WO2003027230A2 - Proteines de matrice extracellulaire et d'adhesion cellulaire - Google Patents

Proteines de matrice extracellulaire et d'adhesion cellulaire Download PDF

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WO2003027230A2
WO2003027230A2 PCT/US2002/024649 US0224649W WO03027230A2 WO 2003027230 A2 WO2003027230 A2 WO 2003027230A2 US 0224649 W US0224649 W US 0224649W WO 03027230 A2 WO03027230 A2 WO 03027230A2
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polynucleotide
polypeptide
seq
sequence
amino acid
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WO2003027230A3 (fr
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Neil Burford
Bridget A. Warren
Brendan M. Duggan
Patricia M. Lehr-Mason
Thomas W. Richardson
Henry Yue
Ian J. Forsythe
Vicki S. Elliott
Jennifer A. Griffin
Ann E. Gorvad
Yalda Azimzai
Deborah A. Kallick
Yuming Xu
Cynthia D. Honchell
Mariah R. Baughn
Kimberly J. Gietzen
Sally Lee
Narinder K. Walia
Y. Tom Tang
Danniel B. Nguyen
Shanya D. Becha
Soo Yeun Lee
Jayalaxmi Ramkumar
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Incyte Genomics, Inc.
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Priority to AU2002332454A priority Critical patent/AU2002332454A1/en
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Publication of WO2003027230A3 publication Critical patent/WO2003027230A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention relates to novel nucleic acids, cell adhesion and extracellular matrix proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of irnmune system disorders, neurological disorders, developmental disorders, connective tissue disorders, genetic disorders, and cell proliferative disorders, including cancer.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and cell adhesion and extracellular matrix proteins.
  • the surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the ECM.
  • the interaction of the cell with its surroundings profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. Families of cell adhesion molecules include the cadherins, integrins, lectins, neural cell adhesion proteins, and some members of the proline-rich proteins.
  • Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms.
  • cadherin molecules cooperate to form focal contacts, or adhesion plaques, between adjacent epithelial cells.
  • the cadherin family includes the classical cadherins and protocadherins.
  • Classical cadherins include the E-cadherin, N-cadherin, and P-cadherin subfamilies.
  • E-cadherin is present on many types of epithelial cells and is especially important for embryonic development.
  • N-cadherin is present on nerve, muscle, and lens cells and is also critical for embryonic development.
  • P-cadherin is present on cells of the placenta and epidermis.
  • cadherins are involved in a variety of cell-cell interactions (Suzuki, S.T. (1996) J. Cell Sci. 109:2609-2611).
  • the intracellular anchorage of cadherins is regulated by their dynamic association with catenins, a family of cytoplasmic signal transduction proteins associated with the actin cytoskeleton.
  • the anchorage of cadherins to the actin cytoskeleton appears to be regulated by protein tyrosine phosphorylation, and the cadherins are the target of phosphorylation-induced junctional disassembly (Aberle, H. et al. (1996) J. Cell Biochem. 61:514-523).
  • Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to the internal cytoskeleton. Integrins are composed of two noncovalently associated transmembrane glycoprotein subunits called ⁇ and ⁇ . At least 8 different ⁇ subunits ( ⁇ l-B8) and at least 12 different ⁇ subunits have been identified ( ⁇ l-oc8, L, oM, cxX, and Hb). Individual ⁇ subunits are capable of associating with different ⁇ subunits, suggesting a possible mechanism for specifying integrin function and ligand binding affinity. Members of the ⁇ subunit family are generally of 90-110 kilodaltons (kD) in molecular weight and share about 40-48% arrrino acid sequence homology.
  • kD kilodaltons
  • cysteines distributed among four repeating units are also conserved. Some variation in these conserved features is observed among some of the more divergent ⁇ subunit family members. Members of the subunit family are generally 150-200 kilodaltons in molecular weight and are not as well conserved as the ⁇ subunit family. All contain seven repeating domains of 24-45 amino acids spaced about 20-35 amino acids apart. The N-termini each contain 3-4 divalent cation binding sites. (For review, see Pigott, R. and C. Power (1994) The Adhesion Molecule Facts Book, Academic Press, San Diego, CA, pp. 9- 12.)
  • Integrins function as receptors that specifically recognize and bind to ECM proteins such as fibronectin, fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand factor, and collagen. Some integrins recognize a specific motif, the RGD sequence, at the C-termini of the ECM proteins they bind. Integrins also bind to immunoglobulin superfamily proteins such as ICAM-1, -2, and -3 and NCAM-1.
  • FAK focal adhesion kinase
  • MAPK mitogen-activated protein kinase
  • 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). 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 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).
  • RTKs growth factor receptor tyrosine kinases
  • LAD leukocyte adhesion deficiency
  • RNA encoding the ⁇ 2 subunit defects in platelet integrin are correlated with Glanzmann's thrombasthemia, a bleeding disorder characterized by insufficient platelet aggregation.
  • Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K.
  • Lectins are further classified into subfamilies based on carbohydrate-binding specificity and other criteria.
  • the galectin subfamily includes lectins that bind ⁇ -galactoside carbohydrate moieties in a thiol-dependent manner (reviewed in Hadari, Y.R. et al. (1998) J. Biol. Chem. 270:3447-3453).
  • Galectins are widely expressed and developmentally regulated.
  • Galectins contain a characteristic carbohydrate recognition domain (CRD).
  • the CRD comprises about 140 amino acids and contains several stretches of about 1 - 10 amino acids which are highly conserved among all galectins.
  • a particular 6-amino acid motif within the CRD contains conserved tryptophan and arginine residues which are critical for carbohydrate binding.
  • the CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation. Secondary structure predictions indicate that the CRD forms several ⁇ -sheets.
  • Galectins play a number of roles in diseases and conditions associated with cell-cell and cell- matrix interactions. For example, certain galectms associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice. Moreover, anti-galectin antibodies inhibit processes associated with cell transformation, such as cell aggregation and anchorage-independent growth (see, for example, Su, Z.-Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7252-7257).
  • Selectins comprise a specialized lectin subfamily involved primarily in inflammation and leukocyte adhesion (Reviewed in Lasky, supra). Selectins mediate the recruitment of leukocytes from the circulation to sites of acute inflammation and are expressed on the surface of vascular endothelial cells in response to cytokine signaling. Selectins bind to specific ligands on the leukocyte cell membrane and enable the leukocyte to adhere to and migrate along the endothelial surface. Binding of selectin to its ligand leads to polarized rearrangement of the actin cytoskeleton and stimulates signal transduction within the leukocyte (Brenner, B. et al. (1997) Biochem. Biophys. Res. Commun.
  • NCAPs Neural cell adhesion proteins
  • NCAPS genes encoding NCAPS are linked with neurological diseases, including hereditary neuropathy, Charcot-Marie-Tooth disease, Dejerine-Sottas disease, X-linked hydrocephalus, MASA syndrome (mental retardation, aphasia, shuffling gait and adducted thumbs), and spastic paraplegia type I.
  • expression of NCAP is not restricted to the nervous system.
  • LI for example, is expressed in melanoma cells and hematopoietic tumor cells where it is implicated in cell spreading and migration, and may play a role in tumor progression (Montgomery, A.M. et al. (1996) J. Cell Biol. 132:475-485).
  • NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et al., supra). They are generally linked to the plasma membrane through a tiansmembrane 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.
  • GPI glycosyl-phosphatidylinositol
  • NCAPs fall into three subgroups: simple-type, complex-type, and mixed-type.
  • Simple-type NCAPs contain one or more variable or constant immunoglobulin domains, but lack other types of domains.
  • Members of the simple-type subgroup include Schwann cell myelin protein (SMP), limbic system-associated membrane protein (LAMP), opiate-binding cell-adhesion molecule (OBCAM), and myelin-associated glycoprotein (MAG).
  • SMP Schwann cell myelin protein
  • LAMP limbic system-associated membrane protein
  • OBCAM opiate-binding cell-adhesion molecule
  • MAG myelin-associated glycoprotein
  • the complex-type NCAPs contain fibronectin type HI domains in addition to the immunoglobulin domains.
  • the complex-type subgroup includes neural cell-adhesion molecule (NCAM), axonin-1, Fll, Bravo, and LI.
  • NCAM neural cell-adhesion molecule
  • 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 ⁇ (GGF ⁇ .) and acetylcholine receptor-inducing factor (ARIA), and the semaphorin/collapsin family such as semaphorin B and collapsin.
  • NGF nerve growth factor
  • NT4 neurotropin 4
  • GGF ⁇ glial growth factor ⁇
  • ARIA acetylcholine receptor-inducing factor
  • semaphorin/collapsin family such as semaphorin B and collapsin.
  • Semaphorms 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).
  • NCAP subfamily includes cell adhesion proteins expressed on distinct subpopulations of brain neurons.
  • Members of the NCAP-LON subgroup possess three immunoglobulin domains and bind to cell membranes through GPI anchors.
  • Kilon (a kindred of NCAP-LON), for example, is expressed in the brain cerebral cortex and hippocampus (Funatsu, N. et al. (1999) J. Biol. " Chem. 274:8224-8230). J-mmunostaining localizes Kilon to the dendrites and soma of pyramidal neurons.
  • Kilon has three C2 type irnmunoglobulin-like domains, six predicted glycosylation sites, and a GPI anchor.
  • 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.
  • LFA-1 Leukocyte function-associated antigen 1
  • ICAM intercellular adhesion molecules
  • 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 arnino acid content.
  • PRPs have short domains which are rich in proline. These proline-rich regions are associated with protein-protein interactions.
  • PRPs proline-rich synapse-associated proteins
  • PSD postsynaptic density
  • ProSAP family 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.
  • SH3 domain ankyrin repeats at the N-terminus
  • PDZ domain a PDZ domain
  • SAM domain 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 HLA-B-associated transcript 2 protein
  • 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 (PROSLTE 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, CP. 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 (Pouting, CP. et al. (1999) Curr. Biol. 9:R585-R588).
  • LRR Leucine rich repeats
  • LRR motifs are short motifs found in numerous proteins from a wide range of species. LRR motifs are of variable length, most commonly 20-29 amino acids, and multiple repeats are typically present in tandem. LRR motifs are important for protein/protein interactions and cell adhesion, and LRR proteins are involved in cell/cell interactions, morphogenesis, and development (Kobe, B. and J. Deisenhofer (1995) Curr. Opin. Struct. Biol. 5:409-416).
