WO2002002634A2 - Molécules de matrice extracellulaire et d'adhésion cellulaire - Google Patents

Molécules de matrice extracellulaire et d'adhésion cellulaire Download PDF

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Publication number
WO2002002634A2
WO2002002634A2 PCT/US2001/021067 US0121067W WO0202634A2 WO 2002002634 A2 WO2002002634 A2 WO 2002002634A2 US 0121067 W US0121067 W US 0121067W WO 0202634 A2 WO0202634 A2 WO 0202634A2
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WIPO (PCT)
Prior art keywords
polynucleotide
seq
polypeptide
amino acid
sequence
Prior art date
Application number
PCT/US2001/021067
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English (en)
Other versions
WO2002002634A3 (fr
Inventor
Y. Tom Tang
Henry Yue
Yalda Azimzai
Ann He
Sajeev Batra
Terence P. Lo
Danniel B. Nguyen
John D. Burrill
Gregory A. Marcus
Kurt A. Zingler
Ameena R. Gandhi
Preeti Lal
Liam Kearney
Neil Burford
Monique G. Yao
Narinder K. Chawla
Vicki S. Elliot
Chandra S. Arvizu
Farrah A. Khan
Mariah R. Baughn
April J. A. Hafalia
Jennifer L. Policky
Janice Au-Young
Yan Lu
Mark L. Borowsky
Dyung Aina M. Lu
Jayalaxmi Ramkumar
Junming Yang
Rajagopal Gururajan
Bridget A. Warren
Kimberly Gietzen
Yuming Xu
Deborah A. Kallick
Ernestine A. Lee
Kavitha Thangavelu
Angelo M. Delegeane
Sally Lee
Original Assignee
Incyte Genomics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Incyte Genomics, Inc. filed Critical Incyte Genomics, Inc.
Priority to US10/312,352 priority Critical patent/US20040053824A1/en
Priority to CA002413186A priority patent/CA2413186A1/fr
Priority to AU2001273151A priority patent/AU2001273151A1/en
Priority to EP01952392A priority patent/EP1383892A2/fr
Priority to JP2002507885A priority patent/JP2004528003A/ja
Publication of WO2002002634A2 publication Critical patent/WO2002002634A2/fr
Publication of WO2002002634A3 publication Critical patent/WO2002002634A3/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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to nucleic acid and amino acid sequences of extracellular matrix and cell adhesion molecules and to the use of these sequences in the diagnosis, treatment, and prevention of genetic, immune/inflammatory, developmental, neurological, connective tissue, and cell proUferative disorders, including cancer and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of extracellular matrix and cell adhesion molecules.
  • 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 suppoitive 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. (1 96) 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.
  • 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.
  • 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 HI 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). The RGD sequence is recognized by the integrin family of cell surface receptors and is also found in other ECM proteins. (Reviewed in Alberts, et al. (1994) Molecular Biology of the Cell. Garland Publishing, New York, NY, 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, 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 transforming growth factor ⁇ , suggesting a role for proteoglycans in cell-cell communication. (Reviewed in Alberts, supra, pp. 973-978.)
  • DPP Dentin phosphoryn
  • odontoblasts Gu, K., et al. (1998) Eur. J. Oral Sci. 106:1043- 1047. DPP is believed to nucleate or modulate the formation of hydroxyapatite crystals.
  • Mucins are highly glycosylated glycoproteins that are the major structural component of the mucus gel. The physiological functions of mucins are cytoprotection, mechanical protection, maintenance of viscosity in secretions, and cellular recognition.
  • MUC6 is a human gastric mucin that is also found in gall bladder, pancreas, seminal vesicles, and female reproductive tract (Toribara, N.W., et al. (1997) J. Biol. Chem. 272:16398-16403). The MUC6 gene has been mapped to human chromosome 11 (Toribara, N.W., et al. (1993) J. Biol. Chem. 268:5879-5885).
  • Hemomucin is a novel Drosophila surface mucin that may be involved in the induction of antibacterial effector molecules (Theopold, U., et al. (1996) J. Biol. Chem. 217:12708-12715).
  • Olfactomedin was originally identified as the major component of the mucus layer surrounding the chemosensory dendrites of olfactory neurons.
  • Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs.
  • the TIGR/myocilin protein, an olfactomedin-related protein expressed in the eye, is associated with the pathogenesis of glaucoma (Kulkarni, N.H. et al. (2000) Genet. Res. 76:41-50).
  • Ankyrin (ANK) repeats mediate protein-protein interactions associated with diverse intracellular functions.
  • ANK repeats are composed of about 33 amino acids that form a helix-turn- helix core preceded by a protruding "tip.” These tips are of variable sequence and may play a role in protein-protein interactions.
  • the helix-turn-helix region of the ANK repeats stack on top of one another and are stabilized by hydrophobic interactions (Yang, Y. et al. (1998) Structure 6:619-626).
  • Sushi repeats also called short consensus repeats (SCR), are found in a number of proteins that share the common feature of binding to other proteins.
  • the sushi domain is important for heparin binding.
  • Sushi domains contain basic amino acid residues, which may play a role in binding (OleszewsM, 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. There is close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin (Kohda, D. et al. (1996) Cell, Vol. 86, 767-775).
  • Multidomain or mosaic proteins play an important role in the diverse functions of the extracellular matrix (Engel, J. et al. (1994) Development (Camb.) S35-42).
  • 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, C.G. New Biol (1990) May;2(5):410-9).
  • 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).
  • a number of proteins that contain calcium-binding EGF-like domain signature sequences are involved in growth and differentiation. Examples include bone mo ⁇ hogenic protein 1 , which induces the formation of cartilage and bone; crumbs, which is a Drosophila epithelial development protein; Notch and a number of its homologs,. which are involved in neural growth and differentiation, and transforming growth factor beta-1 binding protein (Expasy PROSITE document PDOC00913; Soler, C. and Carpenter, G., in Nicola, N.A. (1994) The Cytokine Facts Book, Oxford University Press, Oxford, UK, pp 193-197). EGF-like domains mediate protein-protein interactions for a variety of proteins.
  • EGF-like domains in the ECM glycoprotein fibulin-1 have been shown to mediate both self-association and binding to fibronectin (Tran, H. et al. (1997) J. Biol. Chem. 272:22600-22606).
  • Point mutations in the EGF-like domains of ECM proteins have been identified as the cause of human disorders such as Marfan syndrome and pseudochondroplasia (Maurer, P. et al. (1996) Curr. Opin. Cell Biol. 8:609-617).
  • the CUB domain is an extracellular domain of approximately 110 amino acid residues found mostly in developmentally regulated proteins.
  • the CUB domain contains four conserved cysteine residues and is predicted to have a structure similar to that of immunoglobulins.
  • Vertebrate bone mo ⁇ hogenic protein 1, which induces cartilage and bone formation, and fibropellins I and IH 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 Bonaldo, P. (1991) Blood 77:2305-2315). Crystal structure analysis of an integrin vWFA-like module shows a classic "Rossmann" fold and suggests a metal ion-dependent adhesion site for binding protein ligands (Lee, J.-O. et al. (1995) Cell 80:631-638).
  • 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 share 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 Tucker, R. P. (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).
  • Adhesion- Associated Proteins Adhesion- Associated Proteins
  • the surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the ECM.
  • the interaction of the cell with its surroundings profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. Families of cell adhesion molecules include the cadherins, integrins, lectins, neural cell adhesion proteins, and some members of the proline-rich proteins.
  • Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. These proteins share multiple repeats of a cadherin-specific motif, and the repeats form the folding units of the cadherin extracellular domain. Cadherin molecules cooperate to form focal contacts, or adhesion plaques, between adjacent epithelial cells.
  • the cadherin family includes the classical cadherins and protocadherins.
  • Classical cadherins include the E-cadherin, N-cadherin, and P-cadherin subfamilies. E-cadherin is present on many types of epithelial cells and is especially important for embryonic
  • 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 ⁇ . integrins function as receptors that play a role in signal transduction. For example, binding of integrin to its extracellular ligand may stimulate changes in intracellular calcium levels or protein kinase activity (Sjaastad, M.D. and Nelson, W.J. (1997) BioEssays 19:47-55). At least ten cell surface receptors of the integrin family recognize the ECM component fibronectin, which is involved in many different biological processes including cell migration and embryogenesis (Johansson, S. et al. (1997) Front. Biosci. 2D126-D146).
  • Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K. and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9:237-264). This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L. A. (1991) J. Cell. Biochem. 45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).
  • Lectins are further classified into subfamilies based on carbohydrate-binding specificity and other criteria.
  • the galectin subfamily includes lectins that bind ⁇ -galactoside carbohydrate moieties in a thiol-dependent manner (reviewed in Hadari, Y. R. et al. (1 98) J. Biol. Chem. 270:3447-3453).
  • Galectins are widely expressed and developmentally regulated.
