WO2002059312A2 - Cell adhesion proteins - Google Patents

Cell adhesion proteins Download PDF

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Publication number
WO2002059312A2
WO2002059312A2 PCT/US2001/049206 US0149206W WO02059312A2 WO 2002059312 A2 WO2002059312 A2 WO 2002059312A2 US 0149206 W US0149206 W US 0149206W WO 02059312 A2 WO02059312 A2 WO 02059312A2
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WIPO (PCT)
Prior art keywords
polynucleotide
polypeptide
seq
cadhp
amino acid
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PCT/US2001/049206
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English (en)
French (fr)
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WO2002059312A3 (en
Inventor
Brendan M. Duggan
Yuming Xu
Ernestine A. Lee
Sally Lee
Dyung Aina M. Lu
Bridget A. Warren
Henry Yue
Kimberly J. Gietzen
Cynthia D. Honchell
Neil Burford
Mariah R. Baughn
Tom Y. Tang
Jennifer L. Jackson
Ameena R. Gandhi
Deborah A. Kallick
Olga Bandman
Richard C. Graul
Narinder K. Chawla
Yan Lu
Jayalaxmi Ramkumar
Monique G. Yao
Preeti G. Lal
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Incyte Genomics, Inc.
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Application filed by Incyte Genomics, Inc. filed Critical Incyte Genomics, Inc.
Priority to JP2002559797A priority Critical patent/JP2004537972A/ja
Priority to US10/451,010 priority patent/US20040082761A1/en
Priority to EP01997091A priority patent/EP1385955A2/en
Priority to CA002431144A priority patent/CA2431144A1/en
Publication of WO2002059312A2 publication Critical patent/WO2002059312A2/en
Publication of WO2002059312A3 publication Critical patent/WO2002059312A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to nucleic acid and amino acid sequences of cell adhesion proteins and to the use of these sequences in the diagnosis, treatment, and prevention of immune system disorders, neurological disorders, developmental disorders, and cell proliferative disorders, including cancer, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of cell adhesion proteins.
  • the surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the extracellular matrix (ECM).
  • ECM extracellular matrix
  • the interaction of the cell with its surroundings profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. Families of cell adhesion molecules include the cadherins, integrins, lectins, neural cell adhesion proteins, and some members of the proline-rich proteins.
  • Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. These proteins share multiple repeats of a cadherin-specific motif, and the repeats form the folding units of the cadherin extracellular domain. Cadherin molecules cooperate to form focal contacts, or adhesion plaques, between adjacent epithelial cells.
  • the cadherin family includes the classical cadherins and protocadherins.
  • Classical cadherins include the E-cadherin, N-cadherin, and P-cadherin subfamilies. E-cadherin is present on many types of epithelial cells and is especially important for embryonic development.
  • N-cadherin is present on nerve, muscle, and lens cells and is also critical for embryonic development.
  • P-cadherin is present on cells of the placenta and epidermis. Recent studies report that protocadherins are involved in a variety of cell-cell interactions (Suzuki, S.T. (1996) J. Cell Sci. 109:2609-2611).
  • the intracellular anchorage of cadherins is regulated by their dynamic association with catenins, a family of cytoplasmic signal transduction proteins associated with the actin cytoskeleton.
  • cadherins The anchorage of cadherins to the actin cytoskeleton appears to be regulated by protein tyrosine phosphorylation, and the cadherins are the target of phosphorylation-induced junctional disassembly (Aberle, H. et al. (1996) J. CeU. Biochem. 61:514-523).
  • Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to the internal cytoskeleton. Integrins are composed of two noncovalently associated transmembrane glycoprotein subunits called ⁇ and ⁇ . 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. 2:D126-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. CeU 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. CeU. Biochem. 45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).
  • Lectins are further classified into subfamiUes based on carbohydrate-binding specificity and other criteria.
  • the galectin subfamily includes lectins that bind ⁇ -galactoside carbohydrate moieties in a thiol-dependent manner (reviewed inHadari, Y. R. et al. (1998) J. Biol. Chem. 270:3447-3453).
  • Galectins are widely expressed and developmentaUy regulated.
  • Galectins contain a characteristic carbohydrate recognition domain (CRD).
  • the CRD comprises about 140 amino acids and contains several stretches of about 1 - 10 amino acids which are highly conserved among all galectins.
  • a particular 6-amino acid motif within the CRD contains conserved tryptophan and arginine residues which are critical for carbohydrate binding.
  • the CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation. Secondary structure predictions indicate that the CRD forms several ⁇ -sheets.
  • Galectins play a number of roles in diseases and conditions associated with ceU-ceU and ceU- matrix interactions. For example, certain galectins associate with sites of inflammation and bind to ceU surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice. Moreover, anti-galectin antibodies inhibit processes associated with ceU transformation, such as ceU aggregation and anchorage-independent growth (see, for example, Su, Z.-Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93 J252-7257).
  • 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 ceUs in response to cytokine signaUng. Selectins bind to specific ligands on the leukocyte ceU 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.
  • NCAPs Neural ceU adhesion proteins
  • NCAP neuropeptide kinase kinase
  • NCAP neuropeptide kinase
  • axonal fasciculation axonal fasciculation
  • pathfmding synaptic target-recognition
  • synaptic formation 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,
  • NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et al, supra). They are generaUy linked to the plasma membrane through a transmembrane domain and/or a glycosyl-phosphatidylinositol (GPI) anchor. The GPI linkage can be cleaved by GPI phosphoUpase C. Most NCAPs consist of an extiaceUular region made up of one or more immunoglobuUn domains, a membrane spanning domain, and an intraceUular region. Many NCAPs contain post-translational modifications including covalently attached oligosaccharide, glucuronic acid, and sulfate.
