264, 1081-1186, 1089-1366, 1089-1591, 1098-1264, 1098-1360, 1101-1264,

1106-1554, 1111-1577, 1121-1585, 1122-1260,

1128-1372, 1128-1388, 1128-1395, 1130-1404, 1132-1437, 1148-1386, 1150-1393,

1155-1467, 1160-1262, 1161-1434, 1162-1409, 1162-1433,

1164-1252, 1168-1425, 1184-1769, 1186-1450, 1190-1521, 1195-1437, 1215-1476,

1217-1472, 1217-1513, 1228-1489, 1230-1470,

1230-1756, 1233-1808, 1237-1490, 1237-1502, 1238-1755, 1243-1788, 1260-1549,

1261-1536, 1275-1476, 1275-1505, 1275-1518, 1275-1540,

1279-1755, 1280-1755, 1283-1635, 1290-1553, 1295-1564, 1297-1546, 1297-1548,

1297-1595, 1298-1564, 1300-1590, 1300-1592,

1302-1537, 1304-1518, 1304-1571, 1304-1593, 1308-1572, 1308-1801, 1317-1592,

1319-1761, 1331-1568, 1333-1591, 1334-1592, 1336-1577,

1337-1836, 1343-1826, 1347-1820, 1351-1633, 1353-1722, 1354-1820, 1355-1844,

1356-1595, 1356-1837, 1364-1820, 1364-1835,

1365-1620, 1365-1839, 1367-1637, 1369-1820, 1373-1849, 1376-1578, 1376-1849,

1377-1506, 1378-1820, 1379-1580, 1379-1845, 1381-1810,

1383-1677, 1386-1815, 1395-1632, 1395-1664, 1395-1820, 1399-1820, 1416-1820,

1422-1600, 1431-1627, 1440-1714, 1442-1822,

1444-1551,

1444-1825, 1444-1871, 1447-1820, 1449-1741, 1450-1874, 1452-1563, 1452-1679,

1453-1815, 1454-1826, 1455-1820, 1456-1713, 1459-1819,

1467-1764, 1478-1730, 1484-1820, 1486-1690, 1486-1826, 1488-1726, 1493-1749,

1512-1805, 1530-1849, 1541-1819, 1546-1821,

1550-1651, 1556-1834, 1566-1821, 1571-1802, 1584-1803, 1584-1804, 1584-1820,

1592-1804, 1607-1822, 1608-1817, 1617-1740, 1626-1821,

1627-1874, 1632-1827, 1633-1820, 1637-1821

57/7512812CB1/
1-256, 1-335, 1-428, 1-2292, 23-303, 23-561, 23-651, 25-269,

2292
25-770, 60-637, 131-951, 131-958, 131-1009, 131-1041, 209-719, 291-904,

348-923, 360-882, 403-1014, 403-1075, 403-1124, 419-704, 449-594, 461-1114,

498-1124, 511-1135, 524-1196, 550-1142, 584-681, 598-1136,

672-1121, 724-1288, 756-1427, 766-1365, 776-1565, 787-1410, 804-1445,

852-1162, 867-1362, 877-1568, 894-1430, 919-1402, 925-1689,

935-1490, 1023-1722, 1055-1786, 1092-1606, 1161-1799, 1191-1799,

1206-1799, 1227-1492, 1227-1799, 1231-1799, 1241-1799,

1282-1774, 1283-1821, 1288-1439, 1462-1725, 1490-2292, 1494-1576,

1550-1790, 1941-2219, 1947-2230, 1947-2237, 1947-2242, 1949-2258

58/7512826CB1/
1-129, 1-138, 1-201, 1-206, 1-219, 1-229, 1-231, 1-245,

2755
1-247, 1-259, 1-260, 1-265, 1-266, 1-267, 1-270, 1-277, 1-279,

1-286, 1-297, 1-304,

1-373, 1-385, 1-454, 1-462, 1-470, 1-481, 1-499, 1-520, 1-527,

1-529, 1-534, 1-538, 1-540, 1-563, 1-598, 1-602, 1-627, 1-628,

1-647, 1-663,

1-664, 1-677, 1-726, 1-780, 1-804, 1-817, 1-840, 1-2755, 3-199,

3-260, 3-264, 3-827, 4-298, 4-326, 4-525, 4-562, 4-672, 6-140,

6-457, 18-662,

37-572, 42-292, 50-742, 79-692, 101-897, 103-532, 108-576, 125-402,

126-581, 141-594, 148-701, 152-728, 166-403, 166-827, 167-894,

174-894, 184-426, 185-894, 197-826, 201-454, 207-492, 207-748, 207-838,

211-480, 214-552, 218-894, 222-983, 225-564, 232-831,

272-900, 286-499, 298-902, 298-1051, 303-859, 310-887, 343-897, 355-977,

357-639, 357-836, 357-859, 357-957, 361-504, 374-1016, 381-634,

387-982, 388-890, 390-983, 428-981, 436-867, 444-975, 458-974, 459-719,

473-965, 475-894, 476-4211, 491-970, 497-794, 497-1051,

498-733, 514-1036, 530-968, 537-887, 539-1035, 541-820, 541-903,

556-1167,

561-1042, 563-858, 567-1047, 582-844, 612-1211, 612-1440, 623-1196,

623-1328, 635-1232, 642-886, 643-1202, 646-896, 646-911, 647-1245,

650-1425, 652-894, 652-1177, 696-1244, 702-1012, 702-1263, 702-1300,

711-1292, 714-962, 714-1403, 723-958, 737-1325, 741-1317,

741-1326, 744-1157, 745-1317, 773-977, 786-1056, 812-1366, 817-1524,

828-1406, 831-1451, 869-1135, 891-1049, 895-1392, 896-1416,

903-1196, 903-1493, 904-1160, 905-1187, 907-1173, 916-1194, 916-1470,

925-1549, 936-1523, 946-1214, 947-1374, 952-1333, 953-1176,

959-1256, 969-1223, 969-1549, 975-1271, 1006-1300, 1006-1549, 1008-1251,

1015-1529, 1017-1465, 1029-1523, 1031-1324, 1036-1325,

1036-1549, 1041-1332, 1051-1481, 1055-1316, 1062-1549, 1066-1306,

1066-1313, 1072-1315, 1072-1529, 1075-1338, 1089-1529,

1106-1352, 1125-1549, 1130-1422, 1143-1329, 1155-1404, 1157-1381,

1158-1410, 1205-1472, 1207-1458, 1209-1385, 1228-1487, 1238-1512,

1272-1507, 1280-1548, 1320-1549, 1323-2033, 1331-1549, 1361-1549,

1362-1524, 1362-1549, 1404-1549, 1549-1702, 1549-1721,

1549-1734, 1549-1779, 1549-1806, 1549-1829, 1549-1831, 1549-2030,

1549-2243, 1550-2279, 1551-1740, 1551-2034, 1551-2037, 1552-1661,

1567-2117, 1568-2105, 1568-2223, 1590-2162, 1591-1852, 1595-1879,

1598-2139, 1600-2279, 1628-2139, 1631-1904, 1635-2148,

1646-2204, 1649-2135, 1661-1942, 1664-1908, 1664-2228, 1664-2646,

1665-1900, 1665-1918, 1665-2192, 1666-1803, 1666-1909, 1666-1935,

1666-1960, 1666-1967, 1666-2021, 1666-2291, 1666-2313, 1666-2339,

1667-1900, 1668-1875, 1669-2367, 1676-1942, 1680-2009,

1692-1879, 1692-2193, 1693-2509, 1694-2275, 1700-1935, 1705-2220,

1705-2312, 1706-2002, 1710-2342, 1710-2473, 1711-2072, 1721-2056,

1726-2273, 1727-1993, 1728-2279, 1733-2364, 1736-1927, 1739-2045,

1746-1975, 1746-1996, 1755-1974, 1755-2009, 1755-2043,

1755-2461, 1755-2481, 1758-2247, 1758-2323, 1758-2391, 1758-2412,

1761-2028, 1769-2033, 1771-2078, 1771-2252, 1773-2056, 1774-2056,

1774-2348, 1774-2420, 1781-2082, 1784-2021, 1784-2041, 1784-2049,

1784-2349, 1790-2330, 1793-2471, 1802-2085, 1802-2093,

1802-2414,

1804-2408, 1804-2533, 1805-2056, 1805-2101, 1805-2500, 1805-2612,

1814-2035, 1817-2091, 1819-2099, 1819-2277, 1819-2350, 1821-2385,

1823-2120, 1828-2104, 1828-2278, 1836-2129, 1839-2511, 1840-2105,

1840-2375, 1844-2291, 1851-2116, 1852-2021, 1856-2329,

1857-2134, 1861-2180, 1864-2454, 1865-2439, 1873-2173, 1877-2012,

1877-2161, 1878-2544, 1885-2119, 1893-2111, 1893-2490, 1899-2167,

1900-2400, 1903-2563, 1903-2571, 1910-2177, 1911-2460, 1911-2580,

1912-2171, 1912-2183, 1916-2403, 1917-2197, 1918-2216,