  • the human ISLR (immunoglobulin superfamily containing leucine-rich repeat) protein contains a C2-type irnmunoglobulin domain as well as LRR motifs.
  • the ISLR gene is linked to the critical region for Bardet-Biedl syndrome, a developmental disorder of which the most common feature is retinal dystrophy (Nagasawa, A. et al. (1999) Genomics 61:37-43).
  • the sterile alpha motif (SAM) domain is a conserved protein binding domain, approximately 70 amino acids in length, and is involved in the regulation of many developmental processes in eukaryotes.
  • SAM domain can potentially function as a protein interaction module through its ability to form homo- or hetero-oligomers with other SAM domains (Schultz, J. et al. (1997) Protein Sci. 6:249-253).
  • Extracellular Matrix Proteins Extracellular Matrix Proteins
  • the extracellular matrix is a complex network of glycoproteins, polysaccharides, proteoglycans, and other macromolecules that are secreted from the cell into the extracellular space.
  • the ECM remains in close association with the cell surface and provides a supportive meshwork that profoundly influences cell shape, motility, strength, flexibility, and adhesion. In fact, adhesion of a cell to its surrounding matrix is required for cell survival except in the case of metastatic tumor cells, which have overcome the need for cell-ECM anchorage. This phenomenon suggests that the ECM plays a critical role in the molecular mechanisms of growth control and metastasis. (Reviewed in Ruoslahti, E. (1996) Sci. Am. 275:72-77.) Furthermore, the ECM 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, KJ. et al. (1994) Harrison's Principles of Internal Medicine. McGraw-Hill, Inc., New York, NY, pp. 2105-2117; and Creighton, T.E. (1984) Proteins, Structures and Molecular Principles. W.H. Freeman and Company, New York, NY, pp. 191-197.)
  • the gene coding for the mouse alphal(XUI) collagen chain, coll3al, is approximately 135 kb in size and contains 42 exons varying between 8 base pairs (bp) and 836 bp. It has multiple transcription initiation points, ranging between 470 and 548 bp upstream from the initiation methionine. It has many 27 -bp exons, which is half the ancestral 54-bp size characteristic of fibrillar collagen genes. The other exons vary between 8 and 144 bp, including instances of 36-, 45- and 54-bp exons.
  • 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 PubHshing, New York, NY, pp. 984-986). The fibulin proteins connect elastic fibers and are though to promote the formation and stabilization of the fiber.
  • 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 HI fibronectin repeat. The type IU 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.
  • some type HI fibronectin repeats contain a characteristic tripeptide consisting of Arginine-Glycine-Aspartic acid (RGD).
  • RGD Arginine-Glycine-Aspartic acid
  • 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).
  • 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 tiansforming growth factor ⁇ , suggesting a role for proteoglycans in cell-cell communication (reviewed in Alberts et al., supra, pp. 973-978).
  • 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 is a major component of the dentin ECM.
  • DPP is a proteoglycan mat 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 gallbladder, 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 ⁇ GR/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 detern ining the invasive potential of brain tumor cells (Gary, S.C.
  • Multidomain or mosaic proteins play an important role in the diverse functions of the extracellular matrix (Engel, J. et al. (1994) Development (Camb.):S35-S42).
  • ECM proteins are frequently characterized by the presence of one or more domains which may contain a number of potential intracellular disulfide bridge motifs.
  • domains which match the epidermal growth factor (EGF) tandem repeat consensus are present within several known extracellular proteins that promote cell growth, development, and cell signaling.
  • This signature sequence is about forty amino acid residues in length and includes six conserved cysteine residues, and a calcium-binding site near the N-terminus of the signature sequence.
  • the main structure is a two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet.
  • Subdomains between the conserved cysteines vary in length (Davis, CG. (1990) New Biol. 5:410-419).
  • Post-translational hydroxylation of aspartic acid or asparagine residues has been associated with EGF-like domains in several proteins (Prosite PDOC00010 Aspartic acid and asparagine hydroxylation site).
  • EGF-like domain signature sequences 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 teansfo ⁇ ning growth factor beta-1 binding protein (Expasy PROSITE document PDOC00913; Soler, C. and G. Carpenter, in Nicola, N.A. (1994) The Cvtokine 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.
  • the Slit protein first identified in Drosophila, is critical in central nervous system midline formation and potentially in nervous tissue histogenesis and axonal pathfinding. Itoh et al. have identified mammalian homologues of the slit gene (human Slit-1, Slit-2, Slit-3 and rat Slit-1). The encoded proteins are putative secreted proteins containing EGF-like motifs and leucine-rich repeats, both are conserved protein-protein interaction domains. Slit-1, -2, and -3 mRNAs are expressed in the brain, spinal cord, and thyroid, respectively (Itoh, A. et al. (1998) Brain Res. Mol. Brain Res. 62:175- 186).
  • the Slit family of proteins are indicated to be functional ligands of glypican-1 in nervous tissue and suggests that their interactions may be critical in certain stages during central nervous system histogenesis (Liang, Y. et al. (1999) J. Biol. Chem. 274:17885-17892).
  • 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 HI 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 CUB domain profile).
  • 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 stracture 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).
  • TSR thrombospondin type 1 repeat
  • the TSR is approximately 60 amino acids in length and contains six conserved cysteine residues. Motifs within TSR domains are involved in mediating 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 Fibrinogen beta and gamma chains C- terminal domain signature).
  • E2 is a 32 kd human T-cell surface glycoprotein involved in spontaneous rosette formation with erythrocytes. It is involved in T cell adhesion processes and is the product of the MIC2 gene, the only pseudoautosomal gene to be described in man.
  • Xg(a-) female individuals have no E2 molecule on the surface of their red cells, in contrast with Xg(a+) individuals, but have the molecule in their cytoplasm, in the form of the 28 kd precursor (Gelin, C et al. (1989) EMBO J. 8:3253-3259).
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • 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.
  • Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers.
  • Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium.
  • metastasis has already occurred.
  • Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and liver.
  • the decision to treat with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to staging of lung cancer could positively affect patient outcome.
  • Non Small Cell Lung Carcinoma Squamous cell carcinomas
  • Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands.
  • Squamous cell carcinomas typically arise in proximal airways.
  • the histogenesis of squamous cell carcinomas may be related to chronic inflammation and injury to the bronchial epithehum, leading to squamous metaplasia.
  • SCLC Small Cell Lung Carcinoma
  • Lung cancer cells accumulate numerous genetic lesions, many of which are associated with cytologically visible chromosomal aberrations.
  • the high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earliest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common.
  • Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2.
  • thrombospondin- 1, fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentially expressed in lung cancers.
  • Wang et al. 2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentially overexpresssed in squamous cell carcinoma compared with normal lung epitihelium.
  • the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 and cytokeratin 13.
  • 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. The molecular events that lead to ovarian cancer are poorly understood. Some of the known aberrations include mutation of p53 and microsatellite instability. Since gene expression patterns are likely to vary when normal ovary is compared to ovarian tumors, examination of gene expression in these tissues to identify possible markers for ovarian cancer is particularly relevant to improving diagnosis, prognosis, and treatment of this disease. Colon Cancer
  • Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year. Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes (Potter, J.D. (1999) J. Natl. Cancer Institute 91:916-932). Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et al. (1990) National Institutes of Health, DHHS PublNo. (NTH)90-2789).
  • Colon cancer is causally related to both genes and the environment.
  • Several molecular pathways have been linked to the development of colon cancer, and the expression of key genes in any of these pathways may be lost by inherited or acquired mutation or by hypermethylation.
  • There is a particular need to identify genes for which changes in expression may provide an early indicator of colon cancer or a predisposition for the development of colon cancer.
  • DNA methyltransferase the enzyme that performs DNA methylation
  • histologically normal mucosa from patients with colon cancer or the benign polyps that precede cancer, and this increase continues during the progression of colonic neoplasms (Wafik, S. et al. (1991) Proc. Natl. Acad. Sci. USA 88:3470-3474).
  • CpG islands G+C rich areas of genomic DNA termed "CpG islands” that are important for maintenance of an "open” transcriptional conformation around genes, and that hypermethylation of these regions results in a "closed” conformation that silences gene transcription. It has been suggested that the silencing or downregulation of differentiation genes by such abnormal methylation of CpG islands may prevent differentiation in immortalized cells (Anteguera, F. et al. (1990) Cell 62:503-514). Familial Adenomatous Polyposis (FAP) is a rare autosomal dominant syndrome that precedes colon cancer and is caused by an inherited mutation in the adenomatous polyposis coli (APC) gene.
  • FAP Familial Adenomatous Polyposis
  • FAP is characterized by the early development of multiple colorectal adenomas that progress to cancer at a mean age of 44 years.
  • the APC gene is a part of the APC- ⁇ -catenin-Tcf (T-cell factor) pathway. Impairment of this pathway results in the loss of orderly replication, adhesion, and migration of colonic epithelial cells that results in the growth of polyps.
  • a series of other genetic changes follow activation of the APC- ⁇ -catenin-Tcf pathway and accompanies the transition from normal colonic mucosa to metastatic carcinoma.
  • HNPCC Hereditary nonpolyposis Colorectal Cancer
  • loss of MMR activity contributes to cancer progression through accumulation of other gene mutations and deletions, such as loss of the BAX gene which controls apoptosis, and the TGF ⁇ receptor H gene which controls cell growth. Because of the potential for irreparable damage to DNA in an individual with a DNA MMR defect, progression to carcinoma is more rapid than usual.
  • ulcerative colitis is a minor contributor to colon cancer
  • affected individuals have about a 20-fold increase in risk for developing cancer.
  • Progression is characterized by loss of the p53 gene which may occur early, appearing even in histologically normal tissue.
  • the progression of the disease from ulcerative colitis to dysplasia/carcinoma without an intermediate polyp state suggests a high degree of mutagenic activity resulting from the exposure of proliferating cells in the colonic mucosa to the colonic contents.
  • Breast cancer is a genetic disease commonly caused by mutations in cellular disease. Mutations in two genes, BRCAl 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 noninherited mutations that occur in breast epithelial cells.
  • EGF epidermal growth factor
  • EGFR epidermal growth factor
  • EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motihty, chemotaxis, secretion and differentiation.
  • a human secreted frizzled protein mRNA that is downregulated in breast tumors includes a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix Gla protein which is overexpressed is human breast carcinoma cells; Drgl 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 S 100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithehal 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.
  • Prostate cancer is a common malignancy in men over the age of 50, and the incidence increases with age. In the US, there are approximately 132,000 newly diagnosed cases of prostate cancer and more than 33,000 deaths from the disorder each year.