  • Galectins contain a characteristic carbohydrate recognition domain (CRD).
  • the CRD is about 140 amino acids and contains several stretches of about 1 - 10 amino acids which are highly conserved among all galectins.
  • a particular 6-amino acid motif within the CRD contains conserved tryptophan and arginine residues which are critical for carbohydrate binding.
  • the CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation. Secondary structure predictions indicate that the CRD forms several ⁇ -sheets.
  • Galectins play a number of roles in diseases and conditions associated with cell-cell and cell- matrix interactions. For example, certain galectins associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice.
  • 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
  • NCAP participates in neuronal cell migration, cell adhesion, neurite outgrowth, axonal fasciculation, pathf ⁇ nding, synaptic target-recognition, synaptic formation, myelination and regeneration. NCAPs are expressed on the surfaces of neurons associated with learning and memory. Mutations in genes encoding NCAPS are linked with neurological diseases, including hereditary neuropathy Charcot-Marie-Tooth disease, Dejerine-Sottas disease, X-linked hydrocephalus, MASA syndrome (mental retardation, aphasia, shuffling gait and adducted thumbs), and spastic paraplegia type I. In some cases, expression of NCAP is not restricted to the nervous system.
  • 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 et al. (1996) J. Cell Biol. 132:475-485).
  • NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et al., supra). They are generally linked to the plasma membrane through a transmembrane domain and/or a glycosyl-phosphatidyiinositol (GPI) anchor. The GPI linkage can be cleaved by GPI phospholipase C. Most NCAPs consist of an extracellular region made up of one or more immunoglobulin domains, a membrane spanning domain, and an intracellular region. Many NCAPs contain post-translational modifications including covalently attached oligosaccharide, glucuronic acid, and sulfate. NCAPs fall into three subgroups: simple-type, complex-type, and mixed-type.
  • Simple-type NCAPs contain one or more variable or constant immunoglobulin domains, but lack other types of domains.
  • Members of the simple-type subgroup include Schwann cell myelin protein (SMP), limbic system-associated membrane protein (LAMP), opiate-binding cell-adhesion molecule (OBCAM), and myelin-associated glycoprotein (MAG).
  • 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.
  • 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 H (GGFH) and acetylcholine receptor-inducing factor (ARIA), and the semaphorin/collapsin family such as semaphorin B and colrapsin.
  • NGF nerve growth factor
  • NT4 neurotropin 4
  • GGFH glial growth factor H
  • ARIA acetylcholine receptor-inducing factor
  • semaphorin/collapsin family such as semaphorin B and colrapsin.
  • Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropttm, 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 suggested as having roles in protein-protein interactions and are suggested to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J.A.
  • NCAP subfamily includes cell adhesion proteins expressed on distinct subpopulations of brain neurons.
  • Members of the NCAP-LON subgroup possess three immunoglobulin domains and bind to cell membranes through GPI anchors.
  • Kilon (a kindred of NCAP-LON), for example, is expressed in the brain cerebral cortex and hippocampus (Funatsu et al. (1999) J. Biol. Chem. 274:8224-8230). Immunostaining localizes Kilon to the dendrites and soma of pyramidal neurons.
  • Kilon has three C2 type immunoglobulin-like domains, six predicted glycosylation sites, and a GPI anchor. Expression of Kilon is developmentally regulated. It is expressed at higher levels in adult brain in comparison to embryonic and early postnatal brains. Confocal microscopy shows the presence of Kilon in dendrites of hypothalamic magnocellular neurons secreting
  • Arginine vasopressin regulates body fluid homeostasis, extracellular osmolarity and intravascular volume.
  • Oxytocin induces contractions of uterine smooth muscle during child birth and of myoepithelal cells in mammary glands during lactation.
  • Kilonis proposed to play roles in the reorganization of dendritic connections during neuropeptide secretion.
  • PRPs proline-rich proteins
  • PRPs are defined by a high frequency of proline, ranging from 20-50% of the total amino acid content. Some PRPs have short domains which are rich in proline. These proline-rich regions are associated with protein-protein interactions.
  • PRPs proline-rich synapse-associated proteins
  • PSD postsynaptic density
  • ProSAP Members of ProSAP contain at the N-terminus six to seven ankyrin repeats, followed by an SH3 domain, a PDZ domain, then by seven proline-rich regions and a SAM domain at the C terminus.
  • Another member of PRP is the HLA-B-associated transcript 2 protein (BAT2) which is rich in proline and include short tracts of polyproline, polyglycine, and charged amino acids.
  • BAT2 also contains four RGD (Arg-Gly-Asp) motifs typical of integrins (Banerji, J. et al. (1990) Proc.
  • 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.
  • 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 metamo ⁇ hosis (Brown, D.D. et al. (1996) Proc. Natl. Acad. Sci. USA 93:1924- 1929).
  • the WSC domain was originally found in the yeast WSC (cell-wall integrity and stress response component) proteins which act as sensors of environmental stress.
  • the WSC domains are extracellular and are thought to possess a carbohydrate binding role (Ponting, C.P.
  • polycystin-1 a human plasma membrane protein. Mutations in polycystin-1 are the cause of the commonest form of autosomal dominant polycystic kidney disease (Ponting, C.P. et al. (1 99) Curr. Biol. 9:R585-R588).
  • Toposome is a cell-adhesion glycoprotein isolated from mesenchyme-blastula embryos. Toposome precursors including vitellogenin promote cell adhesion of dissociated blastula cells.
  • 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 is important for protein/protein interactions and cell adhesion, and LRR proteins are involved in cell cell interactions, mo ⁇ hogenesis, and development (Kobe, B. and Deisenhofer, J. (1995) Curr. Opin. Struct. Biol. 5:409-416).
  • the human ISLR (immunoglobulin superfamily containing leucine-rich repeat) protein contains a C2-type immunoglobulin domain as well as LRR.
  • 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 many 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).
  • ECMCAD extracellular matrix and cell adhesion molecules
  • ECMCAD-1 polypeptides, extracellular matrix and cell adhesion molecules
  • ECMCAD-2 extracellular matrix and cell adhesion molecules
  • ECMCAD-3 extracellular matrix and cell adhesion molecules
  • ECMCAD-4 polypeptides, extracellular matrix and cell adhesion molecules
  • ECMCAD-5 polypeptides, extracellular matrix and cell adhesion molecules
  • ECMCAD-7 polypeptides, extracellular matrix and cell adhesion molecules
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:l-36. In another alternative, the polynucleotide is selected from the group consisting of SEQ DO NO:37-72.
  • the invention 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:l-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36.
  • the invention provides a ceU transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ DD NO: 1-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO: 1 -36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-36.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, b) a naturaUy occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, b) a naturaUy occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, 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 specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionaUy, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ JD NO:37-72, b) a naturaUy occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said ampUfied target polynucleotide or fragment thereof, and, optionaUy, if present, the amount thereof.
  • the invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ JD NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and a pharmaceuticaUy acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
  • the invention additionaUy provides a method of treating a disease or condition associated with decreased expression of functional ECMCAD, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ JD NO:l-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional ECMCAD, comprising administering to a patient in need of such treatment the composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with overexpression of functional ECMCAD, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specificaUy binds to the polypeptide.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, b) a naturaUy occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-36, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-36.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:37-72, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, u) a naturaUy occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:37-72, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:37-72, ii) a naturaUy occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:37-72, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • a target polynucleotide selected from the group consisting of i) a polynucleo
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide sequences of the present invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probabiUty score for the match between each polypeptide and its GenBank homolog is also shown.
  • Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 Usts the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNA library for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with appUcable descriptions, references, and threshold parameters.
  • ECMCAD refers to the amino acid sequences of substantiaUy purified ECMCAD obtained from any species, particularly a mammaUan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of ECMCAD.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of ECMCAD either by directly interacting with ECMCAD or by acting on components of the biological pathway in which ECMCAD participates.
  • An "aUeUc variant” is an alternative form of the gene encoding ECMCAD. AUeUc variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many aUeUc variants of its naturaUy occurring form.
  • altered nucleic acid sequences encoding ECMCAD include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as ECMCAD or a polypeptide with at least one functional characteristic of ECMCAD. Included within this definition are polymo ⁇ hisms which may or may not be readily detectable using a particular oUgonucleotide probe of the polynucleotide encoding ECMCAD, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding ECMCAD.
  • 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 functionaUy equivalent ECMCAD.
  • DeUberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubiUty, hydrophobicity, hydrophiUcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of ECMCAD is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged , amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophiUcity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophiUcity values may include: leucine, isoleucine, and vaUne; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence refer to an oUgopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules.
  • amino acid sequence is recited to refer to a sequence of a naturaUy occurring protein molecule
  • amino acid sequence and Uke terms are not meant to Umit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amination relates to the production of additional copies of a nucleic acid sequence.