1918-2505, 1920-2567, 1921-2168, 1925-2478, 1925-2511, 1926-2174,

1927-2160, 1928-2167, 1928-2460, 1930-2217, 1931-2479, 1932-2187,

1936-2205, 1936-2210, 1936-2460, 1937-2460, 1939-2240, 1940-2507,

1941-2182, 1941-2460, 1943-2579, 1946-2456, 1946-2739,

1949-2352, 1952-2567, 1960-2225, 1960-2410, 1961-2056, 1961-2263,

1961-2447, 1961-2460, 1964-2578, 1966-2197, 1967-2592, 1968-2718,

1971-2527, 1972-2152, 1972-2754, 1974-2402, 1974-2489, 1978-2459,

1979-2252, 1979-2256, 1984-2211, 1984-2754, 1988-2592,

1990-2466,

1996-2420, 1996-2477, 1997-2544, 2002-2275, 2003-2592, 2008-2285,

2009-2271, 2015-2273, 2015-2290, 2017-2270, 2017-2271, 2020-2755,

2027-2687, 2034-2282, 2035-2460, 2039-2327, 2041-2342, 2041-2460,

2042-2326, 2056-2460, 2056-2560, 2056-2747, 2057-2368,

2057-2460, 2058-2328, 2059-2299, 2059-2739, 2060-2460, 2064-2638,

2067-2467, 2069-2318, 2069-2458, 2071-2333, 2072-2263, 2072-2266,

2073-2364, 2073-2476, 2074-2738, 2076-2588, 2083-2362, 2084-2476,

2084-2694, 2088-2340, 2088-2352, 2088-2556, 2089-2442,

2094-2460, 2096-2385, 2096-2389, 2098-2324, 2100-2460, 2102-2398,

2102-2710, 2102-2753, 2103-2342, 2104-2390, 2107-2460, 2109-2346,

2114-2423, 2117-2300, 2117-2368, 2117-2386, 2121-2337, 2124-2375,

2131-2369, 2131-2386, 2135-2754, 2137-2680, 2137-2747,

2140-2617, 2141-2163, 2141-2320, 2141-2460, 2141-2476, 2142-2460,

2144-2695, 2144-2755, 2146-2460, 2147-2363, 2147-2460, 2152-2402,

2154-2439, 2154-2445, 2156-2257, 2156-2460, 2158-2755, 2159-2402,

2159-2413, 2159-2426, 2159-2460, 2159-2592, 2161-2460,

2162-2453,

2163-2439, 2171-2455, 2176-2439, 2176-2442, 2176-2463, 2184-2432,

2187-2755, 2192-2458, 2192-2481, 2195-2753, 2198-2473, 2200-2420,

2200-2460, 2203-2480, 2204-2470, 2205-2460, 2207-2389, 2208-2460,

2214-2438, 2215-2360, 2215-2497, 2216-2460, 2219-2501,

2221-2385, 2221-2460, 2221-2479, 2221-2500, 2228-2460, 2229-2739,

2231-2514, 2234-2445, 2234-2501, 2234-2504, 2234-2512, 2236-2725,

2237-2460, 2240-2532, 2244-2486, 2249-2473, 2251-2449, 2252-2533,

2252-2541, 2253-2410, 2254-2460, 2265-2468, 2266-2460,

2269-2474, 2278-2460, 2278-2532, 2297-2517, 2299-2722, 2303-2458,

2304-2570, 2305-2753, 2310-2588, 2311-2473, 2317-2460, 2317-2486,

2318-2644, 2322-2460, 2323-2755, 2324-2474, 2332-2460, 2340-2603,

2341-2555, 2343-2460, 2345-2736, 2346-2460, 2347-2460,

2348-2754, 2349-2628, 2351-2615, 2356-2605, 2359-2530, 2361-2597,

2367-2460, 2371-2462, 2373-2460, 2378-2463, 2384-2460, 2388-2460,

2391-2583, 2397-2737, 2398-2755, 2402-2473, 2406-2639, 2409-2460,

2563-2644

59/7512908CB1/
1-554, 9-1708, 11-729, 19-828, 28-642, 30-260, 32-304,

1708
35-266, 36-272, 37-754, 38-799, 44-736, 45-245, 47-640, 51-187,

56-355, 72-312,

111-395, 165-839, 221-432, 221-458, 250-533, 250-610, 250-673,

255-516, 256-757, 259-533, 260-1123, 262-995, 263-576, 265-521, 273-895,

311-929, 316-973, 317-919, 332-606, 336-929, 340-603, 340-774,

340-869, 340-1007, 340-1095, 340-1103, 348-608, 348-614, 350-781,

350-962, 359-1059, 368-742, 368-863, 382-667, 389-670, 392-638,

392-639, 393-984, 400-946, 400-987, 401-680, 413-781, 420-682,

425-1120, 434-687, 437-1055, 448-1100, 460-884, 464-1017, 479-742,

480-741, 486-729, 486-768, 491-1068, 498-738, 501-752, 507-802,

509-719, 511-764, 533-846, 536-1124, 540-765, 544-774, 552-1115,

562-824, 601-878, 601-910, 604-1019, 606-844, 608-1124, 613-1116,

613-1121, 618-910, 627-882, 627-890, 629-850, 649-926, 649-934,

650-898, 650-913, 680-992, 685-847, 695-952, 705-834, 727-964,

727-1040,

727-1116, 728-989, 729-995, 753-1059, 759-908, 759-1017, 775-982,

775-1124, 776-1032, 789-1060,

799-1044, 804-1087, 819-1051, 824-1060, 825-1124, 828-1124, 856-961,

868-1124, 873-1079, 873-1124, 877-1124, 885-1075, 885-1119,

888-1124, 900-1109, 901-1124, 905-1068, 905-1124, 906-1114, 916-1124,

933-1036, 939-1108, 972-1122, 1071-1597, 1115-1597, 1122-1461,

1123-1343, 1123-1421, 1123-1496, 1123-1533, 1123-1557, 1123-1574,

1123-1582, 1123-1593, 1123-1594, 1123-1603, 1124-1594,

1124-1595, 1124-1599, 1125-1597, 1126-1327, 1126-1594, 1127-1590,

1127-1594, 1128-1603, 1131-1594, 1131-1599, 1131-1603, 1132-1594,

1133-1594, 1133-1603, 1134-1392, 1134-1603, 1138-1594, 1139-1594,

1140-1594, 1142-1594, 1152-1594, 1154-1563, 1154-1578,

1154-1596, 1154-1603, 1157-1594, 1157-1603, 1159-1578, 1161-1345,

1162-1585, 1162-1597, 1162-1603, 1163-1578, 1163-1601, 1164-1594,

1165-1578, 1173-1602, 1174-1585, 1175-1594, 1175-1597, 1177-1578,

1177-1594, 1182-1594, 1183-1416, 1183-1601, 1185-1578,

1186-1389, 1186-1423, 1186-1603, 1187-1594, 1189-1603, 1199-1442,

1199-1592, 1199-1593, 1200-1634, 1203-1526, 1204-1593, 1204-1594,

1209-1456, 1214-1326, 1220-1603, 1225-1486, 1227-1603, 1232-1598,

1235-1507, 1238-1544, 1240-1585, 1241-1594, 1242-1516, 1244-1594,

1253-1585, 1255-1528, 1259-1575, 1259-1594, 1262-1585, 1263-1602,

1263-1603, 1268-1594, 1268-1603, 1272-1594, 1274-1576,

1284-1585, 1284-1603, 1285-1549, 1296-1603, 1316-1584, 1345-1594,

1354-1602, 1371-1603, 1387-1594, 1392-1597, 1396-1603, 1401-1594,

1409-1594, 1427-1534, 1430-1603, 1436-1578, 1442-1602, 1448-1597,

1453-1594, 1470-1704

60/7512909CB1/
1-554, 9-1637, 11-729, 19-828, 28-642, 30-260, 32-304, 35-266,

1637
36-272, 37-754, 38-799, 44-736, 45-245, 47-640, 51-187, 56-355, 72-312,

111-395, 165-839, 221-432, 221-458, 250-533, 250-610, 250-673, 255-516,

256-757, 259-533, 260-1132, 262-995, 263-576, 265-521, 273-895,

311-929, 316-973, 317-919, 332-606, 336-929, 340-603, 340-774, 340-869,

340-1007, 340-1095, 340-1103, 348-608, 348-614, 350-781,

350-962, 359-1059, 368-742, 368-863, 382-667, 388-1260, 389-670,

392-638, 392-639, 393-984, 400-946, 400-987, 401-680, 413-781,

420-682, 425-1120, 434-687, 437-1055, 448-1100, 460-884, 464-1017,

479-742, 480-741, 486-729, 486-768, 491-1068, 498-738, 501-752,

507-802, 509-719, 511-764, 533-846, 536-1160, 540-765, 544-774, 551-1196,

552-1201, 562-824, 601-878, 601-910, 604-1019, 606-844,