  • cancer 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.
  • prostatic cancers Over 95 percent of prostatic cancers are adenocarcinomas which originate in the prostatic acini. The remaining 5 percent are divided between squamous cell and transitional cell carcinomas, both of which arise in tihe prostatic ducts or other parts of the prostate gland.
  • prostate cancer develops through a multistage progression ultimately resulting in an aggressive, metastatic phenotype.
  • the initial step in tumor progression involves the hyperprohferation of normal luminal and/or basal epithehal cells that become hyperplastic and evolve into early-stage tumors.
  • the early-stage tumors are localized in the prostate but eventually may metastasize, particularly to the bone, brain or lung. About 80% of these tumors remain responsive to androgen treatment, an important hormone controlling the growth of prostate epithehal cells.
  • cancer growth becomes androgen-independent and there is currently no known treatment for this condition.
  • PSA prostate specific antigen
  • PSA is a tissue-specific serine protease almost exclusively produced by prostatic epithehal cells.
  • the quantity of PSA correlates with the number and volume of the prostatic epithehal 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.
  • PSA levels are also influenced by factors such as inflammation, androgen and other growth factors, some scientists maintain that changes in PSA levels are not useful in detecting individual cases of prostate cancer.
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF ⁇ Tumor Growth Factor alpha
  • TGF- ⁇ family of growth factors are generally expressed at increased levels inhuman cancers and the high expression levels in many cases correlates with advanced stages of malignancy and poor survival (Gold, L.I. (1999) Crit. Rev. Oncog. 10:303-360).
  • LNCap androgen-dependent stage of prostate cancer
  • PC3 and DU-145 the androgen-independent, hormone refractory stage of the disease
  • Senescence is a normal mechanism of tumor suppression, a homeostatic device that evolved to limit cell prohferation and protect the organism against cancer.
  • the prohferative lifespan of most normal human cells, even in ideal growth conditions, is limited by mtrinsic inhibitory signals that induce cell cycle arrest after a preset number of cell divisions.
  • This process of "replicative senescence” is activated in many cell types by the progressive deletion of the specialized ends of chromosomes — telomeres — which act as molecular "clocks”. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the contiol of tumor suppression mechanisms.
  • Senescent cells have recently been shown to accumulate with age in human tissues.
  • tissue microenvhOnment is disrupted by the accumulation of dysfunctional senescent cells.
  • mutation accumulation may synergize with the accumulation of senescent cells, leading to the increased risk for developing cancer that is a hallmark of mammalian aging.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, genetic disorders, and cell prohferative disorders, including cancer.
  • CADECM 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,” and “CADECM-22,” and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions.
  • CADECM purified polypeptides, cell adhesion and extracellular matrix proteins
  • 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 utihzing 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:l- 22, 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: 1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-22.
  • 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:l-22, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an arnino acid sequence selected from the group consisting of SEQ ID NO:l-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consistmg of SEQ ID NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ TD NO:l-22.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-22. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:23-44.
  • 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: 1-22, 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: 1-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22.
  • 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 consistmg of SEQ ED NO: 1-22, 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 TD NO: 1-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22.
  • 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:l-22, 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: 1-22, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22.
  • 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:23-44, 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 DD NO:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:23-44, 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 consistmg of SEQ ID NO:23-44, 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.
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • 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:23-44, 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:23-44, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to tihe polynucleotide of b), and e) an RNA equivalent of a)-d).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polyn
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
  • compositions comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, 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 ED NO: 1-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and a pharmaceutically acceptable excipient.
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:l-22.
  • 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 ED NO:l-22, 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: 1-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-22.
  • 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:l-22, b) a polypeptide comprising a naturahy 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:l-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-22.
  • 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 ED NO: 1-22, b) a polypeptide comprising a naturahy 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 ED NO: 1-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-22.
  • 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 consistmg of SEQ TD NO: 1-22, b) a polypeptide comprising a naturahy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consistmg of SEQ ID NO: 1-22, c) a biologicahy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-22, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from tihe group consisting of SEQ ID NO: 1-22.
  • 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:23-44, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of tihe 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 consistmg of SEQ ED NO:23-44, ii) a polynucleotide comprising a naturahy 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:23-44, hi) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of
  • 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 consistmg of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:23-44, ii) a polynucleotide comprising a naturahy 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:23-44, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of h), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention.
  • the probabihty scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 hsts the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA libraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide embodiments, along with allele frequencies in different human populations.
  • 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.
  • agonist refers to a molecule which intensifies or mimics the biological activity of CADECM.
  • Agonists may include proteins, nucleic acids, carbohydrates, smah 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 ahehc variants of its naturahy occurring form.
  • ahehc variants Common mutational changes which give rise to ahehc variants are generahy 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 tihe same as CADECM or a polypeptide with at least one functional characteristic of CADECM.
  • 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 ahehc 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 arnino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubihty, hydrophobicity, hydrophihcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of CADECM is retained.
  • negatively charged arnino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophihcity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophihcity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an ohgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturahy occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturahy 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. Amphfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amphfication technologies well known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of CADECM.
  • Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smah molecules, or any other compound or composition which modulates the activity of CADECM either by directly interacting with CADECM or by acting on components of the biological pathway in which CADECM participates.
  • antibody refers to intact immunoglobulin molecules as 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 smah peptides of interest as the immunizing antigen.
  • the polypeptide or ohgopeptide used to immunize an animal e.g. , a mouse, a rat, or a rabbit
  • an animal e.g. , a mouse, a rat, or a rabbit
  • an animal e.g. , a mouse, a rat, or a rabbit
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic dete ⁇ ninant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions maybe double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2 -OH group of a ribonucleotide may be replaced by 2 -F or 2 -NH j ), which may improve a desired property, e.g. , resistance to nucleases or longer hfetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers maybe specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnoi.
  • Intramer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • 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 naturahy occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a polynucleotide having a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; ohgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or ohgonucleotides 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 ceh, the complementary antisense molecule base-pairs with a naturahy occurring nucleic acid sequence produced by the ceh 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.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturahy occurring molecule.
  • immunologicalahy active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic CADECM, or of any ohgopeptide 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,
  • 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 maybe employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components
  • 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
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the stracture and especiahy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha hehcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a 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.
  • 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 preferentiahy selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ED NO:23-44 can comprise a region of unique polynucleotide sequence that specificahy identifies SEQ ED NO:23-44, for example, as distinct from any other sequence in tihe genome from which the fragment was obtained.
  • a fragment of SEQ ED NO:23-44 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amphfication technologies and in analogous methods that distinguish SEQ ED NO:23-44 from related polynucleotides.
  • the precise length of a fragment of SEQ ID NO:23-44 and the region of SEQ ED NO:23-44 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: 1-22 is encoded by a fragment of SEQ ED NO:23-44.
  • a fragment of SEQ ED NO: 1-22 can comprise a region of unique arnino acid sequence that specificahy identifies SEQ ED NO:l-22.
  • a fragment of SEQ ID NO:l-22 can be used as an immunogenic peptide for the development of antibodies that specificahy recognize SEQ ID NO: 1-22.
  • the precise length of a fragment of SEQ ID NO: 1-22 and the region of SEQ ID NO: 1-22 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, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences 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 ahgnment 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 tihe CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence ahgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wl). 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).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Ahgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Ahgnment Search Tool
  • 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.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences” tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters maybe, for example:
  • 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 ah encode substantially the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence ahgnment are weh-known. Some ahgnment 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.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) withblastp set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62
  • Percent identity maybe measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of a fragment taken from a larger, defined 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 maybe measured.
  • "Human artificial chromosomes" are linear microchromosomes which may contain
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that tihe 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 deteimining 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 skih in tihe art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
  • Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55 °C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions wih be readily apparent to those of ordinary skih in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex maybe formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobihzed on a sohd support (e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which cehs or their nucleic acids have been fixed).
  • sohd support e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which cehs or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or ohgopeptide fragment of CADECM which is capable of ehciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or ohgopeptide fragment of CADECM which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of CADECM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CADECM.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in tihe same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an ohgonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubihty to the composition. PNAs preferentiahy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the ceh.
  • 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 wih vary by ceh 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, ahehc or related nucleic acids.
  • Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primmers are short nucleic acids, usually DNA ohgonucleotides, which maybe annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amphfication (and identification) of a nucleic acid, e.g. , by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typicahy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Ohgonucleotides for use as primers are selected using software known in the art for such purpose.
  • OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
  • Similar primer selection programs have incorporated additional features for expanded capabihties.
  • the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) 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 pubhc from the Whitehead Institote/MIT Center for Genome Research, Cambridge MA) ahows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the pubhc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence ahgnments, 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 ohgonucleotides and polynucleotide fragments.
  • ohgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuhy or partiahy complementary polynucleotides in a sample of nucleic acids. Methods of ohgonucleotide 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 accomphshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid maybe part of a vector that is used, for example, to transform a ceh.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia viras, 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 usuahy 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 ah occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing CADECM, nucleic acids encoding CADECM, or fragments thereof may comprise a bodily fluid; an extract from a ceh, chromosome, organehe, or membrane isolated from a ceh; a ceh; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificahy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smah 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 wih reduce the amount of labeled A that binds to the antibody.
  • 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 naturahy associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, 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 ceh type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient ceh. Transformation may occur under natural or artificial conditions according to various methods weh 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 ceh. The method for transformation is selected based on the type of host ceh being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, hpofection, and particle bombardment.
  • 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 weh 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 hmited to animals and plants, in which one or more of the cehs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weh known in the art.
  • the nucleic acid is introduced into the ceh, directly or indirectly by introduction into a precursor of the ceh, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertihzation, 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 et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%o, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an
  • a splice variant may have significant identity to a reference molecule, but wih generahy have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotides that vary from one species to another. The resulting polypeptides wih generahy 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.
  • SNPs single nucleotide polymorphisms
  • 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 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 over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include new human ceh 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, genetic disorders, and ceh prohferative disorders, including cancer.
  • CADECM ceh adhesion and extracellular matrix proteins
  • 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 ED 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 ED 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 polypeptide and polynucleotide embodiments. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptides shown in column 3.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ED NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probabihty 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, ah of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2
  • FIG. 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison Wl).
  • 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 apphed.