  • GPI glycosyl-phosphatidylinositol
  • 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 ceU myelin protein (SMP), limbic system-associated membrane protein (LAMP), opiate-binding ceU-adhesion molecule (OB CAM), and myelin-associated glycoprotein (MAG).
  • SMP Schwann ceU myelin protein
  • LAMP limbic system-associated membrane protein
  • OB CAM opiate-binding ceU-adhesion molecule
  • MAG myelin-associated glycoprotein
  • the complex-type NCAPs contain fibronectin type HI domains in addition to the immunoglobuUn domains.
  • the complex-type subgroup includes neural ceU-adhesion molecule (NCAM), axonin-1, Fll, Bravo, and Ll.
  • NCAPs contain a combination of immunoglobulin domains and other motifs such as tyrosine kinase and epidermal growth factor-Uke 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 gUal growth factor ⁇ (GGFTI) and acetylcholine receptor-inducing factor (ARIA), and the semaphorin/coUapsin family such as semaphorin B and coUapsin.
  • NGF nerve growth factor
  • NT4 neurotropin 4
  • GGFTI gUal growth factor ⁇
  • ARIA acetylcholine receptor-inducing factor
  • semaphorin/coUapsin family such as semaphorin B and coUapsin.
  • 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. AU semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extraceUular region of neuropiUns consists of three different domains: CUB, discoidin, and MAM domains.
  • Plexins are neuronal ceU surface molecules that mediate ceU adhesion via a homophiMc binding mechanism in the presence of calcium ions. Plexins have been shown to be expressed in the receptors and neurons of particular sensory systems (Ohta, . et al. (1995) CeU 14:1189-1199).
  • Plexins which themselves contain complete semaphorin domains, may be both the ancestors of classical semaphorins and binding partners for semaphorins (Winberg, M.L. et al (1998) Cell 95:903-916).
  • NCAP subfamily includes ceU adhesion proteins expressed on distinct subpopulations of brain neurons.
  • Members of the NCAP-LON subgroup possess three immunoglobuUn domains and bind to ceU membranes through GPI anchors.
  • Kilon (a kindred of NCAP-LON), for example, is expressed in the brain cerebral cortex and hippocampus (Funatsu, N. et al. (1999) J. Biol. Chem. 274:8224-8230). Immunostaining locaUzes Kilonto the dendrites and soma of pyramidal neurons.
  • Kilon has three C2 type immunoglobulin-Uke domains, six predicted glycosylation sites, and a GPI anchor.
  • Kilon is developmentaUy 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 magnoceUular neurons secreting neuropeptides, oxytocin or arginine vasopressin (Miyata, S. et al. (2000) J. Comp. Neurol. 424:74-85). Arginine vasopressin regulates body fluid homeostasis, extraceUular osmolality and intravascular volume. Oxytocin induces contractions of uterine smooth muscle during chUd birth and of myoepitheUal ceUs in mammary glands during lactation. In magnoceUular neurons, Kilon is proposed to play roles in the reorganization of dendritic connections during neuropeptide secretion.
  • CeU adhesion proteins also include some members of the proUne-rich proteins (PRPs).
  • PRPs are defined by a high frequency of proUne, ranging from 20-50% of the total amino acid content. Some PRPs have short domains which are rich in proUne. These proUne-rich regions are associated with protein-protein interactions.
  • PRPs proline-rich synapse-associated proteins
  • PSD postsynaptic density
  • ProSAPs Members of the ProSAP family contain six to seven ankyrin repeats at the N-terminus, foUowed by an SH3 domain, a PDZ domain, and seven proUne-rich regions and a SAM domain at the C terminus.
  • Several groups of ProSAPs are important structural constituents of synaptic structures in human brain (Zitzer et al., supra).
  • Another member of the PRP family is the HLA-B -associated transcript 2 protein (B AT2) which is rich in proUne and includes short tracts of polyproMne, polyglycine, and charged amino acids.
  • BAT2 also contains four RGD (Arg-Gly-Asp) motifs typical of integrins (Banerji, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87:2374-2378).
  • Toposome is a ceU-adhesion glycoprotein isolated from mesenchyme-blastula embryos. Toposome precursors including viteUogenin promote ceU adhesion of dissociated blastula ceUs. There are additional specific domains characteristic of cell adhesion proteins. One such domain is the MAM domain, a domain of about 170 amino acids found in the extraceUular region of diverse proteins. These proteins aU share a receptor-Uke architecture comprising a signal peptide, foUowed by a large N-terminal extraceUular domain, a transmembrane region, and an intraceUular domain (PROSITE document PDOC00604 MAM domain signature and profile).
  • MAM domain proteins include zonadhesin, a sperm-specific membrane protein that binds to the zona peUucida of the egg; neuropiUn, a cell adhesion molecule that functions during the formation of certain neuronal circuits, and Xenopus laevis thyroid hormone induced protein B, which contains four MAM domains and is involved in metamorphosis (Brown, D.D. et al. (1996) Proc. Natl. Acad. Sci. USA 93:1924- 1929).
  • the WSC domain was originaUy found in the yeast WSC (ceU-waU integrity and stress response component) proteins which act as sensors of environmental stress.
  • the WSC domains are extraceUular and are thought to possess a carbohydrate binding role (Ponting, C.P. et al. (1999) Curr. Biol. 9:S1-S2).
  • a WSC domain has recently been identified in polycystin-1, a human plasma membrane protein. Mutations in polycystin-1 are the cause of the commonest form of autosomal dominant polycystic kidney disease (Ponting, C.P. et al. (1999) Curr. Biol. 9:R585-R588).