608-1249, 613-1116, 613-1121, 613-1177, 613-1221, 613-1240, 613-1247,

613-1288, 618-910, 627-882, 627-890, 629-850, 636-1157, 649-926,

649-934, 650-898, 650-913, 662-1313, 664-1258, 680-992, 685-847,

695-952, 698-1332, 705-834,

717-1174, 721-1301, 727-964, 727-1040, 727-1116, 728-989, 729-995,

729-1270, 729-1344, 750-1272, 753-1059, 759-908, 759-1017, 769-1326,

769-1377, 775-982, 775-1253, 776-1032, 776-1348, 779-1348, 782-1332,

788-1377, 789-1060, 792-1234, 795-1367, 797-1221, 799-1044,

804-1087, 812-1346, 819-1051, 824-1060, 825-1280, 828-1262, 849-1523,

856-961, 862-1162, 868-1143, 873-1079, 873-1136, 875-1369,

877-1130, 880-1153, 881-1202, 885-1075, 885-1133, 888-1132, 893-1369,

900-1109, 900-1150, 901-1214, 905-1068, 905-1132, 905-1140,

905-1385, 906-1167, 916-1158, 916-1172, 918-1286, 929-1158, 929-1297,

929-1355, 929-1385, 933-1036, 933-1354, 939-1108, 941-1385,

943-1228, 944-1178, 972-1301, 972-1367, 972-4384, 978-1192, 982-1346,

995-1245, 1013-1298, 1016-1260, 1017-1272, 1017-1282,

1029-1205, 1030-1252, 1031-1306, 1039-1289, 1039-1301, 1049-1254,

1054-1322, 1055-1333, 1059-1301, 1064-1354, 1069-1281, 1076-1327,

1086-1384, 1100-1354, 1103-1361, 1106-1343, 1110-1385, 1145-1243,

1176-1385, 1179-1385, 1193-1385, 1383-1526,

1385-1507, 1385-1523, 1385-1531, 1385-1532, 1386-1526, 1399-1633

61/7512769CB1/
1-654, 7-264, 10-302, 11-1866, 12-292, 13-347, 15-241, 16-238,

1866
17-146, 17-310, 25-214, 25-326, 33-331, 34-193, 34-276, 34-285, 34-287,

34-288, 34-291, 34-296, 34-305, 35-213, 35-279, 35-316, 36-145, 36-221,

36-258, 36-273, 36-284, 36-287, 36-306, 36-311, 36-314, 36-316,

36-317, 36-491, 36-581, 36-617, 36-657, 37-289, 37-309, 37-326,

37-347, 39-309, 40-272, 40-347, 41-257, 41-267, 41-272, 41-296, 41-331,

41-347, 42-274, 42-313, 42-340, 44-276, 44-281, 44-286, 44-288,

44-328, 46-296, 46-347, 61-261, 85-247, 154-318, 168-257, 179-837,

190-305,

193-472, 230-292, 346-511, 346-566, 346-580, 346-592, 346-809,

346-821, 352-569, 354-604, 359-630, 361-597, 369-621, 373-922,

379-656, 383-616, 390-580, 391-655, 410-915, 411-647, 420-1012,

420-1074, 432-525, 432-808, 437-730, 439-1072, 441-751, 450-1011,

461-705, 463-1186, 463-1235, 464-695, 467-710, 468-889, 469-984,

470-722, 484-1123, 490-784, 497-1053, 501-782, 514-1362, 521-750,

521-1010, 546-1015, 546-1223, 546-1294, 552-753, 552-911, 552-1291,

552-1331, 557-1084,

557-1362, 560-679, 563-1362, 571-781, 571-1029, 604-1220, 608-876,

610-698, 618-889, 626-1188, 636-846, 637-920, 641-901, 646-1362,

659-1178, 659-1218, 659-1343, 664-799, 669-955, 669-1057, 692-1176,

694-1113, 697-1005, 701-1099, 701-1224, 720-1244, 721-978, 728-1157,

729-977, 732-1329, 740-1335, 742-1349, 742-1426, 747-1424, 752-1385,

771-1049, 781-1508, 788-1286, 791-1154, 791-1298, 792-1154,

793-1298, 797-1063, 798-1507, 799-1065, 799-1101, 799-1591, 815-1507,

817-1019, 841-1222, 842-1794, 856-1170, 869-1506, 877-1539,

878-1508, 879-1017, 879-1172, 879-1508, 882-1539, 884-1464, 888-1103,

903-1352, 905-1513, 906-1526, 929-1060, 931-1249, 937-1546,

938-1196, 941-1455, 959-1175, 960-1237, 972-1281, 977-1402, 980-1291,

984-1247, 986-1722, 987-1211, 987-1258, 992-1226, 993-1278,

995-1599, 999-1269, 1012-1251, 1019-1284, 1019-1728, 1026-1279, 1026-1618,

1027-1312, 1030-1604, 1032-1265, 1032-1508,

1034-1284, 1035-1322, 1037-1325, 1037-1639, 1049-1282, 1049-1312,

1049-1636, 1056-1313, 1063-1747, 1065-1325,

1066-1654, 1069-1312, 1077-1323, 1081-1218, 1082-1365, 1083-1341,

1086-1654, 1095-1353, 1111-1424, 1112-1792, 1118-1569, 1118-1609,

1121-1463, 1121-1576, 1121-1619, 1121-1621, 1130-1639, 1153-1445,

1153-1743, 1159-1445, 1163-1616, 1164-1419, 1165-1455,

1167-1457, 1169-1476, 1171-1448, 1172-1442, 1176-1351, 1178-1399,

1178-1403, 1182-1759, 1188-1799, 1197-1351, 1198-1428, 1199-1458,

1202-1569, 1214-1725, 1219-1701, 1221-1481, 1222-1492, 1222-1515,

1222-1516, 1236-1446, 1239-1507, 1244-1720, 1245-1473,

1246-1542, 1247-1496, 1251-1598, 1252-1502, 1253-1734, 1255-1520,

1256-1760, 1270-1540, 1272-1764, 1276-1515, 1277-1494, 1279-1799,

1284-1794, 1292-1727, 1298-1750, 1301-1477, 1303-1766, 1304-1733,

1305-1748, 1310-1594, 1314-1786, 1323-1614, 1328-1725,

1331-1794, 1332-1579, 1332-1781, 1337-1595, 1337-1742, 1337-1792,

1343-1794, 1348-1795, 1349-1620, 1354-1741, 1358-1536, 1362-1799,

1372-1792, 1372-1794, 1373-1799, 1374-1604, 1374-1791, 1374-1794,

1378-1627, 1381-1792, 1382-1780, 1385-1640, 1388-1799,

1406-1641,

1406-1688, 1409-1799, 1409-1866, 1411-1718, 1413-1794, 1415-1707,

1415-1793, 1432-1676, 1432-1791, 1437-1691, 1441-1617, 1441-1799,

1442-1646, 1443-1793, 1443-1794, 1446-1663, 1449-1799, 1455-1713,

1456-1801, 1467-1794, 1472-1793, 1478-1793, 1480-1793,

1487-1799, 1499-1767, 1507-1769, 1508-1771, 1508-1799, 1508-1801,

1522-1852, 1557-1799, 1601-1794, 1601-1866, 1602-1735, 1602-1793,

1602-1794, 1605-1794, 1606-1678, 1607-1801, 1610-1854, 1621-1799,

1635-1793, 1635-1794, 1650-1793, 1685-1801, 1747-1783

62/7512871CB1/
1-234, 1-235, 1-240, 1-241, 1-265, 1-300, 1-422, 1-444,

1191
1-447, 1-452, 1-494, 1-497, 1-509, 1-511, 1-516, 1-559, 4-535,

5-383, 5-663, 6-234,

6-254, 6-361, 6-412, 6-486, 6-528, 6-585, 6-622, 6-1191, 7-703,

8-251, 9-182, 9-423, 9-591, 12-220, 12-232, 12-273, 14-259, 14-278,

14-701,

15-243, 15-250, 15-546, 16-245, 16-624, 22-662, 27-227, 27-235,

27-252, 28-247, 28-263, 33-245, 33-275, 33-296, 34-184, 34-452,

35-287,

38-606, 39-274, 39-295, 43-246, 43-628, 47-263, 47-301, 47-498,

50-279, 50-476, 54-426, 55-528, 55-684, 63-300, 63-406, 63-498,

63-526,

65-304, 68-611, 75-357, 99-770, 103-259, 103-345, 121-287,

121-406, 123-457, 123-597, 152-770, 157-663, 165-770, 176-554,

196-770,

203-554, 221-464, 221-554, 221-604, 254-469, 256-754, 257-770,

339-494, 343-579, 385-586, 413-622, 414-766, 418-694, 442-672,

442-746, 484-770, 535-746, 723-909, 723-1136, 723-1142, 770-1000,