  • SEQ ED NO:l is 34% identical, from residue H26 to residue S244, to Drosophila sht protein (GenBank ED g4590406), which is a large extracehular matrix protein, as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabihty score is 2.1e- 25, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ED NO:l also has leucine rich repeat domains and leucine rich repeat C- and N- terminal domains, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein famihes/domains. (See Table 3.) Data from BLIMPS analyses, provide further corroborative evidence that SEQ ED NO:l is an extracehular matrix protein.
  • SEQ ID NO:2 is 100% identical, from residue V84 to residue R553, to a human protein which is similar to cadherin and Drosophila fat protein (GenBank ED g4699969), as determined by the Basic Local Ahgnment Search Tool (BLAST).
  • SEQ ED NO:2 also contains a cadherin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains. (See Table 3.) The foregoing provides evidence that SEQ ID NO:2 is a cadherin.
  • SEQ ID NO:4 is 91% identical, from residue Ml to residue K169, to E2 antigen, the MIC2 gene product and a glycoprotein involved in T- ceh adhesion precesses (GenBank ED g30949), as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 1.3e-83, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. Data from other BLAST analyses provide further corroborative evidence that SEQ ID NO:4 is a ceh surface glycoprotein.
  • SEQ ED NO:6 is 100% identical, from residue Ml to residue P398, to cadherin-hke protein VR8 (GenBank ID g9622238) as detennined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 1.0e-214, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ED NO:6 also contains a cadherin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO: 6 is a cadherin.
  • SEQ ED NO:8 is 96% identical, from residue Ml to residue S876, to human protocadherin alpha 13 (GenBank ID g5456904) as determined by the Basic Local Ahgnment Search Tool (BLAST).
  • BLAST Basic Local Ahgnment Search Tool
  • the BLAST probabihty score is 0.0, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ TD NO: 8 also contains a cadherin domain as determined by searching for statisticahy significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:8 is a cadherin-related protein.
  • SEQ ED NO:ll is 57% identical, from residue G36 to residue E777, to human OB-cadherin-1 (GenBank ID gl377894) as dete ⁇ riined by the Basic Local Ahgnment Search Tool (BLAST).
  • SEQ ED NO:l 1 also contains a cadherin cytoplasmic region domain and cadherin domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO: 12 is 150 amino acid residues in length and is 100% identical, from residue Ml to residue D142, to human integrin beta-2 subunit (CD18) (GenBank ED g825636) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 1.9e-75, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ED NO: 12 contains a beta chain integrin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ID NO:12 is an integrin beta-2 subunit.
  • SEQ ID NO:16 is 77% identical, from residue P126 to residue E451 and 80% identical, from residue Ml to T282, to human ICAM-3 (GenBank ED g5705914) as determined by the Basic Local Ahgnment Search Tool (BLAST).
  • the BLAST probabihty scores are l.le-125 and 1.0e-113, respectively, which indicate the probabihty of obtaining the observed polypeptide sequence ahgnments by chance.
  • SEQ ED NO: 16 is locahzed to the plasma membrane, is a ceh adhesion molecule, and is a surface glycoprotein that is a member of the immunoglobulin superfamilys, binds the integrin LFA-1 (ITGB2) and promotes ceh adhesion during immunological and inflammatory reactions, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO: 16 also contains an immunoglobulin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from other BLAST analyses, provide further corroborative evidence that SEQ ID NO: 16 is an ICAM-3, a ligand for LFA-1.
  • SEQ ED NO:21 is 99% identical, from residue K28 to residue Q1993, to human fibronectin precursor (GenBank ID g31397) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 0.0, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ED NO:21 is locahzed to the extracehular matrix, the endoplasmic reticulum and the cytoplasm and is a fibronectin, an extracehular matrix protein, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:21 also contains fibronectin type I, type H and type HI domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:21 is a fibronectin.
  • SEQ ID NO:3, SEQ ID NO:5, SEQ ED NO:7, SEQ ED NO:9-10, SEQ ID NO:13-15, SEQ ID NO: 17-20, and SEQ DD NO:22 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ DD NO: 1-22 are described in Table 7.
  • the full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 hsts the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ED) 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 amphfication technologies that identify SEQ ED NO:23-44 or that distinguish between SEQ ID NO:23-44 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificahy, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST”).
  • the polynucleotide fragments described in column 2 maybe 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").
  • polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as ⁇ _XXXX X_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and Ir ⁇ Tis the number of the prediction generated by the algorithm, and N 12 ⁇ 3 _.., if present, represent specific exons that may have been manually edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of tihe 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).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) maybe used in place of the GenBank identifier (i.e., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the fohowing Table hsts examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IN and Example N).
  • Incyte cD ⁇ A coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cD ⁇ A identification numbers are not shown.
  • Table 5 shows the representative cD ⁇ A libraries for those fuh length polynucleotides which were assembled using Incyte cD ⁇ A sequences.
  • the representative cD ⁇ A library is the Incyte cD ⁇ A library which is most frequently represented by the Incyte cD ⁇ A sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cD ⁇ A libraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (S ⁇ Ps) found in polynucleotide embodiments, along with ahele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ED NO:) and the corresponding Incyte project identification number (PHD) 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 ED).
  • 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 (CBI SNP).
  • CADECM variants are one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the CADECM amino acid sequence, and which contains at least one functional or structural characteristic of CADECM.
  • Various embodiments also encompass polynucleotides which encode CADECM.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consistmg of SEQ ID NO:23-44, which encodes CADECM.
  • the polynucleotide sequences of SEQ DD NO:23-44 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.
  • such a variant polynucleotide wih 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:23-44 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:23-44. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of CADECM.
  • a polynucleotide variant of the invention is a sphce variant of a polynucleotide encoding CADECM.
  • a sphce variant may have portions which have significant sequence identity to a polynucleotide encoding CADECM, but wih generahy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
  • a sphce 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 sphce variant wih have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding CADECM.
  • a polynucleotide comprising a sequence of SEQ DD NO:3 and a polynucleotide comprising a sequence of SEQ ED NO: 12 are sphce variants of each other; and a polynucleotide comprising a sequence of SEQ DD NO:4, a polynucleotide comprising a sequence of SEQ DD NO:17, and a polynucleotide comprising a sequence of SEQ DD NO:18 are sphce variants of each other; and a polynucleotide comprising a sequence of SEQ ED NO:9 and a polynucleotide comprising a sequence of SEQ DD NO: 10 are sphce variants of each other; and a polynucleotide comprising a sequence of SEQ DD NO:22 and a polynucleotide comprising a sequence of SEQ ED NO:16 are sphce variants of each
  • polynucleotides which encode CADECM and its variants are generahy capable of hybridizing to polynucleotides encoding naturahy occurring CADECM under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding CADECM or its derivatives possessing a substantiahy different codon usage, e.g. , inclusion of non-naturahy occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturahy occurring sequence.
  • the invention also encompasses production of polynucleotides which encode CADECM and CADECM derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide maybe inserted into any of the many available expression vectors and ceh systems using reagents weh known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding CADECM or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ DD NO:23-44 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. i52:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.” Methods for DNA sequencing are weh known in the art and may be used to practice any of the embodiments of tihe invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in tihe ELONGASE amphfication system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 hquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Applied Biosystems).
  • Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Apphed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art.
  • the resulting sequences are analyzed using a variety of algorithms which are weh known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding CADECM may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amphfy 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 amphfy 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.
  • a third method involves PCR amphfication 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).
  • multiple restriction enzyme digestions and ligations maybe used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C
  • Capihary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capihary 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, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controhed.
  • Capihary electrophoresis is especiahy preferable for sequencing smah DNA fragments which maybe present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode CADECM may be cloned in recombinant DNA molecules that direct expression of CADECM, or fragments or functional equivalents thereof, in appropriate host cehs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiahy 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 generahy 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 ohgonucleotides may be used to engineer the nucleotide sequences.
  • ohgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnoi. 17:793-797; Christians, EC. et al. (1999) Nat. Biotechnoi. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnoi. 14:315-319) to alter or improve the biological properties of CADECM, such as its biological or enzymatic activity or its abihty to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C et al. (1999) Nat. Biotechnoi. 17:793-797; Christians, EC. et al. (1999) Nat
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene maybe 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 naturahy occurring genes in a directed and controllable manner.
  • polynucleotides encoding CADECM may be synthesized, in whole or in part, using one or more chemical methods weh known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • CADECM itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or sohd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp.
  • the peptide may be substantiahy purified by preparative high performance hquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • the composition of the synthetic peptides may be confirmed by arnino acid analysis or by sequencing. (Creighton, supra, pp. 28-53).
  • 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.
  • Methods which are weh 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, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; 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 ceh systems infected with viral expression vectors (e.g., baculoviras); plant ceh 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 ceh systems (Sambrook, supra; Ausubel et al., supra; Nan Heeke, G.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect ceh systems infected with viral expression vectors (e.
  • Expression vectors derived from retroviruses, adenovirases, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or ceh 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; Buher, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Nerma, I.M. and ⁇ . Somia (1997) Nature 389:239-242).
  • the invention is not limited by the host ceh employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding CADECM.
  • routine cloning, subcloning, and propagation of polynucleotides encoding CADECM can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Joha CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRIPT Stratagene, La Joha CA
  • PSPORT1 plasmid Invitrogen.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
  • vectors which direct high level expression of CADECM may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe 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.
  • 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, CA. 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 tihe omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the smah subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl.
  • viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with tihe omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1311).
  • Ceh Differ. 17:85-105 These constracts can be introduced into plant cehs by direct DNA transformation or pathogen-mediated transfection (The McGraw Hih Yearbook of Science and Technology (1992) McGraw Hih, New York NY, pp. 191-196).
  • a number of viral-based expression systems may be utihzed.
  • polynucleotides encoding CADECM maybe hgated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
  • Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses CADECM in host cehs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cehs.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to dehver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and dehvered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
  • CADECM stable expression of CADECM in cell lines
  • polynucleotides encoding CADECM can be transformed into ceh 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.
  • cehs 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 ahows growth and recovery of cehs which successfully express the introduced sequences.
  • Resistant clones of stably transformed cehs may be propagated using tissue culture techniques appropriate to the ceh type.
  • any number of selection systems maybe used to recover transformed ceh lines. These include, but are not limited to, the herpes simplex viras thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cehs, respectively (Wigler, M. et al. (1977) Ceh 11:223-232; Lowy, I. et al. (1980) Ceh 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD which alter cehular requirements for metabolites
  • Visible markers e.g., an ocyanins, green fluorescent proteins (GFP; Clontech), ⁇ - glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding CADECM is inserted within a marker gene sequence, transformed cehs containing polynucleotides encoding CADECM can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding CADECM under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as weh.