  • LRR Leucine rich repeats
  • LRR motifs are short motifs found in numerous proteins from a wide range of species. LRR motifs are of variable length, most commonly 20-29 amino acids, and multiple repeats are typicaUy present in tandem. LRR motifs are important for protein/protein interactions and ceU adhesion, and LRR proteins are involved in cell/ceU interactions, moiphogenesis, and development (Kobe, B. and Deisenhofer, J. (1995) Curr. Opin. Struct. Biol. 5:409-416).
  • the human ISLR (immunoglobuUn superfamily containing leucine-rich repeat) protein contains a C2-type immunoglobuUn domain as weU as LRR motifs.
  • the ISLR gene is Unked 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).
  • SAM sterile alpha motif
  • the SAM domain can potentiaUy function as a protein interaction module through its abiUtyto form homo- or hetero-oUgomers with other SAM domains (Schultz, J. et al. (1997) Protein Sci. 6:249-253).
  • ceU adhesion proteins and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of immune system disorders, neurological disorders, developmental disorders, and ceU 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 ceU adhesion proteins.
  • the invention features purified polypeptides, ceU adhesion proteins, referred to coUectively as "CADHP” and individuaUy as “CADHP-1,” “CADHP-2,” “CADHP-3,” “CADHP-4,” “CADHP- 5,” “CADHP-6,” “CADHP-7,” “CADHP-8,” “CADHP-9,” and “CADHP-10.”
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected fromu e group consisting of SEQ ID NO: 1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1 - 10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting
  • 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:1-10, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-10.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:l 1-20.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably U ked 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 ED NO:l -10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected
  • 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: 1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO : 1 - 10.
  • 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 Unked 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 ED NO: 1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 1 - 10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ EO NO:1-10.
  • 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:l 1-20, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:11-20, 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 ED NO:l 1-20, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO: 11-20, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, 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 ED NO:l 1-20, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l 1-20, 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) ampUfying said target polynucleotide or fragment thereof using polymerase chain reaction ampUfication, 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: 1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO : 1 - 10, and a pharmaceuticaUy acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-10.
  • the invention additionaUy provides a method of treating a disease or condition associated with decreased expression of functional CADHP, 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 ED NO:1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:1-10.
  • 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 CADHP, 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: 1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO : 1 - 10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:1-10.
  • 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 CADHP, 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 ED NO:1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:1-10, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:1-10.
  • 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: 1-10, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-10, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-10.
  • 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 polynucleotide sequence selected from the group consisting of SEQ BD NO : 11 -20, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • 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:l 1-20, U) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO :11-20, Ui) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO: 11-20, n) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:l 1 -20, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide 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 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probabiUty scores for the matches between each polypeptide and its homolog(s) are 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 Ubrary for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA Ubraries 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.
  • a reference to "a host ceU” includes a pluraUty of such host ceUs
  • a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
  • aU technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skiU in the art to which this invention belongs.
  • CADHP refers to the amino acid sequences of substantiaUy purified CADHP 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 CADHP.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of CADHP either by directly interacting with CADHP or by acting on components of the biological pathway in which CADHP participates.
  • aUeUc variant is an alternative form of the gene encoding CADHP.
  • AUeUc variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
  • a gene may have none, one, or many aUeUc variants of its naturaUy occurring form.
  • Common mutational changes which give rise to aUeUc variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding CADHP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as CADHP or a polypeptide with at least one functional characteristic of CADHP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oUgonucleotide probe of the polynucleotide encoding CADHP, and improper or unexpected hybridization to aUeUc variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding CADHP.
  • 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 CADHP.
  • DeUberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophiUcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of CADHP 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. Where “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.
  • “AmpUfication” 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.
  • the term "antagonist” refers to a molecule which inhibits or attenuates the biological activity of CADHP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of CADHP either by directly interacting with CADHP or by acting on components of the biological pathway in which CADHP 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 CADHP polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oUgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eUcit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oUgonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Ubraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-Uke molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or '-NHj), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specificaUy cross-Unked to their cognate Ugands, e.g., by photo-activation of a cross-Unker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • RNA aptamer refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (BUnd, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-Uke molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone Unkages such as phosphorothioates, melhylphosphonates, 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. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capabiMty of the natural, recombinant, or synthetic CADHP, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5 -AGT-3' pairs with its complement, 3'-TCA-5'.
  • 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 CADHP or fragments of CADHP 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 uncaUed bases, extended using the XL-PCR kit (AppUed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha heUcal conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • the term “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.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
  • a "fragment” is a unique portion of CADHP or the polynucleotide encoding CADHP 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 pmposes 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 : 11 -20 comprises a region of unique polynucleotide sequence that specificaUy identifies SEQ ED NO: 11-20, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ED NO: 11-20 is useful, for example, in hybridization and ampUfication technologies and in analogous methods that distinguish SEQ ED NO :11-20 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ED NO : 11 -20 and the region of SEQ ED NO : 11 -20 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ED NO:1-10 is encoded by a fragment of SEQ ED NO:l 1-20.
  • a fragment of SEQ ED NO:1-10 comprises a region of unique amino acid sequence that specificaUy identifies SEQ ED NO:1-10.
  • a fragment of SEQ ED NO:1-10 is useful as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ED NO:1-10.
  • the precise length of a fragment of SEQ ED NO:1-10 and the region of SEQ ED NO:1-10 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a “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 "full length” polypeptide sequence.
