WO2002063006A2 - Receptors and membrane-associated proteins - Google Patents

Receptors and membrane-associated proteins Download PDF

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Publication number
WO2002063006A2
WO2002063006A2 PCT/US2002/003868 US0203868W WO02063006A2 WO 2002063006 A2 WO2002063006 A2 WO 2002063006A2 US 0203868 W US0203868 W US 0203868W WO 02063006 A2 WO02063006 A2 WO 02063006A2
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WIPO (PCT)
Prior art keywords
polynucleotide
seq
polypeptide
sequence
amino acid
Prior art date
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PCT/US2002/003868
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English (en)
French (fr)
Other versions
WO2002063006A3 (en
Inventor
Yalda Azimzai
Henry Yue
Li Ding
Danniel B. Nguyen
Ameena R. Gandhi
Neil Burford
Kavitha Thangavelu
Vicki S. Elliott
Jayalaxmi Ramkumar
Monique G. Yao
Preeti G. Lal
Tom Y. Tang
Anita Swarnakar
Bridget A. Warren
Narinder K. Chawla
Jennifer L. Policky
Yunming Xu
Cynthia D. Honchell
Janice Au-Young
Mariah R. Baughn
Brendan M. Duggan
Dyung Aina M. Lu
Kimberly J. Gietzen
Jennifer L. Jackson
Bridget E. RAUMANN
Yan Lu
Stephanie K. Kareht
Uyen K. Tran
Thomas W. Richardson
Brook M. EMERLING
April J. A. Hafalia
John D. Burrill
Gregory A. Marcus
Kurt A. Zingler
Amy E. Kable
Ann E. GORVARD
Original Assignee
Incyte Genomics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Incyte Genomics, Inc. filed Critical Incyte Genomics, Inc.
Priority to CA002437571A priority Critical patent/CA2437571A1/en
Priority to US10/467,595 priority patent/US20040166501A1/en
Priority to JP2002562743A priority patent/JP2005504503A/ja
Priority to EP02720945A priority patent/EP1432801A2/en
Publication of WO2002063006A2 publication Critical patent/WO2002063006A2/en
Publication of WO2002063006A3 publication Critical patent/WO2002063006A3/en

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Definitions

  • This invention relates to nucleic acid and amino acid sequences of receptors and membrane- associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cardiovascular, neurological, gastrointestinal, lipid metabolism, cell proliferative, autoirr iune/inflarnmatory, metabolic, developmental, and endocrine disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of receptors and membrane-associated proteins.
  • Eukaryotic organisms are distinct from prokaryotes in possessing many intracellular membrane-bound compartments such as organelles and vesicles. Many of the metabolic reactions which distinguish eukaryotic biochemistry from prokaryotic biochemistry take place within these compartments. In particular, many cellular functions require very stringent reaction conditions, and the organelles and vesicles enable compartmentalization and isolation of reactions which might otherwise disrupt cytosolic metabolic processes.
  • the organelles include mitochondria, smooth and rough endoplasmic reticula, sarcoplasmic reticulum, and the Golgi body.
  • the vesicles include phagosomes, lysosomes, endosomes, peroxisomes, and secretory vesicles.
  • Organelles and vesicles are bounded by single or double membranes.
  • Biological membranes surround organelles, vesicles, and the cell itself.
  • Membranes are highly selective permeability barriers made up of lipid bilayer sheets composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins.
  • Membranes contain ion pumps, ion channels, and specific receptors for external stimuli which transmit biochemical signals across the membranes. These membranes also contain second messenger proteins which interact with these pumps, channels, and receptors to amplify and regulate transmission of these signals.
  • Plasma Membrane Proteins are highly selective permeability barriers made up of lipid bilayer sheets composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins.
  • Membranes contain ion pumps, ion channels, and specific receptors for external stimuli which transmit biochemical signals across the membranes. These membranes also
  • Transmembrane proteins are characterized by extracellular, txansmen ⁇ brane, and intracellular domains. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an ⁇ -helical conformation. TM proteins are classified as bitopic (Types I and II) proteins, which span the membrane once, and polytopic (Types HI and TV) (Singer, S. J. (1990) Annu. Rev. Cell Biol. 6:247-96) proteins, which contain multiple membrane-spanning segments.
  • TM proteins that act as cell-surface receptor proteins involved in signal transduction include growth and differentiation factor receptors, and receptor-interacting proteins such as Drosophila pecanex and frizzled proteins, LIV-1 protein, NF2 protein, and GNS1/SUR4 eukaryotic integral membrane proteins.
  • TM proteins also act as transporters of ions or metabolites, such as gap junction channels (connexins) and ion channels, and as cell anchoring proteins, such as lectins, integrins, and fibronectins.
  • TM proteins function as vesicle and organeUe-forming molecules, such as calveolins; or cell recognition molecules, such as cluster of differentiation (CD) antigens, glycoproteins, and mucins.
  • CD cluster of differentiation
  • hydrophilic molecules across membranes is facilitated by the presence of channel proteins which form aqueous pores which can perforate a lipid bilayer.
  • Many channels consist of protein complexes formed by the assembly of multiple subunits, at least one of which is an integral membrane protein that contributes to formation of the pore. In some cases, the pore is constructed to allow selective passage of only one or a few molecular species.
  • Distinct types of membrane channels that differ greatly in their distribution and selectivity include: (1) aquaporins, which transport water; (2) protein-conducting channels, which transport proteins across the endoplasmic reticulum membrane; (3) gap junctions, which facilitate diffusion of ions and small organic molecules between neighboring cells; and (4) ion channels, which regulate ion flux through various membranes.