770-1022, 770-1104, 770-1185, 770-1189, 770-1191, 772-1185, 773-1188,

774-1044, 776-1185, 778-955, 778-1130, 778-1183, 782-1179,

789-1185, 794-1181,

796-1170, 796-1185, 800-1183, 801-1103, 804-1191, 807-1177,

808-1041, 808-1164, 808-1185, 810-1191, 811-1021, 811-1028, 813-1045,

815-1069, 818-1053, 818-1190, 820-1181, 822-1191, 840-1191,

847-1182, 853-1147, 884-1181, 889-1181, 890-1191, 892-1083, 902-1150,

908-1181, 919-1179, 923-1142, 959-1145, 959-1191, 961-1142,

962-1164, 964-1191, 972-1185, 993-1089, 996-1116, 1050-1184, 1056-1181,

1077-1176

TABLE 5

Polynucleotide

Representative

SEQ ID NO:
Incyte Project ID:
Library

32
7506690CB1
LIVRTUE01

33
7506536CB1
BRAINOT09

34
7506537CB1
BRAINOT09

35
7506655CB1
ARTANOT06

36
7506656CB1
PROSTUT09

37
7510567CB1
KIDNTUT13

38
7506072CB1
PROSTMY01

39
7511354CB1
EOSIHET02

40
7511643CB1
COLTDIT04

42
7511507CB1
NEURDNV05

43
7511819CB1
THYMNOR02

44
7511338CB1
MYEPTXT01

45
7511425CB1
CONNNOT01

46
7511534CB1
EOSIHET02

47
7511648CB1
BMARNOT03

48
7511600CB1
TESTTUT02

49
7511783CB1
LUNGFET05

50
7512383CB1
BRAITUT12

51
7512813CB1
PLACFER01

52
7512842CB1
UTRSTDT01

53
90190613CB1
BRAXNOT01

54
7511894CB1
PROSTMT01

55
3604804CB1
BRAIFER05

56
7512568CB1
BEPINOT01

57
7512812CB1
LUNGDIS03

58
7512826CB1
ENDCNOT04

59
7512908CB1
BRAUNOR01

60
7512909CB1
BRAUNOR01

61
7512769CB1
EPIGNOT01

62
7512871CB1
LEUKNOT03

TABLE 6

Library
Vector
Library Description

ARTANOT06
pINCY
Library was constructed using RNA isolated from aortic adventitia tissue

removed from a 48-year-old Caucasian male.

BEPINOT01
PSPORT1
Library was constructed using RNA isolated from a bronchial epithelium

primary cell line derived from a 54-year-old Caucasian male.

BMARNOT03
pINCY
Library was constructed using RNA isolated from the left tibial bone marrow

tissue of a 16-year-old Caucasian male during a partial left tibial ostectomy with free skin graft.

Patient history included an abnormality of the red blood cells. Previous surgeries included bone

and bone marrow biopsy, and soft tissue excision. Family history included osteoarthritis.

BRAIFER05
pINCY
Library was constructed using RNA isolated from brain tissue removed from a

Caucasian male fetus who was stillborn with a hypoplastic left heart at 23 weeks' gestation.

BRAINOT09
pINCY
Library was constructed using RNA isolated from brain tissue removed from a

Caucasian male fetus, who died at 23 weeks'gestation.

BRAITUT12
pINCY
Library was constructed using RNA isolated from brain tumor tissue removed

from the left frontal lobe of a 40-year-old Caucasian female during excision of a cerebral

meningeal lesion. Pathology indicated grade 4 gemistocytic astrocytoma.

BRAUNOR01
pINCY
This random primed library was constructed using RNA isolated from striatum,

globus pallidus and posterior putamen tissue removed from an 81-year-old Caucasian female

who died from a hemorrhage and ruptured thoracic aorta due to atherosclerosis. Pathology

indicated moderate atherosclerosis involving the internal carotids, bilaterally; microscopic infarcts

of the frontal cortex and hippocampus, and scattered diffuse amyloid plaques and neurofibrillary

tangles, consistent with age. Grossly, the leptomeninges showed only mild thickening and

hyalinization along the superior sagittal sinus. The remainder of the leptomeninges was thin

and contained some congested blood vessels. Mild atrophy was found mostly in the frontal poles

and lobes, and temporal lobes, bilaterally. Microscopically, there were pairs of Alzheimer type II

astrocytes within the deep layers of the neocortex. There was increased satellitosis around

neurons in the deep gray matter in the middle frontal cortex.

The amygdala contained rare diffuse

plaques and neurofibrillary tangles. The posterior hippocampus contained a

microscopic area of cystic cavitation with hemosiderin-laden macrophages surrounded

by reactive gliosis. Patient history included sepsis, cholangitis, post-operative

atelectasis, pneumonia CAD, cardiomegaly due to left ventricular hypertrophy, splenomegaly,

arteriolonephrosclerosis, nodular colloidal goiter, emphysema, CHF, hypothyroidism, and

peripheral vascular disease.

BRAXNOT01
pINCY
Library was constructed using RNA isolated from cerebellar tissue

removed from a 70-year-old male. Patient history included

chronic obstructive airways disease and left ventricular failure.

COLTDIT04
pINCY
Library was constructed using RNA isolated from diseased transverse

colon tissue removed from a 16-year-old Caucasian male

during partial colectomy, temporary ileostomy, and colonoscopy.

Pathology indicated innumerable (greater than 100)

adenomatous polyps with low-grade dysplasia involving the entire colonic

mucosa in the setting of familial polyposis coli.

Family history included benign col on neoplasm, benign hypertension,

cerebrovascular disease, breast cancer, uterine cancer,

and type II diabetes.

CONNNOT01
pINCY
Library was constructed using RNA isolated from mesentery fat

tissue obtained from a 71-year-old Caucasian male during a

partial colectomy and permanent colostomy. Family history included atherosclerotic

coronary artery disease, myocardial infarction, and extrinsic asthma.

ENDCNOT04
pINCY
Library was constructed using RNA isolated from coronary

artery endothelial cell tissue removed from a 3-year-old Caucasian male.

EOSIHET02
PBLUESCRIPT
Library was constructed using RNA isolated from peripheral

blood cells apheresed from a 48-year-old Caucasian male. Patient

history included hypereosinophilia. The cell population was determined to be

greater than 77% eosinophils by Wright's staining.

EPIGNOT01
pINCY
Library was constructed using RNA isolated from epiglottic tissue

removed from a 71-year-old male during laryngectomy with

right parathyroid biopsy. Pathology for the associated tumor tissue indicated

recurrent grade 1 papillary thyroid carcinoma.