  • Homology refers to sequence sirrdlarity 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.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AUgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to aUgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21 -2000) set at default parameters. Such default parameters may be, for example:
  • 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 andjiydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are 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 skiUin 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
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skiU 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 CADHP 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 CADHP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • miciOarray refers to an arrangement of a pluraUty of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • array element refers 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 CADHP.
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of CADHP.
  • 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 CADHP may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biocherriicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic miUeu of CADHP.
  • Probe refers to nucleic acid sequences encoding CADHP, 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 purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • OUgonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oUgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. 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 MFT 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 aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of 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.
  • 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 CADHP, nucleic acids encoding CADHP, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition.
  • the interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will 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 capiUaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular 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 ceUs 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 fransgenic techniques weU known in the art.
  • the nucleic acid is introduced into the ceU, 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 virus.
  • 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 “polymoiphic” variant.
  • a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing 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. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 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 ceU adhesion proteins (CADHP), the polynucleotides encoding CADHP, and the use of these compositions for the diagnosis, treatment, or prevention of immune system disorders, neurological disorders, developmental disorders, and ceU proUferative disorders, including cancer.
  • CADHP ceU adhesion proteins
  • 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 ED), Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ED NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ED) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ED) as shown.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO;) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog.
  • Column 4 shows the probabiUty scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank homologs 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 2 show the polypeptide sequence identification number (SEQ ED NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (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 ED NO:l is 46% identical, over 404 amino acid residues (I119-K522), to the murine, lectin C-type, Kupffer (hepatic sinusoid) ceU receptor (GenBank ED gl 669360, amino acid residues I106-K495), as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 2.1e-90.
  • SEQ ED NO:l is also 45% identical, over 414 amino acid residues (G109- K522), to the rat Kupffer ceU receptor (GenBank ED g205051, amino acid residues G86-R495), as determined by BLAST analysis.
  • the BLAST probabiUty score is 7.2e-88.
  • SEQ ID NO: 1 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.)
  • HMM hidden Markov model
  • SEQ ED NO:2 is 61% identical, over 565 amino acid residues (A8- H569), to murine semaphorin Via (GenBank ID g2623162, amino acid residues A5-H569), as determined by the Basic Local AUgnment Search Tool (BLAST).
  • the BLAST probabiUty score is 6.6e-216.
  • SEQ ED NO:2 also contains a semaphorin 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 ED NO:3 is 44% identical, over 170 amino acid residues (L280-C449), to human integrin-binding protein Del-1 (GenBank ID g2865219, amino acid residues L307-C476), as determined by BLAST analysis.
  • the BLAST probabiUty score is 7.4e-32.
  • SEQ ED NO:3 also contains a CUB domain (characteristic of developmentaUy-regulated proteins) and a f5/8 type C domain (characteristic of secreted proteins such as coagulation factors V and VEH, lactadherin, neuropiUn-1, hemocytin, spondin, and discoidin I) 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 ED NO:4 is 34% identical (over 329 amino acid residues) to human fibuUn 1, isoform C (GenBank ED g31419), as determined by the Basic LxDcal AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 6.2e-41, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ID NO:4 is also 32% identical to human fibulin 2 (over 304 amino acid residues, GenBank ID g575233) and chicken fibuUn 1, isoform C (over 329 amino acid residues, GenBank ED g2947316), with BLAST probabiUty scores of 1.6e-36 and 1.4e-38, respectively.
  • SEQ ID NO:4 also contains EGF-Uke domains, characteristic of fibuUn polypeptides, 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 ED NO:4 comprises EGF-Uke domains, consistent with the fibuUn family of polypeptides.
  • SEQ ED NO:5 is 72% identical (over 119 amino acid residues) to murine samaphorin (semaphorin) G (GenBank ED gl418942), as determined by BLAST analysis, with a probabiUty score of 6.3e-39.
  • SEQ ED NO:5 also contains a semaphorin 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 ED NO: 6 is 40% identical (over 597 amino acid residues) to murine MEG6, a high-molecular- weight protein with multiple EGF-Uke motifs (GenBank ID g3449294), as determined by BLAST analysis, with a probabiUty score of 3.2e-149.
  • SEQ ID NO:6 also contains EGF-Uke 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.)
  • HMM hidden Markov model
  • Data from BLEVIPS and MOTIFS analyses provide further corroborative evidence that SEQ ED NO:6 comprises EGF-Uke and sushi domains, characteristic of members of the protein class.
  • SEQ ID NO:8 is 98% identical over 1141 amino acid residues to human cadherin-23 (GenBank ED gl 1321508) as determined by the Basic Local Alignment 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 ED NO: 8 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.
  • HMM hidden Markov model
  • SEQ ID NO:8 is a cadherin.
  • SEQ ID NO:7, SEQ ID NO:9, and SEQ ED NO:10 were analyzed and annotated in a similar manner.
  • the algorithms and parameters for the analysis of SEQ BD NO:1-10 are described in Table 7.
  • 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.
  • Column 1 Usts the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ED) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU lengthpolynucleoti.de sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or ampUfication technologies that identify SEQ ED NO:11-20 or that distinguish between SEQ ED NO:l 1-20 and related polynucleotide sequences.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Ubraries or from pooled cDNA Ubraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotide sequences.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i. e. , those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as FL_ XXXXXX_N 1 _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was appUed, and YYYYY is the number of the prediction generated by the algorithm, and N 123 , if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • FLXXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was appUed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and iV referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Ubraries for those 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 CADHP variants.
  • a preferred CADHP 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 CADHP amino acid sequence, and which contains at least one functional or structural characteristic of CADHP.
  • the invention also encompasses polynucleotides which encode CADHP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO: 11 -20, which encodes CADHP.
  • the polynucleotide sequences of SEQ ID NO:l 1-20, 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 CADHP.