  • membrane proteins contain amino acid sequence motifs that serve to localize proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD, NGR, and GSL sequence motifs, von WiUebrand factor A (vWFA) domains, and EGF-like domains. RGD, NGR, and GSL motif-containing peptides have been used as drug delivery agents in targeted cancer treatment of tumor vasculature (Arap, W. et al. (1998) Science, 279:377-380). Membrane proteins may also contain amino acid sequence motifs that serve to interact with extracellular or intracellular molecules, such as carbohydrate recognition domains.
  • Chemical modification of amino acid residue side chains alters the manner in which MPs interact with other molecules, such as membrane phospholipids.
  • Examples of such chemical modifications include the formation of covalent bonds with glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl moieties, ADP-ribose, phosphate, and sulphate groups.
  • RNA encoding membrane proteins may have alternative splice sites which give rise to proteins encoded by the same gene but with different messenger RNA and a ino acid sequences. Splice variant membrane proteins may interact with other ligand and protein isoforms.
  • Receptors The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response. Other receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in the cell. The term may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition and which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of DNA.
  • G-Protein Coupled Receptors The term may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition
  • GPCR G-protein coupled receptors
  • GPCRs include receptors for biogenic amines, lipid mediators of inflammation, peptide hormones, and sensory signal mediators.
  • the structure of these highly-conserved receptors consists of seven hydrophobic transmembrane regions, an extracellular N-terminus, and a cytoplasmic C-terminus. Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops. Cysteine disulfide bridges connect the second and third extracellular loops. A conserved, acidic- Arg-aromatic residue triplet present in the second cytoplasmic loop may interact with G proteins. A GPCR consensus pattern is characteristic of most proteins belonging to this superfamily (ExPASy PROSITE document PS00237; and Watson, S. and S.
  • Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens.
  • Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N-terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terrninal cysteine-rich domain.
  • the extracellular domain contains a short spacer domain, an ⁇ -helical coiled-coil domain, and a triple helical collagenous domain.
  • scavenger receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa.
  • TM4SF transmembrane 4 superfamily
  • TM4SF transmembrane 4 superfamily
  • TM4SF is a multigene family encoding type HI integral membrane proteins (Wright, M.D. and To linson, M.G. (1994) Immunol. Today 15:588-594).
  • TM4SF is comprised of membrane proteins which traverse the cell membrane four times.
  • Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor- associated antigens, and surface proteins of the schistosome parasites (Jankowski, S.A. (1994) Oncogene 9:1205-1211).
  • Members of the TM4SF share about 25-30% amino acid sequence identity with one another.
  • TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation, including development and oncogenesis, and cell motility, including tumor cell metastasis.
  • Expression of TM4SF proteins is associated with a variety of tumors, and the level of expression maybe altered when cells are growing or activated.
  • Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. Cancer 61: 706- 715; Liu, E. et al. (1992) Oncogene 7: 1027-1032). Ion channels
  • Ion channels are found in the plasma membranes of virtually every cell in the body.
  • chloride channels mediate a variety of cellular functions including regulation of membrane potential and absorption and secretion of ions across epithelial membranes.
  • chloride channels When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH (see, e.g., Greger, R. (1988) Annu. Rev. Physiol. 50:111-122).
  • Electrophysiological and pharmacological properties of chloride channels including ion conductance, current- voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
  • Many channels have sites for phosphorylationby one or more protein kinases including protein kinase A, protein kinase C, casein kinase JH, and tyrosine kinases, all of which regulate ion channel activity in cells. Inappropriate phosphorylation of membrane proteins has been correlated with pathological changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle. Proton pumps Proton ATPases are a large class of membrane proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane.
  • the resultant gradient maybe used to transport other ions across the membrane (Na + , K + , or Ci) or to maintain organeUe pH.
  • Proton ATPases are further subdivided into the mitochondrial F- ATPases, the plasma membrane ATPases, and the vacuolar ATPases.
  • the vacuolar ATPases establish and maintain an acidic pH within various vesicles involved in the processes of endocytosis and exocytosis (Mellman, I. et al. (1986) Ann. Rev. Biochem. 55:663-700).
  • Proton-coupled, 12 membrane-spanning domain transporters such as PEPT 1 and PEPT 2 are responsible for gastrointestinal absorption and for renal reabsorption of peptides using an electrochemical H + gradient as the driving force.
  • Another type of peptide transporter, the TAP transporter is a heterodimer consisting of TAP 1 and TAP 2 and is associated with antigen processing. Peptide antigens are transported across the membrane of the endoplasmic reticulum by TAP so they can be expressed on the ceU surface in association with MHC molecules.
  • Each TAP protein consists of multiple hydrophobic membrane spanning segments and a highly conserved ATP-binding cassette (BoU, M. et al. (1996) Proc. Natl. Acad. Sci.
  • Pathogenic microorganisms such as herpes simplex virus, may encode inhibitors of TAP-mediated peptide transport in order to evade immune surveiUance (Marusina, K. and Manaco, JJ. (1996) Curr. Opin. Hematol. 3:19-26).
  • ABC Transporters The ATP-binding cassette (ABC) transporters, also caUed the "traffic ATPases", comprise a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, CF. (1992) Annu. Rev. CeU Biol. 8:67-113). ABC proteins share a similar overaU structure and significant sequence homology.
  • AU ABC proteins contain a conserved domain of approximately two hundred amino acid residues which includes one or more nucleotide binding domains. Mutations in ABC transporter genes are associated with various disorders, such as hyperbilirubinemia ⁇ Dubin- Johnson syndrome, recessive Stargardt's disease, X-linked adrenoluekodystrophy, multidrug resistance, celiac disease, and cystic fibrosis. CeU Adhesion Proteins
  • ceU The surface of a ceU is rich in transmembrane proteoglycans, glycoproteins, glycohpids, and receptors. These macromolecules mediate adhesion with other ceUs and with components of the ECM. The interaction of the ceU with its surroundings profoundly influences ceU shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling ceU proliferation and differentiation, tissue construction, and embryonic development. Families of ceU adhesion molecules include the cadherins, integrins, lectins, neural ceU adhesion proteins, and some members of the proline-rich proteins.