  • AmpUfication is generaUy carried out using polymerase chain reaction (PCR) technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of ECMCAD.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of ECMCAD either by directly interacting with ECMCAD or by acting on components of the biological pathway in which ECMCAD participates.
  • antibody refers to intact immunoglobuUn molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind ECMCAD polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oUgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • chemicaUy e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole Umpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specificaUy to antigenic determinants (particular regions or three-dimensional stractures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eUcit the immune response) for binding to an antibody.
  • antisense refers to any composition capable of base-pairing with the "sense”
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone Unkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides 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.
  • the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capabiUty of the natural, recombinant, or synthetic ECMCAD, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding ECMCAD or fragments of ECMCAD may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabiUzing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (AppUed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVEEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • GELVEEW fragment assembly system GCG, Madison WI
  • Phrap Universality of Washington, Seattle WA
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generaUy maintain (a) the stracture of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha heUcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • a "fragment” is a unique portion of ECMCAD or the polynucleotide encoding ECMCAD which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other pruposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentiaUy selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ED NO:37-72 comprises a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:37-72, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:37-72 is useful, for example, in hybridization and ampUfication technologies and in analogous methods that distinguish SEQ JD NO:37-72 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ JD NO:37-72 and the region of SEQ ID NO:37-72 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended pu ⁇ ose for the fragment.
  • a fragment of SEQ ID NO:l-36 is encoded by a fragment of SEQ ID NO:37-72.
  • a fragment of SEQ ID NO:l-36 comprises a region of unique amino acid sequence that specificaUy identifies SEQ ED NO:l-36.
  • a fragment of SEQ ED NO:l-36 is useful as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ED NO:l-36.
  • the precise length of a fragment of SEQ ID NO:l-36 and the region of SEQ ID NO:l-36 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended pu ⁇ ose for the fragment.
  • a “fuU length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aUgned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize aUgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as inco ⁇ orated into the MEGALIGN version 3.12e sequence aUgnment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WT). CLUSTAL V is described in Higgins, D.G. and P.M. Sha ⁇ (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 AUgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aUgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blasto with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantiaUy the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aUgned using a standardized algorithm.
  • Methods of polypeptide sequence aUgnment are weU-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and_hydrophobicity at the site of substitution, thus preserving the stracture (and therefore function) of the polypeptide.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are Unear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome repUcation, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abiUty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive anneaUng conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for anneaUng of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
  • Permissive anneaUng conditions occur, for example, at 68 °C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (TJ for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of • the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides of the present invention include ' wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Useful variations on these wash conditions wiU be readily apparent to those of ordinary s U in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobiUzed on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • soUd support e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or nucleotide 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 signaUng molecules, which may affect ceUular and systemic defense systems.
  • an "immunogenic fragment” is a polypeptide or oUgopeptide fragment of ECMCAD which is capable of eUciting an immune response when introduced into a Uving organism, for example, a ' mammal.
  • the term "immunogenic fragment” also includes any polypeptide or oUgopeptide fragment of ECMCAD which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a pluraUty of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of ECMCAD. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of ECMCAD.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oUgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-Uke or RNA-Uke material.
  • PNA peptide nucleic acid
  • “Operably Unked” 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 Unked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably Unked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oUgonucleotide of at least about 5 nucleotides in length Unked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubiUty to the composition. PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the ceU.
  • Post-translational modification of an ECMCAD may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic miUeu of ECMCAD.
  • Probe refers to nucleic acid sequences encoding ECMCAD, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acid sequences.
  • Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, Ugands, chemiluminescent agents, and enzymes.
  • Probes are short nucleic acids, usuaUy DNA oUgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for ampUfication (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pmpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • OUgonucleotides for use as primers are selected using software known in the art for such pmpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oUgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 lobases. Similar primer selection programs have inco ⁇ orated additional features for expanded capabiUties.
  • the PrimOU primer selection program (available to the pubUc from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • the Primer3 primer selection program (available to the pubUc from the Whitehead Institute/MtT Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming Ubrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oUgonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the pubUc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oUgonucleotide selection are not Umited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturaUy occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accompUshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, 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 Unked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stabiUty.
  • Reporter molecules are chemical or biochemical moieties used for labeUng a nucleic acid, amino acid, or antibody. Reporter molecules include radionucUdes; 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 sequence, is composed of the same Unear sequence of nucleotides as the reference DNA sequence with the exception that aU occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing ECMCAD, nucleic acids encoding ECMCAD, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturaUy associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, sUdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a "transcript image” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not Umited to, bacteriophage or viral infection, electroporation, heat shock, Upofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of repUcation either as an autonomously repUcating plasmid or as part of the host chromosome, as weU as transiently transformed cells which express the inserted DNA or RNA for Umited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not Umited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the ceU, by way of deUberate genetic manipulation, such as by microinjection or by infection with a recombinant viras.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertiUzation, 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%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "aUeUc" (as defined above), “spUce,” “species,” or “polymo ⁇ hic” variant.
  • a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternative spUcing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
  • a polymo ⁇ hic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymo ⁇ hic variants also may encompass "single nucleotide polymo ⁇ hisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymo ⁇ hisms
  • 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 blas ⁇ with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human extraceUular matrix and ceU adhesion molecules (ECMCAD), the polynucleotides encoding ECMCAD, and the use of these compositions for the diagnosis, treatment, or prevention of genetic, immune/inflammatory, developmental, , neurological, connective tissue, and ceU proUferative disorders, including cancer.
  • ECMCAD extraceUular matrix and ceU adhesion molecules
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • polynucleotide sequence identification number Polynucleotide SEQ DD NO:
  • Incyte polynucleotide consensus sequence number Incyte Polynucleotide ID
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (Genbank ID NO:) of the nearest GenBank homolog. Column 4 shows the probabiUty score for the match between each polypeptide and its GenBank homolog. Column 5 shows the annotation of the GenBank homolog along with relevant citations where appUcable, aU of which are expressly inco ⁇ orated by reference herein. Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and
  • FIG. 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein stracture/function analysis and in some cases, searchable databases to which the analytical methods were appUed.
  • SEQ JD NO:2 is 48% identical over 46% of its length to mouse procoUagen type I alpha chain, (GenBank ID gl 92264) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 6.9e-46, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:2 also contains a coUagen triple heUx repeat, as dete ⁇ nined by searching for statisticaUy significant matches in the PFAM database. (See Table 3.) HMMER and SPSCAN analyses indicate the presence of a signal peptide at the N- terminus of SEQ ED NO:2. Data from BLAST analysis of the PRODOM and DOMO databases, as weU as MOTIFS analysis, provide further corroborative evidence that SEQ ED NO:2 is a ceUular matrix protein associated with ceU adhesion.
  • SEQ ID NO:6 is 64% identical to frog MAM domain protein (GenBank ID gl 234793) as determined by the Basic Local AUgnment Search Tool (BLAST).
  • SEQ ID NO:6 also contains four MAM domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from MOTIFS analysis provide further corroborative evidence that SEQ ID NO:6 is a MAM domain ceU adhesion protein.
  • SEQ ID NO:10 is 80% identical to murine semaphorin B (GenBank ID g854326) as determined by the Basic Local AUgnment Search Tool (BLAST).
  • the BLAST probabiUty score is 6.0e-66, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:10 also contains a sema domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • the BLAST and HMMER analyses provide evidence that SEQ ID NO:10 is a semaphorin.
  • SEQ DD NO : 12 is 44% identical to human cadherin superfamily protein VR4- 11
  • SEQ ID NO: 12 (GenBank ID g9622240) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 9.9e-170, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO: 12 also contains a cadherin domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFTLESCAN analyses provide further corroborative evidence that SEQ ED NO:12 is a cadherin.
  • HMM hidden Markov model
  • SEQ ED NO:14 is 91 % identical to murine neuronal glycoprotein (GenBank DD g200057) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ DD NO: 14 also contains fibronectin type HI and immunoglobuUn domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) The BLAST and HMMER analyses provide evidence that SEQ ID NO:14 is a ceU adhesion molecule. In an alternative example, SEQ DD NO:22 is 79% identical to mouse laminin 5 alpha chain (GenBank DD g2599232) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The ,
  • BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide
  • SEQ DD NO:22 also contains a laminin N-terminal domain, multiple laminin EGF-Uke domains, a laminin B domain, and laminin G domains, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, and MOTIFS analyses provide further corroborative evidence that SEQ DD NO:22 is a laminin.
  • SEQ DD NO:24 is 89% identical to Bos taurus brevican (GenBank DD g452821) as determined by the Basic Local AUgnment Search Tool (BLAST).
  • SEQ DD NO:24 also contains a lectin C-type domain, an exfraceUular Unk domain, an EGF-Uke domain, a sushi domain, and an immunoglobuUn domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ DD NO:31 is 87% identical to amouse semaphorin homolog (GenBank DD glll0599) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 0.0, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:31 also contains a Sema domain and a plexin repeat as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ DD NO:31 is a semaphorin.