  • host cehs that contain the polynucleotide encoding CADECM and that express CADECM may be identified by a variety of procedures known to those of skih in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amphfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
  • Hrrmunological 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 ceh sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceh sorting
  • 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.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CADECM include ohgolabeling, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide.
  • polynucleotides encoding CADECM, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuchdes, enzymes, fluorescent, chen ⁇ luminescent, or chromogenic agents, as weh as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cehs transformed with polynucleotides encoding CADECM may be cultured under conditions suitable for the expression and recovery of the protein from ceh culture.
  • the protein produced by a transformed ceh maybe secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode CADECM may be designed to contain signal sequences which direct secretion of CADECM through a prokaryotic or eukaryotic ceh membrane.
  • a host ceh strain may be chosen for its abihty to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cehs which have specific cehular 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 maybe chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant polynucleotides encoding CADECM may be hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a cliimeric CADECM protein containing a heterologous moiety that can be recognized by a commerciahy available antibody may facihtate the screening of peptide libraries for inhibitors of CADECM activity.
  • Heterologous protein and peptide moieties may also facihtate purification of fusion proteins using commerciahy 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 immobihzed glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciahy available monoclonal and polyclonal antibodies that specificahy 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 fohowing purification.
  • fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commerciahy available kits may also be used to facihtate expression and purification of fusion proteins.
  • synthesis of radiolabeled CADECM maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-mefl ⁇ onine.
  • CADECM, fragments of CADECM, or variants of CADECM may be used to screen for compounds that specificahy bind to CADECM.
  • One or more test compounds may be screened for specific binding to CADECM.
  • 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, ohgonucleotides, proteins (e.g., hgands or receptors), or smah molecules.
  • variants of CADECM can be used to screen for binding of test compounds, such as antibodies, to CADECM, a variant of CADECM, or a combination of CADECM and/or one or more variants CADECM.
  • a variant of CADECM can be used to screen for compounds that bind to a variant of CADECM, but not to CADECM having the exact sequence of a sequence of SEQ ID NO: 1-22.
  • CADECM variants used to perform such screening can have a range of about 50% to about 99% sequence identity to CADECM, with various embodiments having 60%, 70%), 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to CADECM can be closely related to the natural hgand of CADECM, e.g., a hgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (Cohgan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural hgand of a receptor CADECM (Howard, A . et al. (2001) Trends Pharmacol. Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to CADECM can be closely related to the natural receptor to which CADECM binds, at least a fragment of the receptor, or a fragment of the receptor including ah or a portion of the hgand binding site or binding pocket.
  • the compound may be a receptor for CADECM which is capable of propagating a signal, or a decoy receptor for CADECM which is not capable of propagating a signal (Ashkenazi, A. and NM. Divit (1999) Curr. Opin. Ceh Biol. 11:255-260; Mantovani, A. et al. (2001) Trends I-mmunol. 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 CA), 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 IgGi (Taylor, P.C et al. (2001) Curr. Opin. Immunol. 13:611-616).
  • 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.
  • an antibody can be selected such that its binding specificity ahows for preferential identification of specific fragments or variants of CADECM.
  • an antibody can be selected such that its binding specificity ahows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of CADECM.
  • anticalins can be screened for specific binding to CADECM, fragments of CADECM, or variants of CADECM.
  • Anticalins are hgand-binding proteins that have been constructed based on a hpocalin scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177- R184; Skerra, A. (2001) J. Biotechnoi. 74:257-275).
  • the protein architecture of lipocalins can include a beta-barrel having eight antiparahel beta-strands, which supports four loops at its open end.
  • loops form the natural hgand-binding site of the lipocahns, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • screening for compounds which specificahy bind to, stimulate, or inhibit CADECM involves producing appropriate cehs which express CADECM, either as a secreted protein or on the ceh membrane.
  • Preferred cehs include cehs from mammals, yeast, Drosophila, or E. coli.
  • Cehs expressing CADECM or ceh membrane fractions which contain CADECM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either CADECM or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with CADECM, either in solution or affixed to a sohd support, and detecting the binding of CADECM to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay maybe carried out using ceh-free preparations, chemical libraries, or natural product mixtures, and the test com ⁇ ound(s) maybe free in solution or affixed to a sohd support.
  • An assay can be used to assess the abihty of a compound to bind to its natural hgand and/or to inhibit the binding of its natural hgand to its natural receptors.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abihty to bind to its natural hgands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a hgand) to improve or alter its abihty to bind to its natural receptors (C nningham, B.C.
  • CADECM, fragments of CADECM, or variants of CADECM may be used to screen for compounds that modulate the activity of CADECM.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for CADECM activity, wherein CADECM is combined with at least one test compound, and the activity of CADECM in the presence of a test compound is compared with the activity of CADECM in the absence of the test compound. A change in the activity of CADECM in the presence of the test compound is indicative of a compound that modulates the activity of CADECM.
  • test compound is combined with an in vitro or ceh-free system comprising CADECM under conditions suitable for CADECM activity, and the assay is performed.
  • a test compound which modulates the activity of CADECM may do so indirectly and need not come in direct contact with tihe test compound. At least one and up to a plurahty of test compounds may be screened.
  • polynucleotides encoding CADECM or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cehs.
  • ES embryonic stem
  • Such techniques are weh known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES cehs such as the mouse 129/SvJ ceh line, are derived from the early mouse embryo and grown in culture.
  • the ES cehs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cehs are identified and microinjected into mouse ceh 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 cehs derived from human blastocysts.
  • Human ES cehs have the potential to differentiate into at least eight separate ceh lineages including endoderm, mesoderm, and ectodermal ceh types. These ceh lineages differentiate into, for example, neural cehs, 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.
  • knockin technology a region of a polynucleotide encoding CADECM is injected into animal ES cehs, and the injected sequence integrates into the animal ceh genome.
  • Transformed cehs are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress CADECM e.g., by secreting CADECM in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnoi. Annu. Rev. 4:55-74). THERAPEUTICS
  • CADECM a region of CADECM and ceh adhesion and extracehular matrix proteins.
  • the expression of CADECM is closely associated with peripheral blood cehs, lung tissue, brain tissue, brain tumor tissue, tissue from the ileum and jejunum, eosinophils, adrenal tumor tissue, kidney tissue, knee cartilage chondrocytes, pituitary gland tissue, myxoma (tumor) tissue from the atrium, tumor tissue of the penis, esophageal tissue, tumor-associated esophageal tissue, and thymus tissue.
  • examples of tissues expressing CADECM can be found in Table 6 and can also be found in Example XI.
  • CADECM appears to play a role in immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, genetic disorders, and ceh prohferative disorders, including cancer.
  • CADECM In the treatment of disorders associated with increased CADECM expression or activity, it is desirable to decrease the expression or activity of CADECM.
  • CADECM In the treatment of disorders associated with decreased CADECM expression or activity, it is desirable to increase the expression or activity of CADECM.
  • CADECM or a fragment or derivative thereof maybe administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADECM.
  • disorders include, but are not limited to, an immune system disorder, such as acquired immunodeficiency syndrome (ADDS), X-linked agammaglobinemia of Braton, common variable immunodeficiency (CVT), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCDD), 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 spondyhtis, amyloidosis, anemia, asthma, athe
  • ADDS acquired immuno
  • 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.
  • composition comprising a substantiahy purified CADECM in conjunction with a suitable pharmaceutical earner may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADECM including, but not limited to, those provided above.
  • an agonist which modulates the activity of CADECM may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADECM including, but not limited to, those hsted above.
  • an antagonist of CADECM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CADECM.
  • disorders include, but are not limited to, those immune system disorders, neurological disorders, developmental disorders, connective tissue disorders, genetic disorders, and ceh prohferative disorders, including cancer, described above.
  • an antibody which specificahy binds CADECM maybe used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cehs or tissues which express CADECM.
  • a vector expressing tihe complement of the polynucleotide encoding CADECM maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of CADECM including, but not limited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments maybe 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 skih 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 maybe produced using methods which are generahy known in the art.
  • purified CADECM may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specificahy bind CADECM.
  • Antibodies to CADECM may also be generated using methods that are weh known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralizing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnoi. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others maybe immunized by injection with CADECM or with any fragment or ohgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase irnmunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium paiyum are especiahy preferable. It is preferred that the ohgopeptides, peptides, or fragments used to induce antibodies to
  • CADECM have an amino acid sequence consisting of at least about 5 amino acids, and generahy wih consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of CADECM amino acids maybe 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 ceh lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceh 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. Ceh Biol. 62:109-120).
  • 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).
  • techniques described for the production of single chain antibodies maybe 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.
  • fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to ahow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, WD. et al. (1989) Science 246:1275-1281).
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are weh 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 generahy used, but a competitive binding assay may also be employed (Pound, supra).
  • Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques maybe used to assess the affinity of antibodies for CADECM.
  • K a 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 a determined for a preparation of monoclonal antibodies, which are monospecific for a particular CADECM epitope, represents a true measure of affinity.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the CADECM-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are 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 DC; Liddeh, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitabihty of such preparations for certain downstream apphcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generahy employed in procedures requiring precipitation of CADECM-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quahty and usage in various apphcations, are generahy available (Catty, supra; Coligan et al., supra).
  • polynucleotides encoding CADECM may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified ohgonucleotides) to the coding or regulatory regions of the gene encoding CADECM.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified ohgonucleotides
  • antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding CADECM (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
  • Antisense sequences can be dehvered intracehularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cehular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Ahergy Clin. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296).
  • Antisense sequences can also be introduced intracehularly through the use of viral vectors, such as retrovirus and adeno-associated viras vectors (Miller, AD.
  • polynucleotides encoding CADECM maybe 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 (SCDD)-Xl disease characterized by X- hnked 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) Ceh 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene
  • CADECM hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi
  • 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 cehs.
  • Mechanical transfer technologies for use with cehs in vivo or ex vitro include (i) direct D ⁇ A microinjection into individual cehs, (ii) ballistic gold particle dehvery, (hi) hposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of D ⁇ A transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Ceh 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnoi. 9:445-450).
  • Expression vectors that may be effective for the expression of CADECM include, but are not limited to, the PCD ⁇ A 3.1, EP TAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCPJPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Joha CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • CADECM maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -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. Biotechnoi.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • hposome transformation kits e.g., the PERFECT LD?DD TRANSFECTION KIT, available from Invitrogen
  • 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 cehs requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to CADECM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding CADECM under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and ( i) 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
  • Retrovirus vectors are commerciahy available (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad.