  • Vezatin is a ubiquitous protein of adherens ceU-ceU junctions, where it interacts with both yosin VHA and the cadherin-catenins complex (Kussel-Andermann,P. et al. (2000) EMBO J. 19:6020-6029).
  • 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 neuropilins consists of three different domains: CUB, discoidin, and MAM domains.
  • InterceUular communication is essential for the development and survival of multiceUular organisms.
  • CeUs communicate with one another through the secretion and uptake of protein signaling molecules.
  • the uptake of proteins into the ceU is achieved by endocytosis, in which the interaction of signaling molecules with the plasma membrane surface, often via binding to specific receptors, results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol.
  • the secretion of proteins from the ceU is achieved by exocytosis, in which molecules inside of the ceU are packaged into membrane-bound transport vesicles derived from the trans Golgi network. These vesicles fuse with the plasma membrane and release their contents into the surrounding extraceUular space. Endocytosis and exocytosis result in the removal and addition of plasma membrane components, and the recycling of these components is essential to maintain the integrity, identity, and functionality of both the plasma membrane and internal membrane-bound compartments.
  • Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids. These regions concentrate certain signaling molecules, including heterotrimeric and s aU G proteins, Src-family tyrosine kinases, endothelial nitric oxide synthase, G-protein-coupled receptors, and certain tyrosine kinase receptors. This concentration of signaling molecules suggests that these microdomains might function as a site for compartmentalization of signaling events. Lipid rafts may also represent sites for the sequestered localization of certain membrane proteins.
  • proteins with lipid modifications such as glycosylphosphatidylinositol-anchored ceU surface proteins and cytoplasmicaUy oriented proteins with closely spaced myristoylation and palmitoylation, as weU as other hydrophobic integral membrane proteins such as caveolin and flotiUin (Baumann, C. A. et al. (2000) Nature (London) 407:202-207).
  • An essential role in intraceUular signaling pathways is fiUed by second messenger molecules, intermediaries that are activated upon binding of ligands to surface receptors and serve as activators of downstream effector molecules.
  • cyclic nucleotides adenosine 3',5'-cychc monophosphate (cAMP) and guanosine 3 '5 -cyclic monophosphate (cGMP) are critical second messengers in a wide variety of signaling pathways.
  • cAMP and cGMP are generated by the enzymes adenylyl (adenylate) cyclase (AC) and guanylyl (guanylate) cyclase (GC) from ATP and GTP.
  • AC adenylyl
  • GC guanylyl
  • a key step in regulating intraceUular cAMP and cGMP levels is modulation of AC and GC activity.
  • Nogo has been identified as a component of the central nervous system myelin that prevents axonal regeneration in adult vertebrates. Cleavage of the Nogo-66 receptor and other glycophosphatidylinositol-jinked proteins from axonal surfaces renders neurons insensitive to Nogo-66, facilitating potential recovery from CNS damage (Foumier, A.E. et al. (2001) Nature 409:341-346).
  • the sht proteins are extraceUular matrix proteins expressed by ceUs at the ventral midline of the nervous system.
  • Slit proteins are ligands for the repulsive guidance receptor Roundabout (Robo) and thus play a role in repulsive axon guidance (Brose, K. et al. (1999) CeU 96:795-806).
  • Lysosomes are the site of degradation of intraceUular material during autophagy and of extraceUular molecules following endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the tr ⁇ ns-Golgi network. These vesicles fuse with endosomes to form the mature lysosome in which hydrolytic digestion of endocytosed material occurs. Lysosomes can fuse with autophagosomes to form a unique compartment in which the degradation of organeUes and other intraceUular components occurs.
  • Peroxisomes are organeUes independent from the secretory pathway. They are the site of many peroxide-generating oxidative reactions in the ceU. Peroxisomes are unique among eukaryotic organeUes in that their size, number, and enzyme content vary depending upon organism, ceU type, and metabolic needs (Waterham, HR. and Cregg, J.M. (1997) BioEssays 19:57-66).
  • CeU fate during embryonic development is determined by members of the activin/TGF- ⁇ superfamUy, cadherins, IGF- 2, and other morphogens.
  • proliferation, maturation, and redifferentiation of germ ceU and reproductive tissues are regulated, for example, by IGF-2, inhibins, activins, and foUistatins (Petraglia, F. (1997) Placenta 18:3-8; Mather, J.P. et al. (1997) Proc. Soc. Exp. Biol. Med. 215:209-222).
  • TGFbeta Transforming growth factor beta signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor and (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I).
  • TbetaR-I-associated protein-1 TRECAP-1
  • TRECAP-1 TbetaR-I-associated protein-1
  • Retinoic acid receptor alpha (RAR alpha) mediates retinoic-acid induced maturation and has been implicated in myeloid development.
  • Genes induced by retinoic acid during granulocytic differentiation include E3, a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis (Scott, L.M. et al. (1996) Blood 88:2517-2530).
  • MOR The ⁇ -opioid receptor
  • MOR mediates the actions of analgesic agents including morphine, codeine, methadone, and fentanyl as weU as heroin.
  • MOR is functionaUy coupled to a G-protein- activated potassium channel (Mestek A. et al. (1995) J. Neurosci. 15:2396-2406).
  • MOR subtypes exist. Alternative splicing has been observed with MOR-1 as with a number of G protein-coupled receptors including somatostatin 2, dopamine D2, prostaglandin EP3, and serotonin receptor subtypes 5-hydroxytiyptamine4 and 5-hydroxytryptarnine7 (Pan, Y.X. et al. (1999) Mol. Pharm. 56:396-403).