  • 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 CADHP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:l 1 - 20 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:l 1 -20.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CADHP.
  • a polynucleotide variant of the invention is a spUce variant of a polynucleotide sequence encoding CADHP.
  • a spUce variant may have portions which have significant sequence identity to the polynucleotide sequence encoding CADHP, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing of exons during mRNA processing.
  • a spUce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding CADHP over its entire length; however, portions of the spUce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide sequence encoding CADHP. Any one of the spUce variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of CADHP.
  • nucleotide sequences which encode CADHP and its variants are generaUy capable of hybridizing to the nucleotide sequence of the naturaUy occurring CADHP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding CADHP 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 utiUzed 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 CADHP and
  • CADHP derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents weU known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding CADHP or any fragment thereof. Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO: 11-20 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R.
  • 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 CADHP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector.
  • Another method, 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 ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR ampUfication 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 commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • 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 fuU-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.
  • capiUary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer continued.
  • CapiUary electrophoresis is especiaUy preferable for sequencing small DNA fragments which may be present in Umited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode CADHP may be cloned in recombinant DNA molecules that direct expression of CADHP, 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 CADHP.
  • nucleotide sequences of the present invention can be engineered using methods generaUy known in the art in order to alter CADHP-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 No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of CADHP, 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 No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F
  • 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 occurring genes in a directed and controUable manner.
  • sequences encoding CADHP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, 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.)
  • CADHP itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or soUd-phase techniques.
  • solution-phase or soUd-phase techniques See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.
  • Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (AppUed Biosystems).
  • AdditionaUy the amino acid sequence of CADHP, 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 may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences encoding CADHP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding CADHP.
  • Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding CADHP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • a variety of expression vector/host systems may be utiUzed to contain and express sequences encoding CADHP. These include, but are not Umited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect ceU systems infected with viral expression vectors (e.g., baculovirus)
  • plant ceU systems transformed with viral expression vectors e
  • 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.
  • the invention is not Umited by the host cell employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding CADHP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding CADHP can be achieved using a multifunctional E.
  • coU vector such as PBLUESCREPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding CADHP into the vector's multiple cloning site disrupts the lacL gene, aUowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • 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. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
  • vectors which direct high level expression of CADHP 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 CADHP.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of CADHP. Transcription of sequences encoding CADHP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMVused 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.
  • sequences encoding CADHP may be Ugated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses CADHP 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 EB V- 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 constiTicted and deUvered via conventional deUvery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic pu ⁇ oses. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355.)
  • sequences encoding CADHP 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.
  • ceUs 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.
  • 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) Cell 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 chlorsuUuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular 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 CADHP is inserted within a marker gene sequence
  • transformed ceUs containing sequences encoding CADHP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding CADHP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host ceUs that contain the nucleic acid sequence encoding CADHP and that express CADHP may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not Umited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of CADHP using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-Unked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding CADHP include oUgolabeUng, nick translation, end-labeUng, or PCR ampUfication using a labeled nucleotide.
  • the sequences encoding CADHP, 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 CADHP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode CADHP may be designed to contain signal sequences which direct secretion of CADHP through a prokaryotic or eukaryotic ceU 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.
  • 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.
  • CHO, HeLa, MDCK, HEK293, and WI38 Different host cells which have specific ceUular machinery and characteristic mechanisms for post-translational activities
  • ATCC American Type Culture CoUection
  • HEK293, and WI38 Different host cells which have specific ceUular machinery and characteristic mechanisms for post-translational activities
  • ATCC American Type Culture CoUection
  • natural, modified, or recombinant nucleic acid sequences encoding CADHP may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric CADHP protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of CADHP 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 CADHP encoding sequence and the heterologous protein sequence, so that CADHP may be cleaved away from the heterologous moiety foUowing purification.
  • CADHP CADHP
  • TNT rabbit reticulocyte lysate or wheat germ extract system Promega
  • T7, T3, or SP6 promoters T7, T3, or SP6 promoters.
  • Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • CADHP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to CADHP. At least one and up to a pluraUty of test compounds may be screened for specific binding to CADHP.
  • test compounds include antibodies, oUgonucleotides, proteins (e.g., receptors), or smaU molecules.
  • the compound thus identified is closely related to the natural Ugand of CADHP, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • CADHP natural Ugand of CADHP
  • the compound can be closely related to the natural receptor to which CADHP 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 CADHP, either as a secreted protein or on the ceU membrane.
  • Preferred ceUs include ceUs from mammals, yeast, Drosophila, or E. coU.
  • CeUs expressing CADHP or ceU membrane fractions which contain CADHP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either CADHP 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 CADHP, either in solution or affixed to a soUd support, and detecting the binding of CADHP 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 cell-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • CADHP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of CADHP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for CADHP activity, wherein CADHP is combined with at least one test compound, and the activity of CADHP in the presence of a test compound is compared with the activity of CADHP in the absence of the test compound. A change in the activity of CADHP in the presence of the test compound is indicative of a compound that modulates the activity of CADHP.
  • a test compound is combined with an in vitro or ceU-free system comprising CADHP under conditions suitable for CADHP activity, and the assay is performed.
  • a test compound which modulates the activity of CADHP 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 CADHP or their mammaUan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs. Such techniques are weU known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No.
  • mouse ES ceUs such as the mouse 129/SvJ ceU line
  • the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D.
  • 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 CADHP 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 cell Uneages including endoderm, mesoderm, and ectodermal ceU types. These cell Uneages differentiate into, for example, neural cells, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding CADHP 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 CADHP 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.