  • SEQ DD NO:35 is 61 % identical to murine C-type lectin (GenBank DD g4159801) as determined by the Basic Local AUgnment Search Tool (BLAST).
  • BLAST Basic Local AUgnment Search Tool
  • the BLAST probabiUty score is 2.9e-75, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ, ID NO:35 also contains a lectin C-type domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and PROFTLESCAN analyses provide further corroborative evidence that SEQ DD NO:35 is a lectin.
  • SEQ DD NO:l, SEQ HD NO:3-5, SEQ DD NO:7-9, SEQ JD NO:ll, SEQ ID NO:13, SEQ ID NO:15-21, SEQ HD NO:23, SEQ DD NO:25-30, SEQ DD NO:32-34 and SEQ DD NO:36 were analyzed and annotated in a similar manner.
  • SEQ DD NO: 1-36 The algorithms and parameters for the analysis of SEQ DD NO: 1-36 are described in Table 7. As shown in Table 4, the fuU length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Columns 1 and 2 Ust the polynucleotide sequence identification number (Polynucleotide SEQ DD NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide DD) for each polynucleotide of the invention. Column 3 shows the length of each polynucleotide sequence in basepairs.
  • Column 4 Usts fragments of the polynucleotide sequences which are useful, for example, in hybridization or ampUfication technologies that identify SEQ DD NO:37-72 or that distinguish between SEQ DD NO:37-72 and related polynucleotide sequences.
  • Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the fuU length polynucleotide sequences of the invention.
  • Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective fuU length sequences.
  • the identification numbers in Column 5 of Table 4 may refer specificaUy, for example, to Incyte cDNAs along with their corresponding cDNA Ubraries.
  • 7347284H1 is the identification number of an Incyte cDNA sequence
  • LUNLTUE01 is the cDNA Ubrary from which it is derived.
  • Incyte cDNAs for which cDNA Ubraries are not indicated were derived from pooled cDNA Ubraries (e.g., 71699406V1).
  • the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., gl 242437) which contributed to the assembly of the fuU length polynucleotide sequences.
  • the identification numbers in column 5 may refer to coding regions predicted by Genscan analysis of genomic DNA.
  • GNN.g7923864_002 is the identification number of a Genscan-predicted coding sequence, with g7923864 being the GenBank identification number of the sequence to which Genscan was appUed.
  • the Genscan-predicted coding sequences may have been edited prior to assembly.
  • the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon-stretching" algorithm.
  • exon-stretching for example,
  • FL2428715_g6815043_000026_g8052237_l_3_4.edit is the identification number of a "stretched" sequence, with 2428715 being the Incyte project identification number, g6815043 being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was appUed, and g8052237 being the GenBank identification number of the nearest GenBank protein homolog. (See Example V.) In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Ubraries for those fuU length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA Ubrary is the Incyte cDNA Ubrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA Ubraries shown in Table 5 are described in Table 6.
  • the invention also encompasses ECMCAD variants.
  • a preferred ECMCAD variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the ECMCAD amino acid sequence, and which contains at least one functional or structural characteristic of ECMCAD.
  • the invention also encompasses polynucleotides which encode ECMCAD.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ DD NO:37-72, which encodes ECMCAD.
  • the polynucleotide sequences of SEQ DD NO:37-72 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • The. invention also encompasses a variant of a polynucleotide sequence encoding ECMCAD.
  • such a variant polynucleotide sequence wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding ECMCAD.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ DD NO:37- 72 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 ED NO:37-72. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of ECMCAD.
  • nucleotide sequences which encode ECMCAD and its variants are generally capable of hybridizing to the nucleotide sequence of the naturaUy occurring ECMCAD under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding ECMCAD or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-Ufe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of DNA sequences which encode ECMCAD and ECMCAD derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding ECMCAD or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ DD NO:37-72 and fragments thereof under various conditions of stringency.
  • Hybridization conditions including anneaUng and wash conditions, are described in "Definitions.” Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppUed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 Uquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppUed Biosystems).
  • Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppUed Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
  • the resulting sequences are analyzed using a variety of algorithms which are weU known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology. John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
  • the nucleic acid sequences encoding ECMCAD 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.
  • restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector.
  • inverse PCR uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and Ugations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al.
  • primers may be designed using commerciaUy 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.
  • Ubraries When screening for fuU length cDNAs, it is preferable to use Ubraries that have been size-selected to include larger cDNAs. In addition, random-primed Ubraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oUgo d(T) Ubrary does not yield a full-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • CapiUary electrophoresis systems which are commerciaUy available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in Umited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode ECMCAD may be cloned in recombinant DNA molecules that direct expression of ECMCAD, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantiaUy the same or a functionaUy equivalent amino acid sequence may be produced and used to express ECMCAD.
  • the nucleotide sequences of the present invention can be engineered using methods generaUy known in the art in order to alter ECMCAD-encoding sequences for a variety of pmposes including, but not Umited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oUgonucleotides may be used to engineer the nucleotide sequences.
  • oUgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spUce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C-C. et al (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of ECMCAD, such as its biological or enzymatic activity or its abiUty to bind to other molecules or compounds.
  • MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C-C. et al (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et
  • DNA shuffling is a process by which a Ubrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Ubrary 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.
  • fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturaUy occulting genes in a directed and controUable manner.
  • sequences encoding ECMCAD may be synthesized, in whole or in part, using chemical methods weU known in the art.
  • chemical methods See, e.g., Carathers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • ECMCAD itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or soUd-phase techniques.
  • Creighton, T. (1984) Proteins, Structures and Molecular Properties WTL Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.
  • Automated synthesis maybe achieved using the ABI 431 A peptide synthesizer (AppUed Biosystems).
  • AdditionaUy the amino acid sequence of ECMCAD, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance Uquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides maybe confirmed by amino acid analysis or by sequencing.
  • the nucleotide sequences encoding ECMCAD 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.
  • elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 ' untranslated regions in the vector and in polynucleotide sequences encoding ECMCAD.
  • Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding ECMCAD.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • sequences encoding ECMCAD and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162.)
  • ECMCAD expression vector/host systems
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculoviras); plant ceU systems transformed with viral expression vectors (e.g., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect ceU systems infected with viral expression vectors (e.g., baculoviras)
  • plant ceU systems transformed with viral expression vectors e.g., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV
  • Expression vectors derived from retroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids, may be used for deUvery of nucleotide sequences to the targeted organ, tissue, or ceU population.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding ECMCAD.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding ECMCAD can be achieved using a multifunctional E. coh vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORTl plasmid (life Technologies).
  • PBLUESCRIPT Stratagene, La JoUa CA
  • PSPORTl plasmid life Technologies
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of ECMCAD may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of ECMCAD.
  • a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of ECMCAD. Transcription of sequences encoding ECMCAD may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; and Winter, J. et al.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding ECMCAD may be Ugated into an adenoviras 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 viras which expresses ECMCAD in host ceUs.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host ceUs.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs Human artificial chromosomes
  • HACs may also be employed to deUver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and deUvered via conventional deUvery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic pu ⁇ oses.
  • Convention deUvery methods Uposomes, polycationic amino polymers, or vesicles
  • sequences encoding ECMCAD can be transformed into ceU Unes using expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • FoUowing the introduction of the vector cells may be aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the p pose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • ceU Unes any number of selection systems may be used to recover transformed ceU Unes. These include, but are not Umited to, the he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetaboUte, 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 cMorsulfuron and phosphinotricm acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboUtes.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 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 ECMCAD is inserted within a marker gene sequence
  • transformed cells containing sequences encoding ECMCAD can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding ECMCAD under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
  • host ceUs that contain the nucleic acid sequence encoding ECMCAD and that express ECMCAD may be identified by a variety of procedures known to those of skill in the art.
  • a two-site, monoclonal-based immunoassay utiUzing monoclonal antibodies reactive to two non-interfering epitopes on ECMCAD is preferred, but a competitive binding assay may be employed.
  • These and other assays are weU known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. V; CoUgan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ECMCAD include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide.
  • sequences encoding ECMCAD, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the Uke.
  • Host ceUs transformed with nucleotide sequences encoding ECMCAD may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode ECMCAD may be designed to contain signal sequences which direct secretion of ECMCAD through a prokaryotic or eukaryotic cell membrane.
  • a host ceU strain may be chosen for its abiUty to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not Umited to, acetylation, carboxylation, glycosylation, phosphorylation, Upidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • natural, modified, or recombinant nucleic acid sequences encoding ECMCAD may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric ECMCAD protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of ECMCAD activity.
  • Heterologous protein and peptide moieties may also faciUtate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not Umited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmoduUn 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 immobiUzed glutathione, maltose, phenylarsine oxide, calmoduUn, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the ECMCAD encoding sequence and the heterologous protein sequence, so that ECMCAD may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
  • a variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins.