  • the vector is propagated in an appropriate vector producing ceh line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cehs 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 AD. Miher (1988) J. Virol. 62:3802-3806; Duh, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al.
  • VSVg vector producing ceh line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceh lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cehs (e.g., CD4 + T-cehs), and the return of transduced cehs to a patient are procedures weh known to persons skilled in the art of gene therapy and have been weh documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
  • an adenovirus-based gene therapy dehvery system is used to dehver polynucleotides encoding CADECM to cehs which have one or more genetic abnormahties with respect to the expression of CADECM.
  • the construction and packaging of adenovirus-based vectors are weh known to those with ordinary skih 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).
  • Potentiahy useful adenoviral vectors are described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference.
  • Adenovirus vectors for gene therapy 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).
  • a herpes-based, gene therapy dehvery system is used to dehver polynucleotides encoding CADECM to target cehs which have one or more genetic abnormahties with respect to the expression of CADECM.
  • the use of herpes simplex virus (HSV)-based vectors may be especiahy valuable for introducing CADECM to cehs of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are weh known to those with ordinary skih in the art.
  • a replication-competent herpes simplex virus (HSV) type 1-based vector has been used to dehver a reporter gene to the eyes of primates (Liu, X.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex viras strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceh 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.
  • 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 fohowing the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesviras, and the infection of cehs with herpesvirus are techniques weh known to those of ordinary skih in the art.
  • an alphaviras (positive, single-stranded RNA viras) vector is used to dehver polynucleotides encoding CADECM to target cehs.
  • SFV Semliki Forest Virus
  • This subgenomic RNA rephcates to higher levels than the fuh length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for CADECM into the alphaviras 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 cehs.
  • alphaviras infection is typically associated with ceh lysis within a few days
  • the abihty to estabhsh a persistent infection in hamster normal kidney cehs (BHK-21) with a variant of Sindbis virus (SENT) 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 wih ahow the introduction of CADECM into a variety of ceh types.
  • the specific transduction of a subset of cehs in a population may require the sorting of cehs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphaviras cDNA and RNA tiansfections, and performing alphaviras infections, are weh known to those with ordinary skih in the art.
  • Ohgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple hehx base-pairing methodology.
  • Triple hehx pairing is useful because it causes inhibition of the abihty of the double hehx 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. Ca , Molecular and Immunologic Approaches, Futura Pubhshing, Mt. Kisco NY, pp. 163-177).
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, 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, fohowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificahy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding CADECM.
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the ohgonucleotide inoperable.
  • the suitabihty of candidate targets may also be evaluated by testing accessibihty to hybridization with complementary ohgonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding 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 ceh lines, cehs, or tissues.
  • RNA molecules maybe modified to increase intracehular stabihty 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.
  • 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 maybe effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense ohgonucleotides, triple hehx-forming ohgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificahy inhibits expression of the polynucleotide encoding CADECM may be therapeuticahy useful, and in the treatment of disorders associated with decreased CADECM expression or activity, a compound which specificahy promotes expression of the polynucleotide encoding CADECM may be therapeuticahy useful.
  • At least one, and up to a plurahty, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciahy-available or proprietary library of naturahy-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 combinatoriahy 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 permeabihzed ceh, or an in vitro ceh-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding CADECM are assayed by any method commonly known in the art.
  • Typicahy 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.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be canied out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceh line such as HeLa ceh (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
  • a particular embodiment of the present invention involves screening a combinatorial library of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Bra e, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into cehs or tissues are available and equahy suitable for use in vivo, in vitro, and ex vivo.
  • ohgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides
  • vectors For ex vivo therapy, vectors maybe introduced into stem cehs taken from the patient and clonahy propagated for autologous transplant back into that same patient. Dehvery by transfection, by hposome injections, or by polycationic amino polymers may be achieved using methods which are weh known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnoi. 15:462- 466).
  • compositions which generahy comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
  • formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Pubhshing, Easton PA).
  • Such compositions may consist of
  • CADECM antibodies to CADECM, and mimetics, agonists, antagonists, or inhibitors of CADECM.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduhary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • compositions for pulmonary administration may be prepared in hquid or dry powder form. These compositions are generahy aerosohzed immediately prior to inhalation by the patient.
  • aerosol dehvery of fast- acting formulations is weh-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary dehvery has the advantage of adrninistiation without needle injection, and obviates the need for potentiahy 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 weh within the capabihty of those skilled in the art.
  • compositions may be prepared for direct intracellular dehvery of macromolecules comprising CADECM or fragments thereof.
  • hposome preparations containing a ceh-impermeable macromolecule may promote ceh fusion and intracellular dehvery of the macromolecule.
  • CADECM or a fragment thereof may be joined to a short cationic N- tenninal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cehs of ah tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeuticahy effective dose can be estimated initially either in ceh culture assays, e.g., of neoplastic cehs, 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 therapeuticahy 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 maybe determined by standard pharmaceutical procedures in ceh cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticahy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceh culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED S0 with httle 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 wih 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, drag combinations), reaction sensitivities, and response to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-hfe and clearance rate of the particular formulation. Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • antibodies which specificahy 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 cehs or tissues.
  • the antibodies may be used with or without modification, and maybe labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • CADECM CADECM
  • ELISAs RIAs
  • FACS fluorescence-activated cell sorting
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • normal or standard values for CADECM expression are established by combining body fluids or ceh 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 frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.
  • polynucleotides encoding CADECM may be used for diagnostic purposes.
  • the polynucleotides which may be used include ohgonucleotides, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of CADECM may be conelated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of CADECM, and to monitor regulation of CADECM levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding CADECM or closely related molecules 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 amphfication wih determine whether the probe identifies only naturahy occurring sequences encoding CADECM, ahehc 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 TD NO:23-44 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.
  • 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 radionuchdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding CADECM maybe used for the diagnosis of disorders associated with expression of CADECM.
  • disorders include, but are not limited to, an immune system disorder, such as acquired immunodeficiency syndrome (ADDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVT), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCDD), immunodeficiency with thrombocytopenia and eczema (Wiskott- Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Qishing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondyhtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
  • 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 weh known in the art.
  • polynucleotides encoding CADECM maybe used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding CADECM maybe 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. H the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding CADECM in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is established. This maybe accomphshed by combining body fluids or ceh extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CADECM, under conditions suitable for hybridization or amphfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiahy purified polynucleotide is used. Standard values obtained in this manner may be compared -with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabhsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may ahow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
  • ohgonucleotides designed from the sequences encoding CADECM may involve the use of PCR. These ohgomers may be chemically synthesized, generated enzymaticahy, or produced in vitro.
  • Ohgomers wih preferably contain a fragment of a polynucleotide encoding CADECM, or a fragment of a polynucleotide complementary to the polynucleotide encoding CADECM, and wih be employed under optimized conditions for identification of a specific gene or condition.
  • Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • ohgonucleotide primers derived from polynucleotides encoding CADECM maybe used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • ohgonucleotide primers derived from polynucleotides encoding CADECM are used to amphfy 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.
  • the ohgonucleotide primers are fluorescently labeled, which ahows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • Additionahy, sequence database analysis methods, termed in sihco SNP (lsSNP) are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceh anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as hfe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-hpoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weh as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med.
  • CADECM CADECM
  • Methods which may also be used to quantify the expression of CADECM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, PC et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236).
  • the speed of quantitation of multiple samples maybe accelerated by ranning the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • ohgonucleotides or longer fragments derived from any of the polynucleotides described herein maybe 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 maybe 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 maybe selected for a patient based on his/her pharmacogenomic profile.
  • 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 ceh type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceh type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al, "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herein).
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totahty of transcripts or reverse transcripts of a particular tissue or ceh type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or then complements comprise a subset of a plurahty 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, ceh 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 ceh 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 weh as toxicological testing of industrial and naturahy-occuning environmental compounds.
  • Ah 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.
  • the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels conesponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceh type.
  • proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
  • a profile of a ceh's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceh type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visualized in the gel as discrete and uniquely positioned spots, typicahy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generahy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiahy sequenced using, for example, standard methods employing chemical or enzymatic cleavage fohowed by mass spectrometry.
  • the identity of the protein in a spot may be deterinined 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 maybe obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for CADECM to quantify the levels of CADECM expression.
  • the antibodies are used as elements on a 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 maybe 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 maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling maybe more rehable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the arnino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays maybe prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT apphcation WO95/251116; Shalon, D. et al. (1995) PCT apphcation WO95/35505; Heher, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662).
  • Various types of microarrays are weh known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Approach, Oxford University Press, London).
  • nucleic acid sequences encoding CADECM maybe used to generate hybridization probes useful in mapping the naturahy occurring genomic sequence.
  • Either coding or noncoding sequences maybe 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 potentiahy cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries
  • 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 Mendehan Inheritance in Man (OMEVI) 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.
  • RFLP restriction fragment length polymorphis
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often tihe placement of a gene on the chromosome of another mammahan species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580).
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • CADECM its catalytic or immunogenic fragments, or ohgopeptides thereof can be used for screening libraries of compounds in any of a variety of drag screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a sohd support, borne on a ceh surface, or located intracehularly. The formation of binding complexes between CADECM and the agent being tested may be measured.
  • test compounds are synthesized on a sohd substrate.
  • the test compounds are reacted with CADECM, or fragments thereof, and washed.
  • Bound CADECM is then detected by methods weh known in the art.
  • Purified CADECM can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a sohd support.
  • competitive drug screening assays in which neutralizing antibodies capable of binding CADECM specificahy compete with a test compound for binding
  • 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 cunently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • TRIZOL Invitrogen
  • poly(A)+ RNA was isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constracted the corresponding cDNA libraries.
  • cDNA was synthesized and cDNA libraries were constracted 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 was initiated using ohgo d(T) or random primers.
  • Syntlietic ohgonucleotide adapters were hgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid
  • Plasmids obtained as described in Example I were recovered from host cehs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceh lysis. Plasmids were purified using at least one of the fohowing: 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. Fohowing precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophihzation, at 4°C
  • plasmid DNA was amphfied from host ceh lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceh lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weh plates, and the concentration of amphfied plasmid DNA was quantified fluorometricahy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN H fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Incyte cDNA recovered in plasmids as described in Example H were sequenced as fohows. Sequencing reactions were processed using standard methods or Hgh-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA icrodispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) hquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Apphed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al, supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VJH.