KIDNTUT13
pINCY
Library was constructed using RNA isolated from kidney tumor tissue removed from

a 51-year-old Caucasian female during a nephroureterectomy. Pathology indicated a grade 3

renal cell carcinoma. Patient history included depressive disorder, hypoglycemia, and uterine

endometriosis. Family history included calculus of the kidney, colon cancer, and type II diabetes.

LEUKNOT03
pINCY
Library was constructed using RNA isolated from white blood cells of a 27-year-old female

with blood type A+. The donor tested negative for cytomegalovirus (CMV).

LIVRTUE01
PCDNA2.1
This 5′ biased random primed library was constructed using RNA isolated from

liver tumor tissue removed from a 72-year-old Caucasian male during partial hepatectomy. Pathology

indicated metastatic grade 2 (of 4) neuroendocrine carcinoma forming a mass. The patient

presented with metastatic liver cancer. Patient history included benign hypertension, type I diabetes,

prostatic hyperplasia, prostate cancer, alcohol abuse in remission, and tobacco abuse

in remission. Previous surgeries included destruction of a pancreatic lesion, closed prostatic

biopsy, transurethral prostatectomy, removal of bilateral testes and total splenectomy. Patient

medications included Eulexin, Hytrin, Proscar, Ecotrin, and insulin. Family history included

atherosclerotic coronary artery disease and acute myocardial infarction in the mother; atherosclerotic

coronary artery disease and type II diabetes in the father.

LUNGDIS03
pINCY
Library was constructed using diseased lung tissue. 0.76 million clones from a

diseased lung tissue library were subjected to two rounds of subtraction hybridization with

5.1 million clones from a normal lung tissue library. The starting library for

subtraction was constructed using polyA RNA isolated from diseased lung tissue. Patient history

included idiopathic pulmonary disease. Subtractive hybridization conditions were based on the methodologies

of Swaroop et al. (1991) Nucleic Acids Res. 19: 1954; and Bonaldo et al. Genome Res. (1996) 6: 791.

LUNGFET05
PSPORT1
Library was constructed using RNA isolated from lung tissue removed from a Caucasian

female fetus, who died at 20 weeks' gestation from anencephalus.

MYEPTXT01
pINCY
Library was constructed using RNA isolated from a treated K-562 cell line, derived from

chronic myelogenous leukemia precursor cells obtained from a 53-year-old female. The cells were treated

with 5-aza-2′deoxycytidine.

NEURDNV05
PCR2-TOPOTA
Library was constructed using pooled cDNA from different donors. cDNA was generated

using mRNA isolated from pooled skeletal muscle tissue removed from ten 21 to 57-year-old Caucasian male and

female donors who died from sudden death; from pooled thymus tissue removed from nine 18 to

32-year-old Caucasian male and female donors who died from sudden death; from pooled liver tissue removed

from 32 Caucasian male and female fetuses who died at 18-24 weeks gestation due to spontaneous abortion;

from kidney tissue removed from 59 Caucasian male and female fetuses who died at 20-33 weeks

gestation due to spontaneous abortion; and from brain tissue removed from a Caucasian male fetus

who died at 23 weeks gestation due to fetal demise.

PLACFER01
pINCY
The library was constructed using RNA isolated from placental tissue removed from a Caucasian

fetus, who died after 16 weeks' gestation from fetal demise and hydrocephalus. Patient history included umbilical

cord wrapped around the head (3 times) and the shoulders (1 time). Serology was positive for anti-CMV. Family

history included multiple pregnancies and live births, and an abortion.

PROSTMT01
pINCY
Library was constructed using RNA isolated from diseased prostate tissue removed from a

67-year-old Caucasian male during radical prostatectomy with regional lymph node excision. Pathology indicated

adenofibromatous hyperplasia. Pathology for the associated tumor tissue indicated grade 3, Gleason

grade 3 + 3 adenocarcinoma. The patient presented elevated prostate specific

antigen (PSA) and induration. Patient history included hyperlipidemia cerebrovascular disease, and a

depressive disorder. Family history included atherosclerotic coronary artery disease and hyperlipidemia.

PROSTMY01
pINCY
This large size-fractionated cDNA and normalized library was constructed using RNA

isolated from diseased prostate tissue removed from a 55-year-old Caucasian male during closed prostatic

biopsy, radical prostatectomy, and regional lymph node excision. Pathology indicated adenofibromatous hyperplasia.

Pathology for the matched tumor tissue indicated adenocarcinoma Gleason grade 4 forming a predominant

mass involving the left side peripherally with extension into the right posterior superior region. The tumor

invaded the capsule and perforated the capsule to involve periprostatic tissue in the left posterior

superior region. The left inferior posterior and left superior posterior surgical margins are positive.

One left pelvic lymph node is metastatically involved. Patient history included calculus of the kidney.

Family history included lung cancer and breast cancer. The size-selected library was normalized in 1 round

using conditions adapted from Soares et al., PNAS (1994) 91: 9228-9232 and Bonaldo et al.,

Genome Research (1996) 6: 791.

PROSTUT09
pINCY
Library was constructed using RNA isolated from prostate tumor tissue removed from

a 66-year-old Caucasian male during a radical prostatectomy, radical cystectomy, and urinary diversion.

Pathology indicated grade 3 transitional cell carcinoma. The patient presented with prostatic inflammatory

disease. Patient history included lung neoplasm, and benign hypertension. Family history included a malignant

breast neoplasm, tuberculosis, cerebrovascular disease, atherosclerotic coronary artery disease and

lung cancer.

TESTTUT02
pINCY
Library was constructed using RNA isolated from testicular tumor removed from a

31-year-old Caucasian male during unilateral orchiectomy. Pathology indicated embryonal carcinoma.

THYMNOR02
pINCY
The library was constructed using RNA isolated from thymus tissue removed from

a 2-year-old Caucasian female during a thymectomy and patch closure of left atrioventricular fistula.

Pathology indicated there was no gross abnormality of the thymus. The patient presented with congenital

heart abnormalities. Patient history included double inlet left ventricle and a rudimentary right ventricle, pulmonary

hypertension, cyanosis, subaortic stenosis, seizures, and a fracture of the skull base.

Family history included reflux neuropathy.

UTRSTDT01
pINCY
Library was constructed using RNA isolated from uterus tissue removed from a 46-year-old

Caucasian female who died from cardiopulmonary arrest. Patient history included liver and breast cancer.

TABLE 7

Program
Description
Reference
Parameter Threshold

ABI
A program that removes vector sequences and masks
Applied Biosystems, Foster City, CA.

FACTURA
ambiguous bases in nucleic acid sequences.

ABI/
A Fast Data Finder useful in comparing and
Applied Biosystems, Foster City, CA;
Mismatch <50%

PARACEL
annotating amino acid or nucleic acid sequences.
Paracel Inc., Pasadena, CA.

FDF

ABI
A program that assembles nucleic acid sequences.
Applied Biosystems, Foster City, CA.

AutoAssembler

BLAST
A Basic Local Alignment Search Tool useful in
Altschul, S. F. et al. (1990) J. Mol. Biol.
ESTs: Probability value =

sequence similarity search for amino acid and nucleic
215: 403-410; Altschul, S. F. et al. (1997)
1.0E−8 or less; Full

acid sequences. BLAST includes five functions:
Nucleic Acids Res. 25: 3389-3402.
Length sequences: Probability

blastp, blastn, blastx, tblastn, and tblastx.

value = 1.0E−10

or less

FASTA
A Pearson and Lipman algorithm that searches for
Pearson, W. R. and D. J. Lipman (1988) Proc.
ESTs: fasta E value =

similarity between a query sequence and a group of
Natl. Acad Sci. USA 85: 2444-2448; Pearson,
1.06E−6; Assembled ESTs:

sequences of the same type. FASTA comprises as
W. R. (1990) Methods Enzymol. 183: 63-98;
fasta Identity = 95%

least five functions: fasta, tfasta, fastx, tfastx, and
and Smith, T. F. and M. S. Waterman (1981)
or greater and Match

ssearch.
Adv. Appl. Math. 2: 482-489.
length = 200 bases or greater;

fastx E value =

1.0E−8 or less;

Full Length sequences:

fastx score =

100 or greater

BLIMPS
A BLocks IMProved Searcher that matches a
Henikoff, S. and J. G. Henikoff (1991)
Probability value =

sequence against those in BLOCKS, PRINTS,
Nucleic Acids Res. 19: 6565-6572;
1.0E−3 or less

DOMO, PRODOM, and PFAM databases to search
Henikoff, J. G. and S. Henikoff (1996)

for gene families, sequence homology, and structural
Methods Enzymol. 266: 88-105;

fingerprint regions.
and Attwood, T. K. et al. (1997) J. Chem. Inf.