  • synthesis of radiolabeled ECMCAD may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • ECMCAD of the present invention or fragments thereof may be used to screen for compounds that specificaUy bind to ECMCAD. At least one and up to a pluraUty of test compounds may be screened for specific binding to ECMCAD.
  • test compounds include antibodies, oUgonucleotides, proteins (e.g., receptors), or smaU molecules.
  • the compound thus identified is closely related to the natural Ugand of ECMCAD, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • ECMCAD ECMCAD
  • the compound can be closely related to the natural receptor to which ECMCAD binds, or to at least a fragment of the receptor, e.g., the Ugand binding site. In either case, the compound can be rationaUy designed using known techniques.
  • screening for these compounds involves producing appropriate ceUs which express ECMCAD, either as a secreted protein or on the ceU membrane.
  • Preferred ceUs include ceUs from mammals, yeast, Drosophila, or E. coU.
  • CeUs expressing ECMCAD or ceU membrane fractions which contain ECMCAD are then contacted with a test compound and binding, stimulation, or inhibition of activity of either ECMCAD 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 ECMCAD, either in solution or affixed to a soUd support, and detecting the binding of ECMCAD to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • ECMCAD of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of ECMCAD.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for ECMCAD activity, wherein ECMCAD is combined with at least one test compound, and the activity of ECMCAD in the presence of a test compound is compared with the activity of ECMCAD in the absence of the test compound. A change in the activity of ECMCAD in the presence of the test compound is indicative of a compound that modulates the activity of ECMCAD.
  • a test compound is combined with an in vitro or ceU-free system comprising ECMCAD under conditions suitable for ECMCAD activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of ECMCAD may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraUty of test compounds may be screened.
  • polynucleotides encoding ECMCAD or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent Number 5,175,383 and U.S. Patent Number 5,767,337.)
  • mouse ES ceUs such as the mouse 129/SvJ ceU Une, are derived from the early mouse embryo and grown in culture.
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • 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) CUn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding ECMCAD may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceUs have the potential to differentiate into at least eight separate ceU Uneages including endoderm, mesoderm, and ectodermal ceU types. These ceU Uneages differentiate into, for example, neural ceUs, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding ECMCAD 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 ECMCAD is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred Unes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress ECMCAD e.g., by secreting ECMCAD in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • THERAPEUTICS e.g., by secreting ECMCAD in its milk.
  • ECMCAD is closely associated with brain, prostate, atrial myxoma, cerebeUum, cervical dorsal root gangUon, cardiac muscle, mesentery fat, kidney epitheUum, thymus, endotheUum, ovary, placenta, smooth muscle, fallopian tube, breast, cartilage, bladder, rib, colon, spine, gaU bladder, blood granulocytes, submandibular gland, seminal vesicle, and intestine tissues; with tumors of the brain, prostate, rib, and faUopian tube; and with dermal microvascular endotheUal ceUs, hNT2 ceUs derived from a human teratocarcinoma, and 293-EBNA transformed embryonal ceUs derived from kidney epitheUal tissue
  • ECMCAD appears to play a role in genetic, immune/inflammatory, developmental, neurological, connective tissue, and ceU proUferative disorders, including cancer.
  • ECMCAD In the treatment of disorders associated with increased ECMCAD expression or activity, it is desirable to decrease the expression or activity of ECMCAD.
  • ECMCAD In the treatment of disorders associated with decreased ECMCAD expression or activity, it is desirable to increase the expression or activity of ECMCAD,
  • ECMCAD 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 ECMCAD.
  • disorders include, but are not Umited to, a genetic disorder such as adrenoleukodystrophy, Alport's syndrome, choroideremia, Duchenne and Becker muscular dystrophy, Down's syndrome, cystic fibrosis, chronic granulomatous disease, Gaucher's disease, Huntington's chorea, Marian's syndrome, muscular dystrophy, myotonic dystrophy, pycnodysostosis, Refsum's syndrome, retinoblastoma, sickle ceU anemia, thalassemia, Werner syndrome, von WiUebrand's disease, Wilms' tumor, ZeUweger syndrome, peroxisomal acyl-CoA oxidase deficiency, peroxisomal thiolase deficiency, peroxisomal bifunctional protein de
  • Gerstinann-Straussler-Scheinker syndrome fatal famiUal insomnia, nutritional and metaboUc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebeUoretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metaboUc, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, posthe ⁇ etic neuralgia, Tour
  • a vector capable of expressing ECMCAD 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 ECMCAD including, but not Umited to, those described above.
  • a composition comprising a substantiaUy purified ECMCAD in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ECMCAD including, but not Umited to, those provided above.
  • an agonist which modulates the activity of ECMCAD may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ECMCAD including, but not Umited to, those Usted above.
  • an antagonist of ECMCAD may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ECMCAD.
  • disorders include, but are not Umited to, those genetic, immune/inflammatory, developmental, neurological, connective tissue, and ceU proUferative disorders, including cancer described above.
  • an antibody which specificaUy binds ECMCAD may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express ECMCAD.
  • a vector expressing the complement of the polynucleotide encoding ECMCAD may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ECMCAD including, but not Umited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of ECMCAD may be produced using methods which are generaUy known in the art.
  • purified ECMCAD may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind ECMCAD.
  • Antibodies to ECMCAD may also be generated using methods that are weU known in the art. Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • NeutraUzing antibodies i.e., those which inhibit dimer formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with ECMCAD or with any fragment or oUgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Umited 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 BaciUi Calmette-Guerin
  • Corvnebacterium parvum are especially preferable.
  • the oUgopeptides, peptides, or fragments used to induce antibodies to ECMCAD have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of ECMCAD amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to ECMCAD may be prepared using any technique which provides for , the production of antibody molecules by continuous ceU Unes in culture. These include, but are not Umited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-ceU hybridoma technique
  • the EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.R et al. (1984) Mol. CeU Biol. 62:109-120.
  • chimeric antibodies such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • techniques developed for the production of “chimeric antibodies” such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce ECMCAD-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn Ubraries or panels of highly specific binding reagents as disclosed in the Uterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for ECMCAD may also be generated.
  • such fragments include, but are not Umited to, F(ab 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression Ubraries may be constracted to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for ECMCAD.
  • Affinity is expressed as an association constant, I , which is defined as the molar concentration of ECMCAD-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
  • I association constant
  • the K, determined for a preparation of monoclonal antibodies, which are monospecific for a particular ECMCAD epitope represents a true measure of affinity.
  • High-affinity antibody preparations with K ranging from about IO 9 to IO 12 L/mole are preferred for use in immunoassays in which the ECMCAD-antibody complex must withstand rigorous manipulations.
  • polyclonal antibody preparations may be further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstream appUcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of ECMCAD-antibody complexes.
  • Procedures for evaluating antibody specificity, liter, and avidity, and guideUnes for antibody quaUty and usage in various appUcations, are generaUy available. (See, e.g., Catty, supra, and CoUgan et al. supra.)
  • the polynucleotides encoding ECMCAD may be used for therapeutic pu ⁇ oses.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oUgonucleotides) to the coding or regulatory regions of the gene encoding ECMCAD.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
  • antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding ECMCAD. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press Inc., Totawa NJ.)
  • Antisense sequences can be deUvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein.
  • Slater J.E. et al. (1998) J. AUergy CU. Immunol. 102(3):469-475; and Scanlon, K.J. et al.
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retroviras and adeno-associated virus vectors.
  • viral vectors such as retroviras and adeno-associated virus vectors.
  • retroviras See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.
  • Other gene deUvery mechanisms include Uposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • polynucleotides encoding ECMCAD may be used for somatic or germUne gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCHD)-Xl disease characterized by X- Unked 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.
  • a genetic deficiency e.g., in the cases of severe combined immunodeficiency (SCHD)-Xl disease characterized by X- Unked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672
  • ECMCAD hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiUensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma crazi.
  • diseases or disorders caused by deficiencies in ECMCAD are treated by constructing mammaUan expression vectors encoding ECMCAD and introducing these vectors by mechanical means into ECMCAD-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) ballistic gold particle deUvery, (in) Uposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J-L. and H. Recipon (1998) . Curr. Opin. Biotechnol. 9:445-450).
  • Expression vectors that may be effective for the expression of ECMCAD include, but are not
  • ECMCAD may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma viras (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (n) an inducible promoter (e.g., the tetracycUne-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.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma viras (RSV), SV40 virus, thymidine kinase (TK), or ⁇
  • Uposome transformation kits e.g., the PERFECT LEPED TRANSFECTION KIT, available from Invitrogen
  • aUow one with ordinary skiU in the art to deUver polynucleotides to target ceUs in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammaUan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to ECMCAD expression are treated by constructing a retroviras vector consisting of (i) the polynucleotide encoding ECMCAD under the control of an independent promoter or the retroviras long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev- responsive element (RRE) along with additional retroviras s-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retroviras vectors e.g., PFB and PFBNEO
  • Retroviras vectors are commerciaUy available (Stratagene) and are based onpubUshed data (Riviere, I. et al. (1995) Proc.