  • CADHP CADHP
  • a mammal inbred to overexpress CADHP e.g., by secreting CADHP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • CADHP Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of CADHP and ceU adhesion proteins.
  • examples of tissues expressing CADHP can be found in Table 6. Therefore, CADHP appears to play a role in immune system disorders, neurological disorders, developmental disorders, and ceU proUferative disorders, including cancer.
  • CADHP appears to play a role in immune system disorders, neurological disorders, developmental disorders, and ceU proUferative disorders, including cancer.
  • CADHP 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 CADHP.
  • disorders include, but are not Umited to, an immune system disorder, such as acquired immunodeficiency syndrome (ADDS), X-Unked agammaglobinemia of Bruton, common variable immunodeficiency (CVF), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCED), immunodeficiency with tiirombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, am
  • ADDS acquired immuno
  • a vector capable of expressing CADHP 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 CADHP including, but not Umited to, those described above.
  • compositions comprising a substantially purified CADHP 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 CADHP including, but not Umited to, those provided above.
  • an agonist which modulates the activity of CADHP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of CADHP including, but not Umited to, those Usted above.
  • an antagonist of CADHP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CADHP.
  • disorders include, but are not Umited to, those immune system disorders, neurological disorders, developmental disorders, and ceU proUferative disorders, including cancer, described above.
  • an antibody which specificaUy binds CADHP 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 CADHP.
  • a vector expressing the complement of the polynucleotide encoding CADHP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of CADHP 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 freatment 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 CADHP may be produced using methods which are generaUy known in the art.
  • purified CADHP may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specifically bind CADHP.
  • Antibodies to CADHP 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 CADHP 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 dinifrophenol.
  • BCG BaciUi Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • the oUgopeptides, peptides, or fragments used to induce antibodies to CADHP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy will 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 CADHP 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 CADHP may be prepared using any technique which provides for the production of antibody molecules by continuous cell Unes in culture. These include, but are not Umited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EB V-hybridoma technique.
  • the hybridoma technique the human B-ceU hybridoma technique
  • the EB V-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, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120.
  • chimeric antibodies such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • 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 CADHP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries.
  • 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.
  • Antibody fragments which contain specific binding sites for CADHP 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 constructed 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.
  • K association constant
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the CADHP- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with KL ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of CADHP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach. IRL Press, Washington DC; LiddeU, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies. John Wiley & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quaUty and suitabiUty of such preparations for certain downstieam 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 CADHP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, 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 CADHP may be used for therapeutic pmposes.
  • 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 CADHP.
  • 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 confrol regions of sequences encoding CADHP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press Inc., Totawa NJ.)
  • Antisense sequences can be deUvered intracellularly 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 CUn. 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 refrovirus and adeno-associated virus vectors.
  • viral vectors such as refrovirus and adeno-associated virus vectors.
  • Other gene deUvery mechanisms include Uposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • Rossi J.J. (1995) Br. Med. BuU. 51(l):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.
  • polynucleotides encoding CADHP 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 (SCED)-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.
  • SCED severe combined immunodeficiency
  • ADA adenosine deaminase
  • diseases or disorders caused by deficiencies in CADHP are treated by constructing mammaUan expression vectors encoding CADHP and introducing these vectors by mechanical means into CADHP-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (n) baUistic gold particle deUvery, (iti) 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 CADHP include, but are not
  • CADHP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 vims, 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 virus (RSV), SV40 vims, thymidine kinase (TK), or ⁇ -actin genes
  • 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 CADHP expression are treated by constructing a refrovirus vector consisting of (i) the polynucleotide encoding CADHP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (n) appropriate RNA packaging signals, and (Ui) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy available (Stratagene) and are based on pubUshed data (Riviere, I. et al. (1995) Proc. Natl. Acad.
  • the vector is propagated in an appropriate vector producing ceU 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 ceU Une
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been well documented (Ranga, U. et al.
  • an adenovirus-based gene therapy deUvery system is used to deUver polynucleotides encoding CADHP to ceUs which have one or more genetic abnormaUties with respect to the expression of CADHP.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art.
  • RepUcation defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al, (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No.
  • Addenovirus 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, EM. 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 CADHP to target ceUs which have one or more genetic abnormaUties with respect to the expression of CADHP.
  • the use of he ⁇ es simplex virus (HSV)-based vectors may be especiaUy valuable for introducing CADHP to ceUs of the central nervous system, for which HS V has a fropism.
  • the construction and packaging of he ⁇ es-based vectors are weU known to those with ordinary sMU in the art.
  • HSV he ⁇ es simplex viras
  • 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the confrol of the appropriate promoter for memeposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby inco ⁇ orated by reference.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding CADHP to target ceUs.
  • SFV SemUki Forest Virus
  • SFV SemUki Forest Virus
  • 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 CADHP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of CADHP-coding RNAs and the synthesis of high levels of CADHP 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 (SESf) 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 alphaviras cDNA and RNA transfections, and performing alphaviras 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 CADHP.
  • 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 foUowing 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 CADHP. 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 intracellular 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' 0-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 CADHP.
  • 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 specifically inhibits expression of the polynucleotide encoding CADHP may be therapeuticaUy useful, and in die treatment of disorders associated with decreased CADHP expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding CADHP may be therapeuticaUy useful.
  • test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary 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 CADHP 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 CADHP 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 CADHP.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; 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. Commun.
  • 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). Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
  • 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 fransfection, 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.)
  • any of the therapeutic methods described above may be appUed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA).
  • Such compositions may consist of CADHP, antibodies to CADHP, and mimetics, agonists, antagonists, or inhibitors of CADHP.