  • Membrane anchors are covalently joined to a protein post-translationaUy and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups.
  • Membrane locaUzation of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction.
  • Synaptobrevins are synaptic vesicle-associated membrane proteins (VAMPs) which were first discovered in rat brain. These proteins were initiaUy thought to be limited to neuronal ceUs and to function in the movement of vesicles from the plasmalemma of one ceU, across the synapse, to the plasmalemma of another ceU. Synaptobrevins are now known to occur and function in constitutive vesicle trafficking pathways involving receptor-mediated endocytotic and exocytotic pathways of many non-neuronal ceU types. This regulated vesicle trafficking pathway may be blocked by the highly specific action of clostridial neurotoxins which cleave the synaptobrevin molecule.
  • VAMPs synaptic vesicle-associated membrane proteins
  • VAMP- IB is involved in subceUular targeting and is an isoform of VAMP- 1 A (Isenmann, S. et al., (1998) Mol. Biol. CeU 9:1649-1660).
  • VAMP-1C to F Four additional splice variants (VAMP-1C to F) have recently been identified. Each variant has variable sequences only at the extreme C-terminus, suggesting that the C-terminus is important in vesicle targeting (Berglund, L. et al., (1999) Biochem. Biophys. Res. Com un. 264:777-780).
  • Lysosomes are the site of degradation of intraceUular material during autophagy, and of extraceUular molecules foUowing endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the fr ⁇ ns-Golgi network. These vesicles fuse with endosomes to form the mature lysosome in which hydrolytic digestion of endocytosed material occurs. Lysosomes can fuse with autophagosomes to form a unique compartment in which the degradation of organeUes and other intraceUular components occurs.
  • Protein sorting by transport vesicles has important consequences for a variety of physiological processes including ceU surface growth, the biogenesis of distinct intraceUular organeUes, endocytosis, and the controUed secretion of hormones and neurotransmitters (Rothman, J.E. and Wieland, F.T. (1996) Science 272:227-234).
  • neurodegenerative disorders and other neuronal pathologies are associated with biochemical flaws during endosomal protein sorting or endosomal biogenesis (Mayer R.J. et al. (1996) Adv. Exp. Med. Biol. 389:261-269).
  • Peroxisomes are organeUes independent from the secretory pathway.
  • Peroxisomes are unique among eukaryotic organeUes in that their size, number, and enzyme content vary depending upon organism, ceU type, and metabolic needs (Waterham, H.R. and Cregg, J.M. (1997) BioEssays 19:57-66).
  • the normal functioning of the eukaryotic ceU requires that aU newly synthesized proteins be correctly folded, modified, and delivered to specific intra- and extraceUular sites.
  • Newly synthesized membrane and secretory proteins enter a ceUular sorting and distribution network during or immediately after synthesis and are routed to specific locations inside and outside of the ceU.
  • the initial compartment in this process is the endoplasmic reticulum (ER) where proteins undergo modifications such as glycosylation, disulfide bond formation, and oligomerization.
  • ER endoplasmic reticulum
  • the modified proteins are then transported through a series of membrane-bound compartments which include the various cisternae of the Golgi complex, where further carbohydrate modifications occur. Transport between compartments occurs by means of vesicle budding and fusion. Once within the secretory pathway, proteins do not have to cross a membrane to reach the ceU surface.
  • the signal for retention in the ER in mammalian ceUs consists of the tetrapeptide sequence, KDEL, located at the carboxyl terminus of resident ER membrane proteins (Munro, S. (1986) CeU 46:291-300). Proteins containing this sequence leave the ERbut are quickly retrieved from the early Golgi cisternae and returned to the ER, while proteins lacking this signal continue through the secretory pathway.
  • ⁇ -amyloid precursor protein involves the putative vesicle transport protein presenilin and may play a role in early-onset Alzheimer's disease (Levy-Lahad, E. et al. (1995) Science 269 :973-977).
  • Mitochondrial Membrane Proteins The mitochondrial electron transport (or respiratory) chain is a series of three enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP then provides the primary source of energy for driving the many energy-requiring reactions of a ceU. Most of the protein components of the mitochondrial respiratory chain are the products of nuclear encoded genes that are imported into the mitochondria, and the remainder are products of mitochondrial genes. Defects and altered expression of enzymes in the respiratory chain are associated with a variety of disease conditions in man, including, for example, neurodegenerative diseases, myopathies, and cancer. Lymphocyte and Leukocyte Membrane Proteins
  • the B-ceU response to antigens is an essential component of the normal immune system. Mature B ceUs recognize foreign antigens through B ceU receptors (BCR) which are membrane- bound, specific antibodies that bind foreign antigens. The antigen/receptor complex is internalized, and the antigen is proteolyticaUy processed. To generate an efficient response to complex antigens, the BCR, BCR-associated proteins, and T ceU response are aU required. Proteolytic fragments of the antigen are complexed with major histocompatabihty complex-II (MHCII) molecules on the surface of the B ceUs where the complex can be recognized by T ceUs.
  • MHCII major histocompatabihty complex-II
  • T ceUs recognize and are activated by the MHCI-antigen complex through interactions with the T ceU receptor/CD3 complex, a T ceU-surface multimeric protein located in the plasma membrane.
  • T ceUs activated by antigen presentation secrete a variety of lymphokines that induce B ceU maturation and T ceU proliferation, and activate macrophages, which kiU target ceUs.
  • Leukocytes have a fundamental role in the inflammatory and immune response, and include monocytes/macrophages, mast ceUs, polymorphonucleoleukocytes, natural killer ceUs, neutrophils, eosinophils, basophils, and myeloid precursors.