  • the vector is propagated in an appropriate vector producing cell Une (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. MiUer (1988) J. Virol, 62:3802-3806; DuU, T. et al, (1998) J. Virol.
  • VPCL vector producing cell Une
  • U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retroviras packaging ceU Unes and is hereby inco ⁇ orated by reference.
  • Propagation of retroviras vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons s iUed in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71 :7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, MX. (1997) J. Virol. 71 :4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
  • an adenoviras-based gene therapy deUvery system is used to deUver polynucleotides encoding ECMCAD to ceUs which have one or more genetic abnormaUties with respect to the expression of ECMCAD.
  • the constraction and packaging of adenoviras-based vectors are weU known to those with ordinary skiU in the art.
  • RepUcation defective adenoviras vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S.
  • Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby inco ⁇ orated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both inco ⁇ orated by reference herein.
  • a he ⁇ es-based, gene therapy deUvery system is used to deUver polynucleotides encoding ECMCAD to target ceUs which have one or more genetic abnormaUties with respect to the expression of ECMCAD.
  • HSV simplex virus
  • the use of he ⁇ es simplex virus (HSV)-based vectors may be especiaUy valuable for introducing ECMCAD to ceUs of the central nervous system, for which HSV has a tropism.
  • HSV simplex virus
  • HSV he ⁇ es simplex viras
  • Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for pmposes including human gene therapy. Also taught by this patent are the constraction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby inco ⁇ orated by reference.
  • an alphavirus (positive, single-stranded RNA viras) vector is used to deUver polynucleotides encoding ECMCAD to target cells.
  • SFV SemUki Forest Virus
  • alphavirus RNA repUcation a subgenomic RNA is generated that normaUy encodes the viral capsid proteins.
  • This subgenomic RNA repUcates to higher levels than the fuU length genomic RNA, resulting in the ove ⁇ roduction 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 ECMCAD into the alphavirus genome in place of the capsid-coding region results in the production of a large number of ECMCAD-coding RNAs and the synthesis of high levels of ECMCAD in vector transduced ceUs.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abiUty to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis viras (SIN) indicates that the lytic repUcation of alphavirases can be altered to suit the needs of the gene therapy appUcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphavirases, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weU known to those with ordinary skiU in the art.
  • OUgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heUx base-pairing methodology.
  • Triple heUx pairing is useful because it causes inhibition of the abiUty of the double heUx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the Uterature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura PubUshing, Mt. Kisco NY, pp. 163-177.)
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of sequences encoding ECMCAD.
  • RNA target Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oUgonucleotide inoperable. The suitabiUty of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides using ribonuclease protection assays.
  • RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemicaUy synthesizing oUgonucleotides such as soUd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding ECMCAD. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into ceU Unes, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stabiUty and half-Ufe. Possible modifications include, but are not Umited 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 Unkages 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 ECMCAD.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not Umited to, oUgonucleotides, antisense oUgonucleotides, triple heUx-forming oUgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encoding ECMCAD may be therapeuticaUy useful, and in the treatment of disorders associated with decreased ECMCAD expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding ECMCAD may be therapeuticaUy useful.
  • At least one, and up to a pluraUty, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary Ubrary of naturaUy-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a ' Ubrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding ECMCAD is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding ECMCAD are assayed by any method commonly known in the art.
  • TypicaUy the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding ECMCAD.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomvces 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 ceU Une such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res.
  • a particular embodiment of the present invention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides,' ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
  • oUgonucleotides such as deoxyribonucleotides,' ribonucleotides, peptide nucleic acids, and modified oUgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. DeUvery by transfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462-466.)
  • compositions which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA).
  • Such compositions may consist of ECMCAD, antibodies to ECMCAD, and mimetics, agonists, antagonists, or inhibitors of ECMCAD.
  • compositions utilized in this invention may be administered by any number of routes including, but not Umited to, oral, intravenous, intramuscular, infra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subUngual, or rectal means.
  • compositions for pulmonary administration may be prepared in Uquid or dry powder form. These compositions are generaUy aerosoUzed immediately prior to inhalation by the patient.
  • aerosol deUvery of fast- acting formulations is weU-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • recent developments in the field of pulmonary deUvery via the alveolar region of the lung have enabled the practical deUvery of drugs such as insuUn to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848).
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pmpose. The determination of an effective dose is weU within the capabiUty of those skilled in the art.
  • SpeciaUzed forms of compositions may be prepared for direct intracellular deUvery of macromolecules comprising ECMCAD or fragments thereof.
  • Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intiaceUular deUvery of the macromolecule.
  • ECMCAD or a fragment thereof may be joined to a short cationic N- terminal portion from the HJV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • the exact dosage wiU be determined by the practitioner, in Ught of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drag combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of deUvery is provided in the Uterature and generaUy available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deUvery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specificaUy bind ECMCAD may be used for the diagnosis of disorders characterized by expression of ECMCAD, or in assays to monitor patients being treated with ECMCAD or agonists, antagonists, or inhibitors of ECMCAD.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for ECMCAD include methods which utiUze the antibody and a label to detect ECMCAD in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • the polynucleotides encoding ECMCAD may be used for diagnostic pmposes.
  • the polynucleotides which may be used include oUgonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of ECMCAD may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of ECMCAD, and to monitor regulation of ECMCAD levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding ECMCAD or closely related molecules may be used to identify nucleic acid sequences which encode ECMCAD.
  • 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 ampUfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding ECMCAD, alleUc 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 ECMCAD encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ HD NO:37-72 or from genomic sequences including promoters, enhancers, and introns of the ECMCAD gene.
  • Means for producing specific hybridization probes for DNAs encoding ECMCAD include the cloning ofpolynucleoti.de sequences encoding ECMCAD or ECMCAD derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commerciaUy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucUdes such as 3 P or 35 S, or by enzymatic labels, such as alkaUne phosphatase coupled to the probe via avidin/biotin coupUng systems, and the Uke.
  • Polynucleotide sequences encoding ECMCAD may be used for the diagnosis of disorders associated with expression of ECMCAD.
  • Such disorders include, but are not Umited to, a genetic disorder such as adrenoleukodystrophy, Alport's syndrome, choroideremia, Duchenne and Becker muscular dystrophy, Down's syndrome, cystic fibrosis, chronic granulomatous disease, Gaucher's disease, Huntington's chorea, Marfan's syndrome, muscular dystrophy, myotonic dystrophy, pycnodysostosis, Refsum's syndrome, retinoblastoma, sickle ceU anemia, thalassemia, Werner syndrome, von WiUebrand's disease, Wilms' tumor, ZeUweger syndrome, peroxisomal acyl- CoA oxidase deficiency, peroxisomal thiolase deficiency, peroxisomal bifunctional protein deficiency, mitochondrial carnitine palmitoyl transferase and carnitine deficiency, mitochondrial very-long-chain acyl-
  • the polynucleotide sequences encoding ECMCAD may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Uke assays; and in microarrays utiUzing fluids or tissues from patients to detect altered ECMCAD expression.
  • Such quaUtative or quantitative methods are weU known in the art.
  • the nucleotide sequences encoding ECMCAD may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding ECMCAD may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding ECMCAD in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in cUnical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabUshed. This maybe accompUshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding ECMCAD, under conditions suitable for hybridization or ampUfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount oftranscri.pt 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 cUnical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earUer thereby preventing the development or further progression of the cancer. Additional diagnostic uses for oUgonucleotides designed from the sequences encoding
  • ECMCAD may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro. OUgomers wiU preferably contain a fragment of a polynucleotide encoding ECMCAD, or a fragment of a polynucleotide complementary to the polynucleotide encoding ECMCAD, and will be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from the polynucleotide sequences encoding ECMCAD may be used to detect single nucleotide polymo ⁇ hisms (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 Umited to, single-stranded conformation polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymo ⁇ hism
  • fSSCP fluorescent SSCP
  • oUgonucleotide primers derived from the polynucleotide sequences encoding ECMCAD are used to ampUfy DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the Uke. SNPs in the DNA cause differences in the secondary and tertiary stractures of PCR products in single-sfranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oUgonucleotide primers are fluorescently labeled, which aUows detection of the ampUmers in high-throughput equipment such as DNA sequencing machines.
  • siUco SNP sequence database analysis methods
  • SNPs sequence database analysis methods, termed in siUco SNP (isSNP)
  • siUco SNP are capable of identifying polymo ⁇ hisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • These computer- based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • ECMCAD ECMCAD
  • Methods which may also be used to quantify the expression of ECMCAD include radiolabeUng or biotinylating nucleotides, coampUfication of a control nucleic acid, and inte ⁇ olating results from standard curves.