Comput. Sci. 37:

HMMER
An algorithm for searching a query sequence against
417-424. Krogh, A. et al. (1994) J. Mol. Biol.
PFAM, INCY, SMART or

hidden Markov model (HMM)-based databases of
235: 1501-1531; Sonnhammer, E. L. L. et al.
TIGRFAM hits:

protein family consensus sequences, such as PFAM,
(1988) Nucleic Acids Res. 26: 320-322;
Probability value =

INCY, SMART and TIGRFAM.
Durbin, R. et al. (1998) Our World View, in
1.0E−3 or less;

a Nutshell, Cambridge Univ. Press, pp. 1-350.
Signal peptide hits:

Score = 0 or greater

ProfileScan
An algorithm that searches for structural and
Gribskov, M. et al. (1988) CABIOS 4: 61-66;
Normalized quality

sequence motifs in protein sequences that match
Gribskov, M. et al. (1989) Methods
specified “HIGH” value for

sequence patterns defined in Prosite.
Enzymol. 183: 146-159; Bairoch, A. et al.
that score ≧ GCG

(1997) Nucleic Acids Res. 25: 217-221.
particular

Prosite motif.

Generally, score =

1.4-2.1.

Phred
A base-calling algorithm that examines automated
Ewing, B. et al. (1998) Genome Res. 8:

sequencer traces with high sensitivity and probability.
175-185; Ewing, B. and

P. Green (1998) Genome

Res. 8: 186-194.

Phrap
A Phils Revised Assembly Program including
Smith, T. F. and M. S. Waterman (1981) Adv.
Score = 120 or greater;

SWAT and CrossMatch, programs based on efficient
Appl. Math. 2: 482-489; Smith, T. F. and
Match length = 56

implementation of the Smith-Waterman algorithm,
M. S. Waterman (1981) J. Mol. Biol. 147:
or greater

useful in searching sequence homology and
195-197; and Green, P., University of

assembling DNA sequences.
Washington, Seattle, WA.

Consed
A graphical tool for viewing and editing Phrap
Gordon, D. et al. (1998) Genome Res. 8:

assemblies.
195-202.

SPScan
A weight matrix analysis program that scans protein
Nielson, H. et al. (1997) Protein Engineering
Score = 3.5 or greater

sequences for the presence of secretory signal
10: 1-6; Claverie, J. M. and S. Audic (1997)

peptides.
CABIOS 12: 431-439.

TMAP
A program that uses weight matrices to delineate
Persson, B. and P. Argos (1994) J. Mol. Biol.

transmembrane segments on protein sequences and
237: 182-192; Persson, B. and P. Argos

determine orientation.
(1996) Protein Sci. 5: 363-371.

TMHMMER
A program that uses a hidden Markov model (HMM)
Sonnhammer, E. L. et al. (1998) Proc. Sixth

to delineate transmembrane segments on protein
Intl. Conf. On Intelligent Systems for Mol.

sequences and determine orientation.
Biol., Glasgow et al., eds., The Am. Assoc.

for Artificial Intelligence (AAAI) Press,

Menlo Park, CA, and MIT Press, Cambridge,

MA, pp. 175-182.

Motifs
A program that searches amino acid sequences for
Bairoch, A. et al. (1997) Nucleic Acids Res.

patterns that matched those defined in Prosite.
25: 217-221; Wisconsin Package Program

Manual, version 9, page M51-59, Genetics

Computer Group, Madison, WI.

TABLE 8

SEQ

Caucasian
African
Asian
Hispanic

ID

EST
CB1
EST

Al-
Amino
Allele 1
Allele 1
Allele 1
Allele 1

NO:
PID
EST ID
SNP ID
SNP
SNP
Allele
Allele 1
lele 2
Acid
frequency
frequency
frequency
frequency

32
7506690
2700047H1
SNP00049844
45
2311
C
C
T
S696
n/a
n/a
n/a
n/a

32
7506690
8014939J2
SNP00049844
341
2312
C
C
T
S696
n/a
n/a
n/a
n/a

33
7506536
1456901H1
SNP00027692
199
790
C
C
G
P231
n/a
n/a
n/a
n/a

33
7506536
1794980H1
SNP00052100
268
1030
G
G
C
G311
n/d
n/a
n/a
n/a

33
7506536
3403559H1
SNP00052099
208
542
G
G
A
R148
n/a
n/a
n/a
n/a

33
7506536
3403559H1
SNP00098207
90
424
C
G
C
A109
n/a
n/a
n/a
n/a

33
7506536
3506466H1
SNP00052100
244
1029
G
G
C
G311
n/d
n/a
n/a
n/a

33
7506536
3743207H1
SNP00052100
33
1026
C
G
C
P310
n/d
n/a
n/a
n/a

34
7506537
1456901H1
SNP00027692
199
603
C
C
G
noncoding
n/a
n/a
n/a
n/a

34
7506537
1794980H1
SNP00052100
268
843
G
G
C
noncoding
n/d
n/a
n/a
n/a

34
7506537
3506466H1
SNP00052100
244
842
G
G
C
noncoding
n/d
n/a
n/a
n/a

34
7506537
3743207H1
SNP00052100
33
839
C
G
C
noncoding
n/d
n/a
n/a
n/a

34
7506537
3752762H1
SNP00098207
98
382
C
G
C
H109
n/a
n/a
n/a
n/a

34
7506537
6334553H1
SNP00052099
126
353
A
G
A
H99
n/a
n/a
n/a
n/a

35
7506655
1311380H1
SNP00097606
7
485
C
C
T
G157
n/a
n/a
n/a
n/a

35
7506655
1378619H1
SNP00062747
123
1704
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
1649327H1
SNP00097606
85
492
C
C
T
Q160
n/a
n/a
n/a
n/a

35
7506655
1678558H1
SNP00062747
185
1682
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
1782173H1
SNP00008154
23
2190
T
T
C
noncoding
n/a
n/a
n/a
n/a

35
7506655
2638672H1
SNP00062747
126
1701
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
2904178H1
SNP00062747
186
1703
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
3069052H1
SNP00062747
169
1700
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
3168038H1
SNP00062747
165
1699
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
3326702H1
SNP00097606
110
484
C
C
T
A157
n/a
n/a
n/a
n/a

35
7506655
3353963H1
SNP00097606
124
475
C
C
T
A154
n/a
n/a
n/a
n/a

35
7506655
3602992H1
SNP00097606
41
482
C
C
T
G156
n/a
n/a
n/a
n/a

35
7506655
3720990H1
SNP00062747
82
1698
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
3902692H1
SNP00106135
237
1310
A
G
A
noncoding
n/a
n/a
n/a
n/a

35
7506655
4564431H1
SNP00097606
97
483
C
C
T
R157
n/a
n/a
n/a
n/a

35
7506655
4648883H1
SNP00062747
184
1702
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
5199479H1
SNP00062747
231
1705
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
6413315H1
SNP00062747
341
1697
A
A
G
noncoding
n/a
n/a
n/a
n/a

35
7506655
6479857H1
SNP00008153
403
973
C
T
C
A320
n/a
n/a
n/a
n/a

35
7506655
684419H1
SNP00098150
15
917
A
A
G
E301
n/d
n/d
n/d
n/d

35
7506655
7039242H1
SNP00008153
192
980
T
T
C
S322
n/a
n/a
n/a
n/a

35
7506655
7081283H1
SNP00130169
64
7
C
C
T
noncoding
n/a
n/a
n/a
n/a

35
7506655
7127858H1
SNP00062747
28
1655
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
1006142H1
SNP00098150
106
918
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
1311380H1
SNP00097606
7
485
C
C
T
G157
n/a
n/a
n/a
n/a

36
7506656
1378619H1
SNP00062747
123
2042
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
1649327H1
SNP00097606
85
492
C
C
T
Q160
n/a
n/a
n/a
n/a

36
7506656
1672772H1
SNP00098150
168
930
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
1678558H1
SNP00062747
185
2020
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
1679821H1
SNP00153045
117
1143
G
G
A
noncoding
n/a
n/a
n/a
n/a