  • Leukocyte membrane proteins include members of the CD antigens, N-CAM, I-CAM, human leukocyte antigen (HLA) class I and HLA class ⁇ gene products, immunoglobulins, immunoglobuhn receptors, complement, complement receptors, interferons, interferon receptors, interleukin receptors, and chemokine receptors.
  • Abnormal lymphocyte and leukocyte activity has been associated with acute disorders such as AIDS, immune hypersensitivity, leukemias, leukopenia, systemic lupus, granulomatous disease, and eosinophiHa.
  • Apoptosis-Associated Membrane Proteins A variety of ligands, receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a ceU. Although some specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are undefined, leaving major aspects of the pathway unknown.
  • arrays When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specificaUy related to a particular genetic predisposition, condition, disease, or disorder.
  • the invention features purified polypeptides, receptors and membrane-associated proteins, referred to coUectively as "REMAP” and individuaUy as "REMAP-1,” “REMAP-2,” “REMAP-3,” “REMAP-4,” “REMAP-5,” “REMAP-6,” “REMAP-7,” “REMAP-8,” “REMAP-9,” “REMAP-10,” “REMAP-11,” “REMAP-12,” “REMAP-13,” “REMAP-14,” “REMAP-15,” “REMAP-16,” “REMAP-17,” “REMAP-18,” “REMAP-19,” “REMAP-20,” “REMAP-21,” “REMAP-22,” “REMAP-23,” “REMAP-24,” “REMAP-25,” “REMAP-26,” “REMAP-27,” “REMAP-28,” “REMAP-29” “REMAP-30,” “REMAP-31,” “REMAP-32,” “REMAP-33,” “REMAP-34,” “REMAP-35,” “REMAP-36,” “REMAP-37,” “REMAP-38,” “REMAP-39,” “REMAP-40,”
  • 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: 1-43, 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:l-43, C) abiologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:l-43.
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an a ino acid sequence selected from the group consisting of SEQ ID NO: 1-43, 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:l- 43, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-43.
  • the polynucleotide is selected from the group consisting of SEQ ID NO:44-86.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, 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:l-43, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group
  • 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: 1-43, 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:l-43, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • the invention provides an isolated antibody which specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-43, 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: 1-43 , c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43.
  • 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:44-86, 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:44-86, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide comprises at least 60 contiguous nucleotides.
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:44-86, 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:44-86, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionaUy, if present, the amount thereof.
  • the probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:44-86, 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:44-86, 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 rnethod comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction arnphfication, and b) detecting the presence or absence of said amplified 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: 1-43, 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: 1-43, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and a pharmaceuticaUy acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43.
  • the invention additionaUy provides a method of treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, b) a polypeptide comprising a naturaUy occurring a ino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43.
  • 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 REMAP, 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: 1-43, 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: 1-43 , c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43.
  • 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 REMAP, comprising adjninistering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, 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:l-43, c) abiologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-43.
  • 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: 1-43, 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: 1-43, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-43.
  • 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 ID NO:44-86, 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:44-86, ii) 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:44-86, hi) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:44-86, ii) 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:44-86, 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 probability 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 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNA library for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
  • REMAP refers to the amino acid sequences of substantiaUy purified REMAP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of REMAP.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of REMAP either by directly interacting with REMAP or by acting on components of the biological pathway in which REMAP participates.
  • an "aUelic variant” is an alternative form of the gene encoding REMAP.
  • AUelic 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 aUelic variants of its naturaUy occurring form.
  • Common mutational changes which give rise to aUelic 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 REMAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as REMAP or a polypeptide with at least one functional characteristic of REMAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding REMAP, and improper or unexpected hybridization to aUehc variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding REMAP.
  • 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 REMAP.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of REMAP 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 hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence refer to an oligopeptide, 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 like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amphfication is generaUy carried out using polymerase chain reaction (PCR) technologies weU known in the art.
  • PCR polymerase chain reaction
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of REMAP.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of REMAP either by directly interacting with REMAP or by acting on components of the biological pathway in which REMAP participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind REMAP polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • chemicaUy Commonly used carriers that are chemicaUy coupled to peptides include bovine serum albumin, fhyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specificaUy to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No.
  • Aptamer compositions maybe double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2 -NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers maybe specificaUy cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker.
  • the term "intrarner” refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M.
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a ceU, the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic REMAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • compositions comprising polynucleotide sequences encoding REMAP or fragments of REMAP may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and maybe associated with a stabilizing agent such as a carbohydrate.
  • the probe In hybridizations, the probe maybe deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry 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 (Applied 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 GELvTEW 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 helical conformation,
  • deletion refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons maybe 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 REMAP or the polynucleotide encoding REMAP 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 purposes maybe 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 a ino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • a fragment of SEQ ID NO:44-86 comprises a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:44-86, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:44-86 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:44-86 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO:44-86 and the region of SEQ ID NO:44-86 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO: 1-43 is encoded by a fragment of SEQ JD NO:44-86.
  • a fragment of SEQ ID NO: 1-43 comprises a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:l-43.
  • a fragment of SEQ ID NO:l-43 is useful as an immunogenic peptide for the development of antibodies that specificaUy recognize SEQ ID NO:l-43.
  • the precise length of a fragment of SEQ ID NO:l-43 and the region of SEQ ID NO:l-43 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 “fuU length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • the "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent > similarity" between aligned polynucleotide sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool caUed “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences” can be accessed and used interactively at http://www.ncbi.njn .nm.gov/gorf ⁇ l2.htrnl. The "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings.
  • blastn with the "BLAST 2 Sequences” tool Version 2.0.12 (A ⁇ ril-21-2000) set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62
  • Percent identity maybe measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, maybe 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 aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are weU-known. Some alignment methods take into account conservative a ino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and_hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above).