  • the polynucleotide sequences derived from Incyte cDNAs were vahdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic prograiriming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubhc 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 Genomics, Palo Alto CA); hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and T ⁇ GRFAM (Haft, D.H.
  • Pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM
  • PROTEOME databases
  • 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 probabihstic approach which analyzes consensus primary structures of gene famihes; see, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce fuh length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cD ⁇ A assemblages to fuh length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cD ⁇ A assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the fuh length polynucleotide sequences were translated to derive the corresponding fuh length polypeptide sequences.
  • a polypeptide may begin at any of the methionine residues of the fuh length translated polypeptide.
  • Fuh length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, INCY, and TTGRFAM; and HMM-based protein domain databases such as SMART.
  • GenBank protein databases Genpept
  • PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM hidden Markov model
  • Prosite Prosite
  • HMM-based protein family databases such as PFAM, INCY, and TTGRFAM
  • HMM-based protein domain databases such as SMART.
  • Fuh length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio Inc., Alameda CA)
  • Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between ahgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuh length sequences and provides apphcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, ah of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, the scores, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. 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.
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
  • the encoded polypeptides were analyzed by querying against PFAM models for ceh adhesion and extracehular matrix proteins. Potential ceh adhesion and extracehular matrix proteins were also identified by homology to Incyte cDNA sequences that had been annotated as ceh adhesion and extracehular matrix proteins.
  • Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • Fuh length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example HI. Alternatively, fuh length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences. V. Assembly of Genomic Sequence Data with cDNA Sequence Data "Stitched" Sequences
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example TV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible sphce variants that were subsequently confirmed, edited, or extended to create a fuh length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity.
  • Partial DNA sequences were extended to fuh length with an algorithm based on BLAST analysis.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IN.
  • a chimeric protein was generated by using the resultant high-scoring segment pahs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pahs
  • GenBank protein homolog the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial D ⁇ A sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of CADECM Encoding Polynucleotides
  • sequences which were used to assemble SEQ ID ⁇ O:23-44 were compared with sequences from the Incyte LEFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ED NO:23-44 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of ah sequences of that cluster, including its particular SEQ DD NO:, to that map location.
  • pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the tenninus of the chromosome's p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceh type or tissue have been bound (Sambrook, supra, ch. 7; Ausubel et al., supra, ch. 4).
  • 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 fohows: 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 quahty in a BLAST ahgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotides encoding CADECM are analyzed with respect to the tissue sources from which they were derived. For example, some fuh length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI). Each cDNA sequence is derived from a cDNA library constructed from a human tissue.
  • Each human tissue is classified into one of the fohowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitaha, male; germ cehs; hemic and immune system; hver; 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 ah categories.
  • each human tissue is classified into one of the fohowing disease/condition categories: cancer, ceh 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 ah 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 LTFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of CADECM Encoding Polynucleotides
  • Fuh length polynucleotides are produced by extension of an appropriate fragment of the fuh length molecule using ohgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the concentration of DNA in each weh was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weh of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan H (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transfened to 384-weh plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wl), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison Wl
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cehs. Transformed cehs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-weh plates in LB/2x carb hquid media.
  • fuh length polynucleotides are verified using the above procedure or are used to obtain 5 'regulatory sequences using the above procedure along with ohgonucleotides designed for such extension, and an appropriate genomic library.
  • SNPs single nucleotide polymorphisms
  • LEFESEQ database Incyte Genomics
  • Sequences from the same gene were clustered together and assembled as described in Example HI, allowing the identification of ah sequence variants in the gene.
  • An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecah enors by requiring a minimum Phred quahty score of 15, and removed sequence ahgnment enors and enors resulting from improper trimming of vector sequences, chimeras, and sphce variants.
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze ahele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprised 194 individuals (97 male, 97 female), ah African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), ah Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Ahele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no ahehc variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ DD NO:23-44 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specificahy described, essentially the same procedure is used with larger nucleotide fragments.
  • Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine tnphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled ohgonucleotides are substantiahy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schueh, Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentiahy washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
  • the linkage or synthesis of anay 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 sohd with a non-porous surface (Schena, M., ed. (1999) DNA Microanays: A Practical Approach, Oxford University Press, London). Suggested substrates include sihcon, sihca, glass shdes, glass chips, and sihcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines weh known to those of ordinary skih 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. Biotechnoi. 16:27-31).
  • Fuh length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microanay. Fragments or ohgomers suitable for hybridization can be selected using software weh known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cehulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l ohgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng ⁇ oly(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 Laboratories, Inc.
  • Anay elements are amphfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amphfied anay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified anay elements are immobilized on polymer-coated glass shdes.
  • Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distihed water washes between and after treatments.
  • Glass shdes are etched in 4% hydrofluoric acid (NWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distihed water, and coated with 0.05% arninopropyl silane (Sigma) in 95% ethanol. Coated shdes are cured in a 110°C oven.
  • Array elements are apphed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the anay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capihary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of anay element sample per shde.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosshnker (Stratagene). Microanays are washed at room temperature once in 0.2% SDS and three times in distihed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered sahne (PBS) (Tropix, Inc., Bedford MA) for 30 rninutes at 60° C fohowed by washes in 0.2% SDS and distihed water as before.
  • PBS phosphate buffered sahne
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixtore is heated to 65° C for 5 udinutes and is ahquoted onto the microanay surface and covered with an 1.8 cm 2 covershp.
  • the anays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope shde.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60° C.
  • the arrays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 rninutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melvihe NY).
  • the shde containing the array is placed on a computer-controhed X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm anay used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentiahy. Emitted light is spht, based on wavelength, into two photomultipher tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) conesponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipher 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 typicahy 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 typicahy 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 conelated with a weight ratio of hybridizing species of 1:100,000.
  • the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipher tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, tihe 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 exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed using the GEMTOOLS program (Incyte Genomics). Expression
  • SEQ ID NO:25 showed differential expression in tumorous tissue versus non- cancerous tissue, as determined by microarray analysis. Matched normal and tumorigenic lung tissue samples are provided by the Roy Castle International Centre for Lung Cancer Research (Liverpool, UK). The expression of SEQ DD NO:25 was decreased at least 2-fold in two of five experiments where lung squamous ceh carcinoma was matched with normal tissue from the same donors. Therefore, in various embodiments, SEQ ID NO:25 can be used for one or more of the fohowing: i) monitoring treatment of lung squamous ceh carcinoma, ii) diagnostic assays for lung squamous ceh carcinoma, and iii) developing therapeutics and/or other treatments for lung squamous ceh carcinoma.
  • SEQ ID NO:26, SEQ DD NO:29, SEQ DD NO:39, and SEQ DD NO:43 showed differential expression in colon cancer tissue, as determined by microanay analysis.
  • the expression level of SEQ DD NO:26 was increased at least 2.8-fold in human colon crypt tissue with adenocarcinoma when matched with normal tissue from the same donor. Matched normal and tumorigenic colon or colon crypt tissue samples and matched normal and tumorigenic ovarian tissue samples are provided by the Huntsman Cancer Institute, (Salt Lake City, UT).
  • SEQ ED NO:29 was increased by at least two fold in colon adenocarcinoma tissue harvested from a 58 year old female donor diagnosed with mucinous adenocarcinoma, when compared to normal colon tissues harvested from grossly uninvolved colon tissue of the same donor.
  • the expression of SEQ ID NO:39 also was increased at least 2.7-fold in human colon crypts from colon adenocarcinoma tissue matched with normal tissue from the same donor.
  • the colon crypts tissue was obtained from an 64- year-old female with moderately weh differentiated colon adenocarcinoma. Normal colon tissue was obtained from grossly uninvolved colon tissue from the same donor.
  • SEQ DD NO:43 also showed differential expression associated with colon cancer, as determined by microanay analysis. Comparisons were made of normal colon to colon tumor tissues from the fohowing donors: a 48-year- old female with a sigmoid colon tumor originating from a metastatic gastric sarcoma; a 56-year-old female with poorly differentiated metastatic adenocarcinoma, possibly of ovarian origin; a 58-year-old female with mucinous adenocarcinoma; and an 83 -year-old female with adenocarcinoma. The expression of SEQ ED NO:43 was increased by at least two-fold in each of the tumor tissues as compared to the matched microscopicahy normal tissue from the same donor.
  • SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:39, and SEQ DD NO:43 can be used for one or more of the fohowing: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.
  • SEQ ED NO:26, SEQ BD NO:27, and SEQ DD NO:39 were differentially expressed in ovarian tumor tissue, as detenrhned by microarray expression analysis. Matched normal and tumorigenic ovarian tissue samples are provided by the Huntsman Cancer Institute, (Salt Lake City, UT). The expression of SEQ D NO:26 was decreased by at least 2.8-fold , in ovarian tumor tissue when matched with normal tissue from the same donor. The expression of SEQ DD NO:27 was decreased by at least 2-fold in ovarian tumor tissue when matched with normal tissue from the same donor.
  • SEQ DD NO:39 was decreased at least 2.8- fold in an ovarian adenocarcinoma when matched with normal tissue from the same donor.
  • the tumorous ovary tissue was obtained from ovarian adenocarcinoma from a 79-year-old female.
  • Normal ovary tissue was obtained from ovary from the same donor. Therefore, in various embodiments, SEQ ED NO:26, SEQ ID NO:27, and SEQ DD NO:39 can be used for one or more of the fohowing: i) monitoring treatment of ovarian cancer, ii) diagnostic assays for ovarian cancer, and hi) developing therapeutics and/or other treatments for ovarian cancer.
  • SEQ DD NO:27 and SEQ DD NO:39 showed differential expression in cancer ceh lines or tumorous tissue versus non-cancerous ceh lines or tissues, as determined by microarray analysis.
  • Primary mammary gland cehs were isolated from a donor with fibrocystic breast disease, and served as a normal or control ceh line.
  • Breast tumor cehs were isolated from the pleural effusion of a donor with late stages of tumor progression and mahgnant transformation.
  • Expression of SEQ ED NO:27 was increased by at least two-fold in the breast tumor ceh line, when compared to expression levels in the control ceh line.
  • the expression of SEQ ED NO:39 was decreased by at least two fold in breast tumor ceh lines that were harvested from donors at different stages of tumor progression and mahgnant transformation when grown in one of two different chemically defined, serum-free media both supplemented with growth factors and growth hormones.