36
7506656
1698338H1
SNP00008153
174
921
T
T
C
noncoding
n/a
n/a
n/a
n/a

36
7506656
1782173H1
SNP00008154
23
2528
T
T
C
noncoding
n/a
n/a
n/a
n/a

36
7506656
1800953H1
SNP00092571
204
1413
C
C
T
noncoding
n/a
n/a
n/a
n/a

36
7506656
193898H1
SNP00098150
31
931
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
2187682H1
SNP00098150
16
929
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
2638672H1
SNP00062747
126
2039
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
2904178H1
SNP00062747
186
2041
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
3069052H1
SNP00062747
169
2038
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
3168038H1
SNP00062747
165
2037
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
3212643H1
SNP00098150
111
927
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
3285931H1
SNP00098150
32
928
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
3321710H1
SNP00092571
149
1412
C
C
T
noncoding
n/a
n/a
n/a
n/a

36
7506656
3326702H1
SNP00097606
110
484
C
C
T
A157
n/a
n/a
n/a
n/a

36
7506656
3353963H1
SNP00097606
124
475
C
C
T
A154
n/a
n/a
n/a
n/a

36
7506656
3602992H1
SNP00097606
41
482
C
C
T
G156
n/a
n/a
n/a
n/a

36
7506656
3642390H1
SNP00153045
2
1140
G
G
A
noncoding
n/a
n/a
n/a
n/a

36
7506656
3690567H1
SNP00008153
203
918
T
T
C
noncoding
n/a
n/a
n/a
n/a

36
7506656
3720990H1
SNP00062747
82
2036
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
3885387H2
SNP00098150
14
917
A
A
G
noncoding
n/d
n/d
n/d
n/d

36
7506656
3902692H1
SNP00106135
237
1648
A
G
A
noncoding
n/a
n/a
n/a
n/a

36
7506656
4564431H1
SNP00097606
97
483
C
C
T
R157
n/a
n/a
n/a
n/a

36
7506656
4648883H1
SNP00062747
184
2040
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
5199479H1
SNP00062747
231
2043
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
5289285H1
SNP00153045
55
1142
G
G
A
noncoding
n/a
n/a
n/a
n/a

36
7506656
6413315H1
SNP00062747
341
2035
A
A
G
noncoding
n/a
n/a
n/a
n/a

36
7506656
7081283H1
SNP00130169
64
7
C
C
T
noncoding
n/a
n/a
n/a
n/a

36
7506656
7127858H1
SNP00062747
28
1993
A
A
G
noncoding
n/a
n/a
n/a
n/a

37
7510567
4218337F6
SNP00148602
37
896
C
C
T
noncoding
n/a
n/a
n/a
n/a

37
7510567
7137262H1
SNP00071130
184
257
G
A
G
G43
0.92
0.92
0.89
0.89

38
7506072
1535170H1
SNP00009199
149
1756
C
C
T
noncoding
n/a
n/a
n/a
n/a

38
7506072
1535170H1
SNP00037178
68
1675
C
C
T
noncoding
n/a
n/a
n/a
n/a

38
7506072
1535170H1
SNP00144912
35
1642
T
T
C
noncoding
n/a
n/a
n/a
n/a

38
7506072
2681553H1
SNP00125000
231
1755
G
A
G
noncoding
n/a
n/a
n/a
n/a

38
7506072
2790256H2
SNP00037177
57
894
G
A
G
noncoding
n/a
n/a
n/a
n/a

38
7506072
2924591H1
SNP00037177
185
898
G
A
G
noncoding
n/a
n/a
n/a
n/a

38
7506072
3817244H1
SNP00144911
198
1458
A
C
A
noncoding
n/a
n/a
n/a
n/a

38
7506072
3880004H1
SNP00144911
226
1460
A
C
A
noncoding
n/a
n/a
n/a
n/a

38
7506072
4069389H1
SNP00009199
251
1754
C
C
T
noncoding
n/a
n/a
n/a
n/a

38
7506072
4069389H1
SNP00144912
137
1640
T
T
C
noncoding
n/a
n/a
n/a
n/a

38
7506072
4163749H1
SNP00009199
32
1755
C
C
T
noncoding
n/a
n/a
n/a
n/a

38
7506072
4183639H1
SNP00037178
174
1673
T
C
T
noncoding
n/a
n/a
n/a
n/a

38
7506072
4709207H1
SNP00144911
224
1459
A
C
A
noncoding
n/a
n/a
n/a
n/a

38
7506072
7027968H1
SNP00125000
402
1757
A
A
G
noncoding
n/a
n/a
n/a
n/a

38
7506072
7374370H1
SNP00053945
541
540
A
A
G
E110
n/d
n/d
n/d
n/d

39
7511354
1658373T6
SNP00015452
2
2260
A
G
A
noncoding
n/a
n/a
n/a
n/a

39
7511354
1872966T6
SNP00015452
37
2275
G
G
A
noncoding
n/a
n/a
n/a
n/a

39
7511354
7168495H1
SNP00132303
109
285
A
A
G
T32
n/a
n/a
n/a
n/a

39
7511354
7168495H1
SNP00132304
126
302
A
A
G
H38
n/a
n/a
n/a
n/a

39
7511354
7422001T1
SNP00015452
42
2237
G
G
A
noncoding
n/a
n/a
n/a
n/a

40
7511643
1335233H1
SNP00070206
135
2035
A
A
C
E659
n/a
n/a
n/a
n/a

40
7511643
1736837F6
SNP00040689
328
2382
A
A
G
noncoding
n/a
n/a
n/a
n/a

40
7511643
1808096F6
SNP00040688
213
2069
T
T
C
G670
n/a
n/a
n/a
n/a

40
7511643
5654454H1
SNP00040689
268
2386
A
A
G
noncoding
n/a
n/a
n/a
n/a

40
7511643
6454710H1
SNP00067649
402
402
G
A
G
E115
0.17
0.13
0.33
0.29

40
7511643
6801870J1
SNP00070206
130
2014
C
A
C
P652
n/a
n/a
n/a
n/a

40
7511643
7007828H1
SNP00010482
335
1999
G
G
A
R647
n/a
n/a
n/a
n/a

41
7511400
4767844H1
SNP00134633
31
569
T
T
C
W87
n/a
n/a
n/a
n/a

41
7511400
4767844H1
SNP00134634
140
678
C
C
T
P123
n/a
n/a
n/a
n/a

42
7511507
1254303F1
SNP00009660
349
1232
C
C
T
I274
0.73
0.73
0.8
0.79

42
7511507
1254303F1
SNP00009661
526
1409
C
C
T
Y333
0.92
0.95
0.97
0.91

42
7511507
1254303F1
SNP00116740
96
979
G
G
A
R190
n/d
n/d
n/d
n/d

42
7511507
1298133H1
SNP00037904
77
2409
A
A
C
noncoding
n/d
n/a
n/a
n/a

42
7511507
1322651T6
SNP00009662
222
2148
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
1414920T6
SNP00009662
241
2160
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
1436024T6
SNP00009662
225
2178
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
1557825F6
SNP00009660
139
1233
C
C
T
H275
0.73
0.73
0.8
0.79

42
7511507
1557825T6
SNP00009662
219
2162
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
1600521T6
SNP00009662
205
2181
G
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
1682522F6
SNP00116740
222
981
G
G
A
G191
n/d
n/d
n/d
n/d

42
7511507
1955727H1
SNP00058560
144
1775
C
C
T
noncoding
n/d
n/a
n/a
n/a

42
7511507
2060177T6
SNP00009662
239
2169
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
2543695T6
SNP00009662
216
2168
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
2734727H1
SNP00116739
191
667
C
C
A
T86
n/d
n/a
n/d
n/d

42
7511507
2890616T6
SNP00009662
191
2198
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
3113405H1
SNP00116738
11
2
C
C
G
noncoding
n/a
n/a
n/a
n/a