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (A ⁇ ril-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example:
  • Gap x drop-off 50
  • 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 linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome replication, 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 ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, ie., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and maybe consistent among hybridization experiments, whereas wash conditions maybe varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (T j for the specific sequence at a defined ionic strength and pH.
  • T j thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • An equation for calculating T m and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specificaUy see volume 2, chapter 9.
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ l.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions wiU be readily apparent to those of ordinary skiU in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary 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 maybe formed in solution (e.g., C 0 t or R ⁇ analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • a solid support e.g., paper, membranes, filters, chips, pins or glass slides, 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 a ino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect ceUular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oligopeptide fragment of REMAP which is capable of enciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of REMAP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microarray refers to an arrangement of a pluraUty of polynucleotides, polypeptides, or other chemical compounds on a substrate.
  • 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 REMAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of REMAP.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the ceU.
  • Post-translational modification of an REMAP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic milieu of REMAP.
  • Probe refers to nucleic acid sequences encoding REMAP, their complements, or fragments thereof, which are used to detect identical, aUelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, ligands, chennluminescent agents, and enzymes.
  • "Primers" are short nucleic acids, usuaUy DNA oligonucleotides, which may be annealed to a target ' polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid 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, maybe used. Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2 nd ed., vol.
  • 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).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrirnOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • Primer3 primer selection program (available to the public from the Whitehead Institute MfT Center for Genome Research, Cambridge MA) aUows the user to input a "nrispriming library," in which sequences to • avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a 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 accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid maybe part of a vector that is used, for example, to transform a ceU.
  • such recombinant nucleic acids may be part of a viral vector, e.g. , based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuchdes; enzymes; fluorescent, chen ⁇ lurninescent, 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 linear 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 REMAP, nucleic acids encoding REMAP, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specificaUy binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 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, slides, 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 limited to, bacteriophage or viral infection, electroporation, heat shock, hpofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the ceUs of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art. The nucleic acid is introduced into the ceU, directly or indirectly by introduction into a precursor of the ceU, by way of deliberate 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 fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook 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 "aUelic” (as defined above), “sphce,” “species,” or “polymorphic” variant.
  • a sphce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity 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 receptors and membrane-associated proteins (REMAP), the polynucleotides encoding REMAP, and the use of these compositions for the diagnosis, treatment, or prevention of cardiovascular, neurological, gastrointestinal, lipid metabolism, ceU proliferative, autoimmune/inflammatory, metabolic, developmental, and endocrine disorders.
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID).
  • Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ JD NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) 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 ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide JD) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank JD NO:) of the nearest GenBank homolog.
  • Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank homolog(s) along with relevant citations where applicable, aU of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2
  • FIG. 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WI).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
  • SEQ JD NO:21 is 52% identical, from residue G14 to residue E585, to the rat, C2 domain- containing, transmembrane protein, GLUT4 (GenBank ID g4193489), as determined by the Basic Local AHgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 6.8e-192, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnrnent by chance.
  • BLAST Basic Local AHgnment Search Tool
  • SEQ ID NO:21 also contains C2 domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and PROFTLESCAN analyses provide further corroborative evidence that SEQ JD NO:21 is a C2 domam-containing transmembrane protein.
  • HMM hidden Markov model
  • SEQ ID NO:27 is 97% identical, from residue Ml to residue K115, to human vesicle associated membrane protein-IB (GenBank ID g3372648) as determined by the Basic Local Ahgnrnent Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 4.2e- 55, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnrnent by chance. SEQ ID NO:27 also contains a synaptobrevin 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 JD NO:27 is a synaptobrevin (note that "synaptobrevin” is another name for the vesicle-associated membrane protein (VAMP) family of membrane trafficking proteins).
  • VAMP vesicle-associated membrane protein
  • SEQ JD NO:30 is 99% identical from residue M323 to residue Y848 (62% identical over the full length of SEQ ID NO:30) to human delayed-rectifier potassium channel alpha subunit (GenBank JD g2815901) as determined by the Basic Local Ahgnrnent Search Tool (BLAST).
  • BLAST Basic Local Ahgnrnent Search Tool
  • the BLAST probabihty score is 9. le-284, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnrnent by chance.
  • SEQ ID NO:30 also contains a potassium channel tetramerization 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 JD NO:37 is 32% identical from residue G477 to residue L683, and 32% identical from residue K41 to residue L216, to human HERC2 (GenBank ID g4079809) as determined by the Basic Local Ahgnrnent Search Tool (BLAST).
  • BLAST Basic Local Ahgnrnent Search Tool
  • the BLAST probabihty score is 3.3e-25, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnrnent by chance.
  • SEQ JD NO:37 also contains a membrane occupation and recognition nexus repeat as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • PROFTLESCAN analyses provide further corroborative evidence that SEQ ID NO:37 is a GTP dissociation factor.
  • Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte JD) 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 length polynucleotide sequences of the invention, and of fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ JD NO:44-86 or that distinguish between SEQ ID NO:44-86 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 libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the 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.
  • polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought togeih&r by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as ⁇ J ⁇ XXXXXJS[ 1 _N 2 ⁇ YYY_ N 3 JSl 4 represents a "stitched" sequence in which XX ⁇ KXX is the identification number of the cluster of sequences to which the algorithm was apphed, and Y ⁇ T ⁇ T is the number of the prediction generated by the algorithm, and N ⁇ disturb , 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.
  • a polynucleotide sequence identified as F XXXXXX_gMAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was apphed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i. e. , gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the foUowing Table hsts examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example TV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA hbraries for those fuU length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA hbraries shown in Table 5 are described in Table 6.
  • the invention also encompasses REMAP variants.