  • compositions utilized in this invention may be administered by any number of routes including, but not Umited to, oral, intravenous, intramuscular, infra-arterial, intramedullary, infrathecal, 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
  • Pulmonary deUvery has the advantage of administration without needle injection, and obviates the need for potentiaUy toxic penetration enhancers.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pmpose.
  • the determination of an effective dose is weU within the capabiUty of those skiUed in the art.
  • SpeciaUzed forms of compositions may be prepared for direct intraceUular deUvery of macromolecules comprising CADHP or fragments thereof.
  • Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule.
  • CADHP or a fragment thereof may be joined to a short cationic N- termrnal 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.
  • ceU culture assays e.g., of neoplastic ceUs
  • animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example CADHP or fragments thereof, antibodies of CADHP, and agonists, antagonists or inhibitors of CADHP, which ameUorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the
  • 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.
  • antibodies which specificaUy bind CADHP may be used for the diagnosis of disorders characterized by expression of CADHP, or in assays to monitor patients being treated with CADHP or agonists, antagonists, or inhibitors of CADHP.
  • Antibodies useful for diagnostic pmposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for CADHP include methods which utilize the antibody and a label to detect CADHP in human body fluids or in extracts of ceUs or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • CADHP CADHP-specific kinase
  • ELISAs ELISAs
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • CADHP expression normal or standard values for CADHP expression are estabUshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to CADHP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of CADHP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • the polynucleotides encoding CADHP 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 CADHP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of CADHP, and to monitor regulation of CADHP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding CADHP or closely related molecules may be used to identify nucleic acid sequences which encode CADHP.
  • 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 will determine whether the probe identifies only naturaUy occurring sequences encoding CADHP, aUeUc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the CADHP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 11-20 or from genomic sequences including promoters, enhancers, and introns of the CADHP gene.
  • Means for producing specific hybridization probes for DNAs encoding CADHP include the cloning of polynucleotide sequences encoding CADHP or CADHP 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 32 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 CADHP may be used for the diagnosis of disorders associated with expression of CADHP.
  • an immune system disorder such as acquired immunodeficiency syndrome (ADDS), X-Unked agammaglobinemia of Braton, common variable immunodeficiency (CN1), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCED), immunodeficiency withthrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ec
  • ADDS acquired immunodeficiency syndrome
  • erythroblastosis fetaUs, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophiUa, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sj ⁇ gren' s syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic pu ⁇ ura, ulcerative coUtis, uveitis, Werner syndrome, compUcations of cancer, hemodialysis, and extraco ⁇ oreal circulation, viral, bacterial, fun
  • the polynucleotide sequences encoding CADHP 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 utilizing fluids or tissues from patients to detect altered CADHP expression.
  • quaUtative or quantitative methods are weU known in the art.
  • the nucleotide sequences encoding CADHP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding CADHP 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 CADHP 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 may be accompUshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding CADHP, under conditions suitable for hybridization or ampUfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual 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
  • CADHP may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymatically, or produced in vitro. OUgomers wiU preferably contain a fragment of a polynucleotide encoding CADHP, or a fragment of a polynucleotide complementary to the polynucleotide encoding CADHP, 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 CADHP 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 CADHP 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 structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oUgonucleotide primers are fluorescently labeled, which aUows detection of the ampUmers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in siUco SNP (isSNP), are capable of identifying polymo ⁇ hisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • Methods which may also be used to quantify the expression of CADHP include radiolabeUng or biotinylating nucleotides, coampUfication of a control nucleic acid, and inte ⁇ olating results from standard curves.
  • radiolabeUng or biotinylating nucleotides 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 crOarray 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.
  • CADHP CADHP
  • fragments of CADHP or antibodies specific for CADHP may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein-protein interactions, drag-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. Patent No.
  • a franscript 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 franscripts 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 natm-aUy-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. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000)
  • 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 freatment.
  • 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 CADHP to quantify the levels of CADHP 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 . and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • 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 freated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified.
  • the amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • 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. (1 95) PCT appUcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and HeUer, M.J.
  • nucleic acid sequences encoding CADHP may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
  • Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constractions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA Ubraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • BACs bacterial chromosome cDNA Ubraries.
  • 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
  • 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 localized 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. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to franslocation, inversion, etc., among normal, carrier, or affected individuals.
  • CADHP its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between CADHP and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different smaU test compounds are synthesized on a soUd substrate. The test compounds are reacted with CADHP, or fragments thereof, and washed. Bound CADHP is then detected by methods weU known in the art. Purified CADHP can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neufraUzing antibodies can be used to capture the peptide and i mobiUze it on a soUd support.
  • nucleotide sequences which encode CADHP 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 LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 3. 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 cenfrifuged 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.
  • RNA was treated with DNase.
  • 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).
  • RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
  • RNA was provided with RNA and constructed the corresponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the
  • UNEZAP 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.
  • the cDNA was size-selected (300- 1000 bp) using SEPHACRYL SI 000, 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., PBLUESCREPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invittogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pENCY (Incyte Genomics), or derivatives thereof.
  • 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 cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell 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
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput insfrumentation 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
  • 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 MEGAB ACE 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 7J). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VTfl.
  • 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; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); 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, and BLOCKS, PRINTS, DOMO, PRODOM
  • PROTEOME databases with sequences from Homo sapiens, Rattus norveg
  • HMM is a probabiUstic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
  • the queries 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 fuU 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 fuU length translated polypeptide.
  • FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, 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. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb.
  • the encoded polypeptides were analyzed by querying against PFAM models for ceU adhesion proteins. Potential ceU adhesion proteins were also identified by homology to Incyte cDNA sequences that had been annotated as ceU adhesion proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases.