42
7511507
3905760H1
SNP00054891
112
1467
T
C
T
L353
n/a
n/a
n/a
n/a

42
7511507
424333R6
SNP00116740
228
980
G
G
A
R190
n/d
n/d
n/d
n/d

42
7511507
4805531H1
SNP00009660
18
1234
C
C
T
A275
0.73
0.73
0.8
0.79

42
7511507
4805531H1
SNP00009661
195
1411
C
C
T
P334
0.92
0.95
0.97
0.91

42
7511507
7644762J1
SNP00009662
111
2149
A
A
G
noncoding
0.97
n/d
n/d
0.99

42
7511507
7708989J1
SNP00009661
241
1410
C
C
T
P334
0.92
0.95
0.97
0.91

42
7511507
7752348J1
SNP00009660
299
1203
C
C
T
H265
0.73
0.73
0.8
0.79

42
7511507
7752348J1
SNP00009661
476
1380
C
C
T
L324
0.92
0.95
0.97
0.91

42
7511507
7752348J1
SNP00116740
46
950
A
G
A
stop180
n/d
n/d
n/d
n/d

42
7511507
7762216J1
SNP00009660
353
1207
C
C
T
T266
0.73
0.73
0.8
0.79

42
7511507
7762216J1
SNP00116740
100
954
G
G
A
D182
n/d
n/d
n/d
n/d

43
7511819
1556245F6
SNP00114741
11
3747
A
A
G
noncoding
n/d
n/a
n/a
n/a

43
7511819
1556245F6
SNP00114742
26
3762
A
A
G
noncoding
0.98
n/a
n/a
n/a

43
7511819
2304978H1
SNP00036174
57
2430
G
G
C
noncoding
n/a
n/a
n/a
n/a

43
7511819
3000013H1
SNP00042735
170
1746
G
A
G
V551
n/a
n/a
n/a
n/a

43
7511819
5043136H1
SNP00114740
126
3305
G
G
C
noncoding
n/a
n/a
n/a
n/a

43
7511819
7761779J1
SNP00036174
419
2399
G
G
C
noncoding
n/a
n/a
n/a
n/a

44
7511338
028327H1
SNP00139433
23
684
C
C
T
noncoding
n/a
n/a
n/a
n/a

44
7511338
1700475T6
SNP00139433
152
685
C
C
T
noncoding
n/a
n/a
n/a
n/a

44
7511338
1843678H1
SNP00008138
183
138
C
C
G
noncoding
n/a
n/a
n/a
n/a

44
7511338
1906256H1
SNP00034004
223
881
C
C
G
noncoding
n/a
n/a
n/a
n/a

44
7511338
1976291T6
SNP00139433
13
837
C
C
T
noncoding
n/a
n/a
n/a
n/a

44
7511338
2061585T6
SNP00116497
29
820
C
C
T
noncoding
n/a
n/a
n/a
n/a

44
7511338
2061585T6
SNP00139433
163
686
C
C
T
noncoding
n/a
n/a
n/a
n/a

44
7511338
2367548H1
SNP00116497
195
818
C
C
T
noncoding
n/a
n/a
n/a
n/a

44
7511338
2502746H1
SNP00125461
103
188
C
C
T
N10
n/a
n/a
n/a
n/a

44
7511338
6016362H1
SNP00050967
37
245
T
T
G
G29
n/a
n/a
n/a
n/a

44
7511338
6828414H1
SNP00050967
505
246
T
T
G
C30
n/a
n/a
n/a
n/a

45
7511425
1007336H1
SNP00027190
48
3075
C
C
G
S982
0.95
n/a
n/a
n/a

45
7511425
1299176T6
SNP00150051
94
3764
C
C
T
noncoding
n/a
n/a
n/a
n/a

45
7511425
1868109H1
SNP00025347
245
2466
G
G
A
E779
n/a
n/a
n/a
n/a

45
7511425
1957925H1
SNP00076280
205
2994
C
C
T
L955
n/a
n/a
n/a
n/a

45
7511425
2692870H1
SNP00076279
70
2081
G
A
G
R651
0.47
n/a
n/a
n/a

45
7511425
2692870H1
SNP00100766
83
2094
T
T
C
C655
n/a
n/a
n/a
n/a

45
7511425
3170918H1
SNP00103142
27
2147
C
C
T
T673
n/d
n/a
n/a
n/a

45
7511425
6710722H1
SNP00141033
33
3510
C
C
T
noncoding
n/a
n/a
n/a
n/a

45
7511425
724339T6
SNP00141033
309
3602
C
C
T
noncoding
n/a
n/a
n/a
n/a

45
7511425
842889T6
SNP00141033
314
3595
C
C
T
noncoding
n/a
n/a
n/a
n/a

45
7511425
8617639H1
SNP00150051
107
3439
G
G
A
A1104
n/a
n/a
n/a
n/a

45
7511425
8617639J1
SNP00150051
473
3464
G
G
A
S1112
n/a
n/a
n/a
n/a

45
7511425
873395T6
SNP00141033
404
3515
C
C
T
noncoding
n/a
n/a
n/a
n/a

46
7511534
1658373T6
SNP00015452
2
2484
A
G
A
noncoding
n/a
n/a
n/a
n/a

46
7511534
1830479T6
SNP00015452
68
2485
G
G
A
noncoding
n/a
n/a
n/a
n/a

46
7511534
1872966T6
SNP00015452
37
2499
G
G
A
noncoding
n/a
n/a
n/a
n/a

46
7511534
7168495H1
SNP00132303
109
285
A
A
G
T32
n/a
n/a
n/a
n/a

46
7511534
7168495H1
SNP00132304
126
302
A
A
G
H38
n/a
n/a
n/a
n/a

46
7511534
7422001T1
SNP00015452
42
2461
G
G
A
noncoding
n/a
n/a
n/a
n/a

47
7511648
4056310T6
SNP00105090
164
2987
C
C
T
V987
n/a
n/a
n/a
n/a

47
7511648
7057433F6
SNP00141841
216
2884
T
T
C
L953
n/a
n/a
n/a
n/a

48
7511600
1275278H1
SNP00020695
89
776
C
C
T
A233
n/a
n/a
n/a
n/a

48
7511600
1480416H1
SNP00020694
79
369
A
A
G
A97
0.95
n/a
n/a
n/a

48
7511600
3016303H1
SNP00136636
173
680
C
C
A
T201
n/d
n/a
n/a
n/a

48
7511600
3088849H1
SNP00020446
65
137
G
A
G
S20
n/d
n/d
n/d
n/a

48
7511600
8020007J1
SNP00020694
480
397
A
A
G
K107
0.95
n/a
n/a
n/a

48
7511600
8020007J1
SNP00136636
169
708
C
C
A
G210
n/d
n/a
n/a
n/a

49
7511783
1456284H1
SNP00140641
213
427
C
C
A
A90
n/a
n/a
n/a
n/a

49
7511783
2110750H1
SNP00053655
125
713
G
G
A
R185
n/a
n/a
n/a
n/a

49
7511783
2138747H1
SNP00050861
186
156
G
G
T
noncoding
0.99
n/a
n/a
n/a

49
7511783
2138747T6
SNP00053655
62
752
G
G
A
noncoding
n/a
n/a
n/a
n/a

49
7511783
2359491T6
SNP00140641
393
433
C
C
A
T92
n/a
n/a
n/a
n/a

50
7512383
1230087T6
SNP00011822
245
2873
G
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1230947H1
SNP00011822
178
2892
G
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1290019H1
SNP00011822
38
3256
C
C
T
noncoding
n/a
n/a
n/a
n/a

50
7512383
1372412T6
SNP00011822
243
2889
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1396849H1
SNP00069830
89
1700
G
G
A
E504
n/a
n/a
n/a
n/a

50
7512383
1482967T6
SNP00011822
241
2886
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1614437F6
SNP00011822
133
2894
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1618583T6
SNP00011822
243
2872
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1619082T6
SNP00011822
239
2868
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1939843R6
SNP00011822
259
2853
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
1993601T6
SNP00011822
245
2867
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
2105564H1
SNP00020343
93
1257
C
C
T
S356
n/a
n/a
n/a
n/a

50
7512383
2105564T6
SNP00011822
242
2882
G
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
2114872T6
SNP00011822
244
2883
G
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
4259235T1
SNP00011822
221
2893
A
G
A
noncoding
n/a
n/a
n/a
n/a

50
7512383
613960R7
SNP00069403
212
474
G
G
A
G95
0.99
n/a
n/a
n/a

50
7512383
7418612T1
SNP00011822
174
2891
A
G
A
noncoding
n/a
n/a
n/a
n/a

Number	Date	Country	Kind
60379840	May 2002	US	national
60381291	May 1992	US	national
60383183	May 2002	US	national
60394146	Jul 2002	US	national

Cell adhesion and extracellular matrix proteins

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

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Priority Claims (4)

PCT Information