  • a preferred REMAP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% a ino acid sequence identity to the REMAP amino acid sequence, and which contains at least one functional or structural characteristic of REMAP.
  • the invention also encompasses polynucleotides which encode REMAP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:44-86, which encodes REMAP.
  • the polynucleotide sequences of SEQ ID NO:44-86 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 REMAP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:44- 86 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:44-86.
  • a polynucleotide variant of the invention is a sphce variant of a polynucleotide sequence encoding REMAP.
  • a sphce variant may have portions which have significant sequence identity to the polynucleotide sequence encoding REMAP, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing.
  • a sphce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to the polynucleotide sequence encoding REMAP over its entire length; however, portions of the sphce 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 REMAP.
  • a polynucleotide comprising a sequence of SEQ ID NO:85 is a sphce variant of a polynucleotide comprising a sequence of SEQ TD NO:84.
  • a polynucleotide comprising a sequence of SEQ ID NO: 86 is a sphce variant of a polynucleotide comprising a sequence of SEQ ID NO:71. Any one of the sphce variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of REMAP.
  • nucleotide sequences which encode REMAP and its variants are generaUy capable of hybridizing to the nucleotide sequence of the naturaUy occurring REMAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding REMAP or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non- naturaUy occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-hfe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of DNA sequences which encode REMAP and
  • REMAP derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence maybe inserted into any of the many available expression vectors and ceU systems using reagents weU known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding REMAP 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:44-86 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 annealing and wash conditions, are described in "Definitions.”
  • Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Apphed Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amphfication system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NN), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 D ⁇ A sequencing system (Apphed Biosystems), the MEGABACE 1000 D ⁇ A 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 REMAP maybe 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.
  • one method which maybe employed, restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Appli ⁇ 2:318-322.)
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amphfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • capture PCR involves PCR amphfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations maybe used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
  • primers may be designed using conrmerciaUy 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.
  • hbraries When screening for fuU length cDNAs, it is preferable to use hbraries that have been size-selected to include larger cDNAs. In addition, random-primed hbraries, which often include sequences containing the 5 'regions of genes, are preferable for situations in which an oligo d(T) library does not yield a fuU-length cDNA. Genomic hbraries may be useful for extension of sequence into 5 'non-transcribed regulatory regions.
  • CapiUary electrophoresis systems which are commerciaUy available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/hght intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode REMAP may be cloned in recombinant DNA molecules that direct expression of REMAP, 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 maybe produced and used to express REMAP.
  • nucleotide sequences of the present invention can be engineered using methods generaUy known in the art in order to alter REMAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide- mediated site-directed mutagenesis maybe used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C.-C. et al. (1999) Nat. 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 REMAP, such as its biological or enzymatic activity or its ability 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.C. e
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene maybe recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturaUy occurring genes in a directed and controUable manner.
  • sequences encoding REMAP may be synthesized, in whole or in part, using chemical methods weU known in the art.
  • chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Sy p. Ser. 7:225-232.
  • REMAP itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solution-phase or solid-phase techniques.
  • AdditionaUy the amino acid sequence of REMAP, 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 liquid 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 a ino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • the nucleotide sequences encoding REMAP 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 REMAP. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding REMAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • Methods which are weU known to those skiUed in the art may be used to construct expression vectors containing sequences encoding REMAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY, ch. 9, 13, and 16.)
  • a variety of expression vector host systems may be utihzed to contain and express sequences encoding REMAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems.
  • 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
  • Expression vectors derived from retioviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for dehvery of nucleotide sequences to the targeted organ, tissue, or ceU population.
  • Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; BuUer, R.M.
  • the invention is not limited by the host ceU employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding REMAP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding REMAP can be achieved using a multifunctional E.
  • coh vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding REMAP into the vector's multiple cloning site disrupts the lacZ gene, aUowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of REMAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of REMAP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter maybe used.
  • Yeast expression systems may be used for production of REMAP.
  • 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 REMAP. Transcription of sequences encoding REMAP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
  • viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1311).
  • plant promoters such
  • CeU Differ. 17:85-105. These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196.)
  • mammalian ceUs a number of viral-based expression systems may be utilized.
  • sequences encoding REMAP may be ligated 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 REMAP in host ceUs.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host ceUs.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
  • liposomes, polycationic amino polymers, or vesicles for therapeutic purposes.
  • REMAP in ceU lines is preferred.
  • sequences encoding REMAP can be transformed into ceU lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfully express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the a inoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • 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 REMAP is inserted within a marker gene sequence
  • transformed ceUs containing sequences encoding REMAP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding REMAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as weU.
  • host ceUs that contain the nucleic acid sequence encoding REMAP and that express REMAP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amphfication, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of REMAP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated ceU sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on REMAP is preferred, but a competitive binding assay may be employed.
  • assays are weU known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. PaulMN, Sect. TV; Cohgan, J.E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, JD. (1998) Irnmunochemical Protocols, Humana Press, Totowa NJ.)
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding REMAP include oligolabehng, nick translation, end-labeling, or PCR amphfication using a labeled nucleotide.
  • the sequences encoding REMAP, or any fragments thereof maybe cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionuchdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with nucleotide sequences encoding REMAP may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode REMAP maybe designed to contain signal sequences which direct secretion of REMAP through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its ability 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 limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture CoUection
  • Manassas VA American Type Culture CoUection
  • natural, modified, or recombinant nucleic acid sequences encoding REMAP may be hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric REMAP protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may facihtate the screening of peptide hbraries for inhibitors of REMAP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), cahnodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of then cognate fusion proteins on immobihzed glutathione, maltose, phenylarsine oxide, cahnodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable incmiunoaffinity 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 REMAP encoding sequence and the heterologous protein sequence, so that REMAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
  • a variety of commerciaUy available kits may also be used to facihtate expression and purification of fusion proteins.