  • radiolabeUng or biotinylating nucleotides coampUfication of a control nucleic acid
  • inte ⁇ olating results from standard curves See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oUgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oUgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymo ⁇ hisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • ECMCAD ECMCAD
  • fragments of ECMCAD or antibodies specific for ECMCAD 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 ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al, "Comparative Gene Transcript Analysis," U.S.
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totaUty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraUty of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU Unes, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU Une.
  • 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 precUnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental compounds.
  • AU compounds induce characteristic gene expression patterns, frequently termed molecular finge ⁇ rints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol.
  • the normaUzation procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in inte ⁇ retation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include aU expressed gene sequences.
  • the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
  • Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type.
  • proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
  • a profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuaUzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for ECMCAD to quantify the levels of ECMCAD expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111 ; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures 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 profiUng may be more reUable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untieated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appUcation W095/251116; Shalon, D. et al, (1995) PCT appUcation WO95/35505; Heller, R.A. et al. (1 97) Proc. Natl. Acad. Sci.
  • nucleic acid sequences encoding ECMCAD may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences.
  • conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constractions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA Ubraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • BACs bacterial chromosome PI constructions
  • single chromosome cDNA Ubraries See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev.
  • nucleic acid sequences of the invention may be used to develop genetic Unkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymo ⁇ hism (RFLP).
  • RFLP restriction fragment length polymo ⁇ hism
  • FISH Fluorescent in situ hybridization
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as Unkage analysis using estabUshed chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely locaUzed by genetic Unkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • Unkage analysis using estabUshed chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan species, such as mouse, may reveal associated markers even if the exact
  • nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to ttanslocation, inversion, etc., among normal, carrier, or affected individuals.
  • ECMCAD its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries 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 soUd support, borne on a cell surface, or located intraceUularly. The formation of binding complexes between ECMCAD and the agent being tested may be measured.
  • Another technique for drag screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different smaU test compounds are synthesized on a soUd substrate.
  • the test compounds are reacted with ECMCAD, or fragments thereof, and washed.
  • Bound ECMCAD is then detected by methods weU known in the art.
  • Purified ECMCAD can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
  • the nucleotide sequences which encode ECMCAD may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not Umited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Ubraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A)+ RNA was isolated using oUgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300- 1000 bp) using SEPHACRYLSIOOO, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were Ugated into compatible restriction enzyme sites of the polyUnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof.
  • PBLUESCRIPT plasmid (Stratagene)
  • PSPORTl plasmid (Life Technologies)
  • PCDNA2.1 plasmid Invitrogen, Carlsbad CA
  • PBK-CMV plasmid (Stratagene)
  • Recombinant plasmids were transformed into competent E. coU ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
  • Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophiUzation, at 4°C.
  • plasmid DNA was ampUfied from host ceU lysates using direct Unk PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycUng steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampUfied plasmid DNA was quantified fluorometricaUy 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 foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppUed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) Uquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or suppUed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (AppUed Biosystems) in conjunction with standard ABI protocols and base caUing software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VDT.
  • the polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, Unker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • PubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases
  • BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • HMM is a probabiUstic approach which analyzes consensus primary stractures of gene famines. See, for example, Eddy, S.R. (1996) Curr. Opin. Stract. Biol. 6:361-365.)
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce fuU length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fi ⁇ T length.
  • FuU length polypeptide sequences were translated to derive the corresponding fuU length polypeptide sequences.
  • a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as inco ⁇ orated into the MEGALIGN multisequence aUgnment program (DNASTAR), which also calculates the percent identity between aUgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides appUcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where appUcable, the scores, probabiUty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiUty value, the greater the identity between two sequences).
  • Genscan is a general-pmpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. KarUn (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. KarUn (1998) Curr. Opin. Stract. 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 extraceUular matrix and ceU adhesion molecules. Potential extraceUular matrix and ceU adhesion molecules were also identified by homology to Incyte cDNA sequences that had been annotated as extraceUular matrix and ceU adhesion molecules.
  • Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc 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 pubUc cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • FuU length polynucleotide sequences were obtained by assembUng Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubUc cDNA sequences using the assembly process described in Example DX Alternatively, fuU length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a full 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 fuU 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 IV.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the franslated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenB ank protein homolog.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubUc human genome databases. Partial DNA 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.
  • sequences which were used to assemble SEQ DD NO:37-72 were compared with sequences from the Incyte LIFESEQ database and pubUc domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID NO:37-72 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 pubUc 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.
  • pubUc 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.
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
  • centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers.
  • 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.
  • Mb megabase
  • 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.
  • SEQ JD NO:47 was mapped to chromosome 3 within the interval from 162.00 to 168.30 centiMorgans.
  • SEQ DD NO:49 was mapped to chromosome 4 within the interval from 63.90 to 88.50 centiMorgans.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quaUty in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% • identity and 100% overlap.
  • polynucleotide sequences encoding ECMCAD are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example ID).
  • Each cDNA sequence is derived from a cDNA Ubrary constructed from a human tissue.
  • Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU Une, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding ECMCAD.
  • cDNA sequences and cDNA Ubrary/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
  • FuU length polynucleotide sequences were also produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide 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 hahpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA Ubraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the parameters for primer pair T7 and SK+ were as foUows: Step 1 : 94 °C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C.
  • the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan 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 aUquot 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, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to reUgation 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 reUgated using T4 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coU ceUs. Transformed ceUs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384- weU plates in LB/2x carb Uquid media.
  • the ceUs were lysed, and DNA was ampUfied by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the foUowing parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4 C C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reampUfied using the same conditions as described above.
  • fuU length polynucleotide sequences are verified using the above procedure or are used to obtain 5' regulatory sequences using the above procedure along with oUgonucleotides designed for such extension, and an appropriate genomic Ubrary.
  • Hybridization probes derived from SEQ DD NO:37-72 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oUgonucleotides, consisting of about 20 base pairs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • OUgonucleotides are designed using state-of-the-art software such as OLIGO 4,06 software (National Biosciences) and labeled by combining 50 pmol of each oUgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
  • An aUquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl D, Eco Rl, Pst I, Xba I, or Pvu H (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & SchueU, Durham NH)- Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saUne sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared.
  • Unkage or synthesis of array elements upon a microarray can be achieved utiUzing photoUthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena (1999), supra).
  • Suggested substrates include siUcon, siUca, glass sUdes, glass chips, and siUcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and Unk elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines weU known to those of ordinary skfil in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • RNA is isolated from tissue samples using the guanidinium thiocyanate method and poIy(A) + RNA is purified using the oUgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-franscriptase, 0.05 pg/ ⁇ l oUgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with
  • 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 labeUng) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
  • reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.
  • the sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampUfied from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampUfied in thirty cycles of PCR from an initial quantity of 1 -2 ng to a final quantity greater than 5 ⁇ g.
  • AmpUfied array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified array elements are immobiUzed on polymer-coated glass sUdes. Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass sUdes are etched in 4% hydrofluoric acid (VWR).
  • Array elements are appUed to the coated glass substrate using a procedure described in US Patent No. 5,807,522, inco ⁇ orated herein by reference.
  • 1 ⁇ l of the array element DNA is loaded into the open CapiUary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per sUde.
  • Microarrays are UV-crossUnked using a STRATALINKER UV-crossUnker (Stratagene).
  • Microarrays are washed at room temperature once in 0.2% SDS and three times in distiUed water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saUne (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2%
  • PBS phosphate buffered saUne
  • Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aUquoted onto the microarray surface and covered with an 1.8 cm 2 coversUp.
  • the arrays are transferred to a wate ⁇ roof chamber having a cavity just sUghtly larger than a microscope sUde.
  • the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60° C.
  • the arrays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried.
  • Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an
  • Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral Unes at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Ught is focused on the array using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the sUde containing the array is placed on a computer-controUed X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiUne laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate, filters positioned between the array and the photomultipUer tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array is typicaUy scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typicaUy caUbrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the caUbration is done by labeUng samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the ou ⁇ ut of the photomultipUer tube is digitized using a 12-bit RTI-835H analog-to-digital (A D) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer.
  • a D analog-to-digital
  • the digitized data are displayed as an image where the signal intensity is mapped using a ' Unear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
  • Sequences complementary to the ECMCAD-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring ECMCAD.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaller or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of ECMCAD.
  • a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oUgonucleotide is designed to prevent ribosomal binding to the ECMCAD-encoding transcript.
  • XII Expression of ECMCAD Expression and purification of ECMCAD 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. Examples of such promoters include, but are not Umited 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 ECMCAD upon induction with isopropyl beta-D- thiogalactopyranoside (EPTG).
  • ECMCAD isopropyl beta-D- thiogalactopyranoside
  • Expression of ECMCAD in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU Unes with recombinant Autographica caUfornica nuclear polyhedrosis viras (AcMNPV), commonly known as baculoviras.