  • Normal or control breast ceh line was described above.
  • Tumorous breast ceh lines were obtained in the fohowing ways.
  • Breast carcinoma cehs were derived in vitro from cehs emigrating from a tomor.
  • breast tumor cehs were isolated from invasive tumor of donors.
  • nonmahgnant or mahgnant primary breast adenocarcinoma cehs were obtained from the pleural effusion of donors.
  • SEQ DD NO:39 was decreased at least 2.7-fold in a breast tomor when matched with normal tissue from the same donor.
  • the tumorous breast tissue was obtained from a 43 -year-old female with invasive lobular carcinoma in situ. Normal breast tissue was obtained from breast from tihe same donor. Therefore, in various embodiments, SEQ DD NO:27 and SEQ DD NO:39 can be used for one or more of the fohowing: i) monitoring treatment of breast cancer, h) diagnostic assays for breast cancer, and i) developing therapeutics and/or other treatments for breast cancer.
  • SEQ ED NO:43 showed differential expression associated with breast cancer, as determined by microarray analysis.
  • the breast carcinoma ceh lines ' include MCF7, a breast adenocarcinoma ceh line derived from the pleural effusion of a 69-year-old female; T-47D, a breast carcinoma ceh line derived from a pleural effusion from a 54-year-old female with an infiltrating ductal carcinoma of the breast; Sk-BR-3, a breast adenocarcinoma ceh line isolated from a mahgnant pleural effusion of a 43-year-old female; BT-20, a breast adenocarcinoma isolated in vitro from cehs emigrating out of thin shces of a tumor mass isolated from a 74-year-old female; MDA-mb-231, a breast tumor ceh line isolated from the pleural effusion of a 51 -year-old female, which forms poorly differentiated adenocarcino
  • the primary mammary epithehal ceh line HMEC was derived from normal human mammary tissue (Clonetics, San Diego, CA). Ah ceh cultures were propagated in a chemicahy-defined medium, according to the suppher's recommendations and grown to 70-80% confluence prior to RNA isolation.
  • the microarray experiments showed that expression of SEQ ED NO:43 was decreased by at least two-fold in ah six ceh lines examined as compared to the primary mammary epithehal ceh line HMEC. Therefore, in various embodiments, SEQ DD NO:43 can be used for one or more of the fohowing: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and hi) developing therapeutics and/or other treatments for breast cancer.
  • SEQ ED NO:27 showed differential expression in senescent (passage 8) and pre-senescent (passage 7) versus non-senescent progenitor PrEC cehs (passage 3), as determined by microanay expression analysis.
  • Senescent cehs accumulate with age in human tissues. The tissue microenvironment may be disrupted by the accumulation of dysfunctional senescent cehs. Thus, mutation accumulation may synergize with the accumulation of senescent cehs, leading to the increased risk for developing cancer, a hallmark of mammalian aging.
  • PrEC are primary prostate epithehal cehs isolated from a normal donor and were grown in the optimal growth media to 70-80% confluence prior to harvesting.
  • SEQ DD NO:27 expression levels differed by at least 2-fold in senescent (passage 8) and pre-senescent (passage 7) PrECs, when compared to non-senescent PrECs. Therefore, in various embodiments, SEQ DD NO:27 can be used for one or more of the fohowing: i) monitoring treatment of cancer and other age-related disorders, ii) diagnostic assays for cancer and other age-related disorders, and hi) developing therapeutics and/or other treatments for cancer and other age-related disorders.
  • SEQ DD NO:43 also showed differential expression in prostate cancer, as determined by microarray analysis.
  • Prostate carcinoma ceh lines at various stages of tumor progression were compared to primary prostate epithehal cehs.
  • the prostate carcinoma ceh lines include: DU145, a prostate carcinoma ceh line with no detectable sensitivity to hormones, isolated from a metastatic site in the brain of a 69-year-old male, that does not express prostate specific antigen; LNCaP, a prostate carcinoma ceh line that expresses androgen receptors and prostate specific antigen and was isolated from a lymph node of a 50-year-old male with metastatic prostate cancer; and PC3, a prostate adenocarcinoma ceh line isolated from a metastatic site in the bone of a 62-year-old male with grade TV prostate adenocarcinoma.
  • SEQ ED NO:43 can be used for one or more of the fohowing: i) monitoring treatment of prostate cancer, ii) diagnostic assays for prostate cancer, and hi) developing therapeutics and/or other treatments for prostate cancer.
  • SEQ ED NO:44 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 abihty to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smah 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 individuahy 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:44 was increased by at least two-fold in blood leukocytes as compared to the reference sample. Therefore, SEQ ID NO:44 can be used as a marker for blood leukocytes.
  • Sequences complementary to the CADECM-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturahy occurring CADECM.
  • ohgonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CADECM.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary ohgonucleotide is designed to prevent ribosomal binding to the CADECM-encoding transcript.
  • CADECM expression and purification of CADECM is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the 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 isopropylbeta-D- tliiogalactopyranoside (IPTG).
  • CADECM in eukaryotic cehs is achieved by infecting insect or mammalian ceh lines with recombinant Autographica califomica nuclear polyhedrosis virus (AcMNPV), commonly known as baculoviras.
  • AcMNPV Autographica califomica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CADECM by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • baculovirus Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cehs 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, Y. et al. (1996) Hum. Gene Ther. 7:1937- 1945).
  • CADECM is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceh lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobihzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
  • the GST moiety can be proteolyticahy cleaved from CADECM at specificahy engineered sites.
  • FLAG an 8-amino acid peptide
  • 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins
  • CADECM function is assessed by expressing the sequences encoding CADECM at physiologicahy elevated levels in mammalian ceh culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter.
  • 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceh line, for example, an endothelial or hematopoietic ceh line, using either hposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cehs from nontransfected cehs and is a rehable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • an automated, laser optics-based technique is used to identify transfected cehs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cehs and other cehular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceh death.
  • CADECM The influence of CADECM on gene expression can be assessed using highly purified populations of cehs transfected with sequences encoding CADECM and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cehs and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cehs are efficiently separated from nontransfected cehs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cehs using methods weh known by those of skih 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
  • PAGE polyacrylamide gel electrophoresis
  • the CADECM arnino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a conesponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skih in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or hydrophilic regions are weh described in the art (Ausubel et al., supra, ch. 11).
  • ohgopeptides of about 15 residues in length are synthesized using an ABI 43 IA peptide synthesizer (Apphed 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.
  • Media containing CADECM are passed over the immunoaffinity column, and the column is washed under conditions that ahow 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.
  • CADECM or biologicahy active fragments thereof, are labeled with 1 5 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
  • Bolton-Hunter reagent Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously anayed in the wehs of a multi-weh plate are incubated with the labeled CADECM, washed, and any wehs with labeled CADECM complex are assayed. Data obtained using different concentrations of CADECM are used to calculate values for the number, affinity, and association of CADECM with the candidate molecules.
  • molecules interacting with CADECM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commerciahy 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 CT) which employs the yeast two-hybrid system in a high-throughput manner to determine ah interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • An assay for CADECM activity measures the expression of CADECM on the ceh surface.
  • cDNA encoding CADECM is transfected into a non-leukocytic ceh line.
  • Ceh 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 irrrmunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of CADECM expressed on the ceh surface.
  • an assay for CADECM activity measures the amount of ceh aggregation induced by overexpression of CADECM.
  • cultored cehs 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 ceh agglutination, or clumping, associated with transfected cehs is compared with that associated with untransfected cehs.
  • the amount of ceh agglutination is a direct measure of CADECM activity.
  • an assay for CADECM activity measures the disruption of cytoskeletal filament networks upon overexpression of CADECM in cultured ceh lines (Rezniczek, G. A. et al. (1998) J. Ceh 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 cehs, such as rat kangaroo PtK2 or rat bladder carcinoma 804G cehs.
  • Actin filaments and intermediate filaments such as keratin and vimentin are visualized by immunofluorescence microscopy using antibodies and techniques weh known in the art.
  • the configuration and abundance of cyoskeletal filaments can be assessed and quantified using confocal imaging techniques.
  • the bundling and cohapse of cytoskeletal filament networks is indicative of CADECM activity.
  • ceh adhesion activity in CADECM is measured in a 96-weh plate in which wehs are first coated with CADECM by adding solutions of CADECM of varying concentrations to the wehs. Excess CADECM is washed off with saline, and the wehs incubated with a solution of 1% bovine serum albumin to block non-specific ceh binding. Ahquots of a ceh suspension of a suitable ceh type are then added to the wehs and incubated for a period of time at 37 °C. Non-adherent cehs are washed off with saline and the cehs stained with a suitable ceh stain such as Coomassie blue.
  • a suitable ceh stain such as Coomassie blue.
  • the intensity of staining is measured using a variable wavelength multi-weh plate reader and compared to a standard curve to determine the number of cehs adhering to the CADECM coated plates.
  • the degree of ceh staining is proportional to the ceh adhesion activity of CADECM in the sample.

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Abstract

Divers modes de réalisation de l'invention concernent des protéines et des polynucléotides de matrice extracellulaire et d'adhésion de cellule humaine (CADECM) qui identifient et qui sont codants pour ces CADECM. Des modes de réalisation de l'invention concernent aussi des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. D'autres modes de réalisation de l'invention enfin concernent des techniques destinées à diagnostiquer, traiter ou prévenir des troubles associés à une expression aberrante de CADECM.
PCT/US2002/024649 2001-08-03 2002-08-02 Proteines de matrice extracellulaire et d'adhesion cellulaire WO2003027230A2 (fr)

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Cited By (7)

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US8921056B2 (en) 2003-10-10 2014-12-30 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt
US8951745B2 (en) 2003-10-10 2015-02-10 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-Spondins) and/or Wnt
US9081011B2 (en) 2003-10-10 2015-07-14 Deutsches Krebsforschungszentrum Compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (R-spondins) and/or Wnt
US8926970B2 (en) 2006-10-20 2015-01-06 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Rspondin antibodies as inhibiting factors of angiogenesis and vaculogenesis
US9226963B2 (en) 2006-10-20 2016-01-05 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Antagonist anti-Rspondin3 antibodies
US10273276B2 (en) 2006-10-20 2019-04-30 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Rspondins as modulators of angiogenesis and vasculogenesis
US10538563B2 (en) 2006-10-20 2020-01-21 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Rspondins as modulators of angiogenesis and vasculogenesis

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