  • 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.
  • 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 in. 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 TV. Partial cDNAs assembled as described in Example HI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a fuU 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.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • 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 D ⁇ A sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
  • sequences which were used to assemble SEQ ED ⁇ O:l 1-20 were compared with sequences from the Incyte L FESEQ database and pubUc domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ED NO: 11-20 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. Inclusion of a mapped sequence in a cluster resulted in the assignment of aU sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • 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
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the pubUc, such as the NCBI "GeneMap'99" World Wide Web site
  • 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 pioduced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding CADHP 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 HE). 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 CADHP.
  • cDNA sequences and cDNA Ubrary/tissue information are found in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII.
  • 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 hai ⁇ in structures and primer-primer dimerizations was avoided.
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the foUowing parameters for primer pair PCI A and PCI B: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68 °C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C.
  • the parameters for primer pair T7 and SK+ 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 well of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • Step 1 94°C, 3 min
  • Step 2 94°C, 15 sec
  • Step 3 60°C, 1 min
  • Step 4 72°C, 2 min
  • Step 5 steps 2, 3, and 4 repeated 29 times
  • Step 6 72°C, 5 min
  • Step 7 storage at 4°C.
  • DNA 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 ED NO:11-20 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeUng 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 following endonucleases: Ase I, Bgl fi, Eco RI, Pst I, Xba I, or Pvu fl (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. X. Microarrays
  • the 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 skiU in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
  • microarray preparation and usage is described in detail below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oUgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-teanscriptase, 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, Lnc.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampUfied from bacterial cells 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 are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distiUed water washes between and after treatments.
  • Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol.
  • Coated sUdes are cured in a 110°C oven.
  • Array elements are appUed to the coated glass substrate using a procedure described in U.S.
  • 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 STRATALESfKER 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% SDS and distiUed water as before.
  • PBS phosphate buffered saUne
  • 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 fransferred 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 comer of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 horns 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.
  • 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., Melville 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 multiline 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, allowing 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 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 CADHP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring CADHP.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of CADHP.
  • 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 CADHP-encoding transcript.
  • CADHP CADHP expression and purification of CADHP is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not 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 CADHP upon induction with isopropyl beta-D- thiogalactopyranoside (EPTG).
  • CADHP in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU Unes with recombinant Autographica caUfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica caUfornica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding CADHP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
  • CADHP is synthesized as a fusion protein with, e.g., glutathione
  • GST S-fransferase
  • FLAG a peptide epitope tag
  • GST a 26-kilodalton enzyme from Schistosoma iaponicum
  • the GST moiety can be proteolyticaUy cleaved from CADHP at specificaUy engineered sites.
  • 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 CADHP obtained by these methods can be used directly in the assays shown in Examples XVI and XVII, where appUcable. XIII. Functional Assays
  • CADHP function is assessed by expressing the sequences encoding CADHP 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 (Invittogen, Carlsbad CA), both of which contain the cytomegaloviras promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic cell 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 nonfransfected 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.
  • Row cytometry (FCM) an automated, laser optics-based technique, is used to identify transfected ceUs expressing GFP or CD64- GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward 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.
  • CADHP The influence of CADHP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding CADHP 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 CADHP and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • the CADHP 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-CADHP activity by, for example, binding the peptide or CADHP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • CADHP is substantiaUy purified by immunoaffinity chromatography using antibodies specific for CADHP.
  • An immunoaffinity column is constracted by covalently coupUng anti-CADHP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupUng, the resin is blocked and washed according to the manufacturer's instructions.
  • CADHP Media containing CADHP are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of CADHP (e.g., high ionic sfrength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/CADHP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and CADHP is collected.
  • Candidate molecules previously arrayed in the weUs of a multi-weU plate are incubated with the labeled CADHP, washed, and any weUs with labeled CADHP complex are assayed. Data obtained using different, concentrations of CADHP are used to calculate values for the number, affinity, and association of CADHP with the candidate molecules.
  • CADHP molecules interacting with CADHP are analyzed using the yeast two-hybrid system as described in Fields, S. and 0. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • CADHP may also be used in the PATHCALLING process (CuraGen Coip., 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 CADHP activity measures the expression of CADHP on the ceU surface.
  • cDNA encoding CADHP 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 CADHP-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 CADHP expressed on the ceU surface.
  • an assay for CADHP activity measures the amount of cell aggregation induced by overexpression of CADHP.
  • cultured cells such as NEH3T3 are transfected with cDNA encoding CADHP contained within a suitable mammaUan expression vector under control of a strong promoter.
  • Cofransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH) is useful for identifying stable transfectants.
  • the amount of cell agglutination, or clumping, associated with transfected ceUs is compared with that associated with untransfected cells.
  • the amount of cell agglutination is a direct measure of CADHP activity.
  • 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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WO2002081510A2 (en) * 2001-01-18 2002-10-17 Curagen Corporation Proteins, polynucleotides encoding them and methods of using the same
EP1317538A1 (en) * 2000-09-13 2003-06-11 Smithkline Beecham Novel compounds

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EP1317538A1 (en) * 2000-09-13 2003-06-11 Smithkline Beecham Novel compounds
EP1317538A4 (en) * 2000-09-13 2005-04-06 Smithkline Beecham NOVEL CONNECTIONS
WO2002081510A2 (en) * 2001-01-18 2002-10-17 Curagen Corporation Proteins, polynucleotides encoding them and methods of using the same
WO2002081510A3 (en) * 2001-01-18 2003-12-04 Curagen Corp Proteins, polynucleotides encoding them and methods of using the same

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EP1385955A2 (en) 2004-02-04

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