  • AcMNPV Autographica caUfornica nuclear polyhedrosis viras
  • the nonessential polyhedrin gene of baculoviras is replaced with cDNA encoding ECMCAD by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates.
  • ECMCAD is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified ECMCAD obtained by these methods can be used directly in the assays shown in Examples XVI and XVH where appUcable. XIII. Functional Assays
  • ECMCAD function is assessed by expressing the sequences encoding ECMCAD at physiologicaUy elevated levels in mammaUan ceU culture systems.
  • cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic ceU Une, using either Uposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward Ught scatter and 90 degree side Ught scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry. Oxford, New York NY.
  • ECMCAD The influence of ECMCAD on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding ECMCAD and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobuUn G (IgG).
  • Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding ECMCAD and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • ECMCAD substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • PAGE polyacrylamide gel electrophoresis
  • the ECMCAD amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions are weU described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-ECMCAD activity by, for example, binding the peptide or ECMCAD to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio- iodinated goat anti-rabbit IgG.
  • XV Purification of Naturally Occurring ECMCAD Using Specific Antibodies
  • ECMCAD Media containing ECMCAD are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of ECMCAD (e.g., high ionic strength buffers in the presence of detergent) .
  • the column is eluted under conditions that disrupt antibody/ECMCAD binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or fhiocyanate ion), and ECMCAD is coUected.
  • ECMCAD or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously arrayed in the weUs of a multi- weU plate are incubated with the labeled ECMCAD, washed, and any weUs with labeled ECMCAD complex are assayed. Data obtained using different concentrations of ECMCAD are used to calculate values for the number, affinity, and association of ECMCAD with the candidate molecules.
  • molecules interacting with ECMCAD are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • ECMCAD may also be used in the PATHCALLING process (CuraGen Co ⁇ ., New Haven ; CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • An assay for ECMCAD activity measures the expression of ECMCAD on the ceU surface.
  • cDNA encoding ECMCAD is transfected into a non-leukocytic ceU Une.
  • CeU surface proteins are labeled with biotin (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed using ECMCAD-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of ECMCAD expressed on the ceU surface.
  • an assay for ECMCAD activity measures the amount of ceU aggregation induced by overexpression of ECMCAD.
  • cultured ceUs such as NJTJ.3T3 are transfected with cDNA encoding ECMCAD contained within a suitable mammaUan 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 ceU agglutination, or clumping, associated with transfected ceUs is compared with that associated with untransfected ceUs.
  • the amount of ceU agglutination is a direct measure of ECMCAD activity.
  • an assay for ECMCAD activity measures the disruption of cytoskeletal filament networks upon overexpression of ECMCAD in cultured ceU Unes (Rezniczek, G. A. et al. (1998) J. CeU Biol. 141 :209-225).
  • cDNA encoding ECMCAD is subcloned into a mammaUan expression vector that drives high levels of cDNA expression.
  • This construct is transfected into cultured ceUs, such as rat kangaroo PtK2 or rat bladder carcinoma 804G ceUs.
  • Actin filaments and intermediate filaments such as keratin and vimentm are visuaUzed by immunofluorescence microscopy using antibodies and techniques weU known in the art.
  • the configuration and abundance of cyoskeletal filaments can be assessed and quantified using confocal imaging techniques.
  • the bundUng and coUapse of cytoskeletal filament networks is indicative of ECMCAD activity.
  • ceU adhesion activity in ECMCAD is measured in a 96-weU microtiter assay in which weUs are first coated with ECMCAD by adding solutions of ECMCAD of varying concentrations to the weUs. Excess ECMCAD is washed off with saUne, and the weUs incubated with a solution of 1 % bovine serum albumin to block non-specific ceU binding. AUquots of a ceU suspension of a suitable ceU type are then added to the micortiter weUs and incubated for a period of time at 37 °C.
  • Non-adhered ceUs are washed off with saUne and the ceUs stained with a suitable ceU stain such as Coomassie blue.
  • the intensity of staining is measured using a variable wavelength microtiter plate reader and compared to a standard curve to deter ⁇ ne the number of ceUs adhering to the ECMCAD coated plates.
  • the degree of ceU staining is proportional to the ceU adhesion activity of ECMCAD in the sample.
  • HMMER PFAM S184 S270 ⁇ 279 N468 N489 N65 P598-S687, P700-S790, P802-S891, S342 S348 S377 N765 N860 P903-S986 S397 S406 S436 N895 N913
  • Immunoglobulin domain HMMER PFAM S442 S449 S507 N931 N956 D43-A102, G137-V198, G242-A299, S512 S549 S558 C339-A388, G424-A481, G514-V579 S572 S588 S617 CONTACTIN CELL ADHESION NEUROFASCIN BLAST_PRODOM S67 S678 S690 GLYCOPROTEIN GP135 IMMUNOGLOBULIN S713 S772 S797 PD001890:
  • TNFR/NGFR motif C2051-C2090 MOTIFS
  • RGD motifs MOTIFS R1722-D1724, R1838-D1840
  • CONNNOTOl pINCY Library was constructed using RNA isolated from mesentery fat tissue obtained from a 71- year-old Caucasian male during a partial colectomy and permanent colostomy. Family history included atherosclerotic coronary artery disease, myocardial infarction, and extrinsic asthma.
  • CONNTUT0 pINCY Library was constructed using RNA isolated from tumorous spinal tissue removed from a 35-year-old Caucasian male during an exploratory laparotomy. Pathology indicated schwannoma with degenerative changes. Patient history included anxiety, depression, neurofibromatosis and benign neoplasm of the scrotum. Previously the patient had a spinal fusion. Family history included brain cancer, liver disease, and multiple sclerosis .
  • ENDCNOT03 pINCY Library was constructed using RNA isolated from dermal microvascular endothelial cells removed from a neonatal Caucasian male.
  • FTUBTUR01 PCDNA2.1 This random primed library was constructed using RNA isolated from fallopian tube tumor tissue removed from an 85-year-old Caucasian female during bilateral salpingo- oophorectomy and hysterectomy. Pathology indicated poorly differentiated mixed endometrioid (80%) and serous (20%) adenocarcinoma, which was confined to the mucosa without mural involvement. Endometrioid carcinoma in situ was also present. Pathology for the associated uterus tumor indicated focal endometrioid adenocarcinoma in situ and moderately differentiated invasive adenocarcinoma arising in an endometrial polyp. Metastatic endometrioid and serous adenocarcinoma was present at the cul-de-sac tumor. Patient history included medullary carcinoma of the thyroid and myocardial infarction.
  • GBLADIT03 pINCY Library was constructed using RNA isolated from diseased gallbladder tissue removed from a 53-year-old Caucasian female during cholecystectomy. Pathology indicated mild chronic cholecystitis and cholelithiasis with approximately 150 mixed stones ranging in size from 0.1 cm to 0.5 cm. The patient presented with abdominal pain and nausea and vomiting. Patient history included hyperlipidema and tobacco and alcohol abuse. Previous surgeries included adenotonsillectomy. Patient medications included Zantac, Provera, Premarin, and calcium. Family history included benign hypertension in the mother and the father.
  • HNT3AZT01 pINC Library was constructed using RNA isolated from the hNT2 cell line (derived from a human teratocarcinoma that exhibited properties characteristic of a committed neuronal precursor). Cells were treated for three days with 0.35 micromolar 5-aza-2'- deoxycytidine (AZ) .
  • AZ 5-aza-2'- deoxycytidine
  • ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
  • fastx score 100 or greater
  • HMM hidden Markov model
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res. 8: 186-194.
  • TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E.L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182.
  • HMM hidden Markov model

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Abstract

La présente invention concerne des molécules humaines de matrice extracellulaire et d'adhésion cellulaire ou 'ECMCAD' (ExtraCellular Matrix and Cell ADhesion) ainsi que des polynucléotides qui identifient et codent les ECMCAD. L'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. L'invention concerne enfin des procédés permettant de diagnostiquer, de traiter ou de prévenir des troubles associés à l'expression aberrante des ECMCAD.
PCT/US2001/021067 2000-06-30 2001-06-29 Molécules de matrice extracellulaire et d'adhésion cellulaire WO2002002634A2 (fr)

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CA002413186A CA2413186A1 (fr) 2000-06-30 2001-06-29 Molecules de matrice extracellulaire et d'adhesion cellulaire
AU2001273151A AU2001273151A1 (en) 2000-06-30 2001-06-29 Human extracellular matrix and cell adhesion polypeptides
EP01952392A EP1383892A2 (fr) 2000-06-30 2001-06-29 Proteines de matricielles extracellulaires et d'adhesions cellulaires humaines
JP2002507885A JP2004528003A (ja) 2000-06-30 2001-06-29 細胞外マトリクスおよび細胞接着分子

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