  • synthesis of radiolabeled REMAP maybe achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled a ino acid precursor, for example, 35 S-methionine.
  • REMAP of the present invention or fragments thereof may be used to screen for compounds that specificaUy bind to REMAP. At least one and up to a plurahty of test compounds may be screened for specific binding to REMAP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or smaU molecules. In one embodiment, the compound thus identified is closely related to the natural ligand of
  • REMAP e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • the compound can be closely related to the natural receptor to which REMAP binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationaUy designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate ceUs which express REMAP, either as a secreted protein or on the ceU membrane.
  • ceUs include ceUs from mammals, yeast, Drosophila, or E. coli. CeUs expressing REMAP or ceU membrane fractions which contain REMAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either REMAP 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 REMAP, either in solution or affixed to a sohd support, and detecting the binding of REMAP to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay maybe carried out using ceU-free preparations, chemical hbraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a sohd support.
  • REMAP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of REMAP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for REMAP activity, wherein REMAP is combined with at least one test compound, and the activity of REMAP in the presence of a test compound is compared with the activity of REMAP in the absence of the test compound. A change in the activity of REMAP in the presence of the test compound is indicative of a compound that modulates the activity of REMAP.
  • a test compound is combined with an in vitro or ceU-free system comprising REMAP under conditions suitable for REMAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of REMAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurahty of test compounds maybe screened.
  • polynucleotides encoding REMAP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.)
  • mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
  • the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceUblastocysts 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 REMAP may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding REMAP 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 REMAP is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress REMAP e.g., by secreting REMAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS
  • REMAP appears to play a role in cardiovascular, neurological, gastrointestinal, lipid metabolism, ceU proliferative, autoimmune/inflammatory, metabohc, developmental, and endocrine disorders.
  • REMAP 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 REMAP.
  • cardiovascular disorders include, but are not limited to, a cardiovascular disorder including blood vessel disorders such as arterio venous fistula, atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and comphcations of thrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, heart disorders such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic end
  • composition comprising a substantiaUy purified REMAP 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 REMAP including, but not limited to, those provided above.
  • an agonist which modulates the activity of REMAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of REMAP including, but not limited to, those hsted above.
  • an antagonist of REMAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of REMAP. Examples of such disorders include, but are not limited to, those cardiovascular, neurological, gastrointestinal, hpid metabohsm, ceU prohferative, autoimmune/inflammatory, metabohc, developmental, and endocrine disorders described above.
  • an antibody which specificaUy binds REMAP may be used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express REMAP.
  • a vector expressing the complement of the polynucleotide encoding REMAP maybe administered to a subject to treat or prevent a disorder associated with increased expression or activity of REMAP including, but not limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of REMAP may be produced using methods which are generaUy known in the art.
  • purified REMAP may be used to produce antibodies or to screen hbraries of pharmaceutical agents to identify those which specificaUy bind REMAP.
  • Antibodies to REMAP may also be generated using methods that are weU known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralizing antibodies i.e., those which inhibit dimer formation are generaUy preferred for therapeutic use.
  • Single chain antibodies may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others may be immunized by injection with REMAP or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Cahnette-Guerin
  • Corvnebacterium parvum are especiaUy preferable.
  • the ohgopeptides, peptides, or fragments used to induce antibodies to REMAP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of REMAP a ino acids maybe fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to REMAP may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique. (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 splicing 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 single chain antibodies may be adapted, using methods known in the art, to produce REMAP-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin hbraries.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening irnmunoglobulin hbraries or panels of highly specific binding reagents as disclosed in the literature.
  • Antibody fragments which contain specific binding sites for REMAP may also be generated.
  • fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression hbraries maybe constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W . et al. (1989) Science 246:1275-1281.)
  • Various immunoassays maybe used for screening to identify antibodies having the desired specificity.
  • K a is defined as the molar concentration of REMAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equihbrium conditions.
  • K a association constant
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular REMAP epitope, represents a true measure of affinity.
  • Hgh-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 REMAP- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to IO 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of REMAP, 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 quahty and suitabihty of such preparations for certain downstream apphcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of REMAP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quahty and usage in various apphcations, are generaUy available. (See, e.g., Catty, supra, and Cohgan et al. supra.)
  • the polynucleotides encoding REMAP may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified ohgonucleotides) to the coding or regulatory regions of the gene encoding REMAP.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified ohgonucleotides
  • antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding REMAP. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.)
  • Antisense sequences can be delivered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cettular sequence encoding the target protein.
  • Slater J.E. et al. (1998) J. AUergy Clin. 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 retrovirus and adeno-associated virus vectors.
  • viral vectors such as retrovirus and adeno-associated virus vectors.
  • Other gene dehvery mechanisms include hposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • polynucleotides encoding REMAP may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • diseases or disorders caused by deficiencies in REMAP are treated by constructing mammalian expression vectors encoding REMAP and introducing these vectors by mechanical means into REMAP-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) ballistic gold particle dehvery, (hi) hposome-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 maybe effective for the expression of REMAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitiogen, Carlsbad CA), PCMV-SCPJPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • REMAP maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • TRANSFECTION KIT available from Invitrogen
  • aUow one with ordinary skiU in the art to deliver 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 mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to REMAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding REMAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (hi) a Rev-responsive element (RRE) along with additional retrovirus as-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy available (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and AD. Mffler (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al.
  • VSVg vector producing ceU line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
  • an adenovirus-based gene therapy dehvery system is used to dehver polynucleotides encoding REMAP to ceUs which have one or more genetic abnormahties with respect to the expression of REMAP.
  • the construction and packaging of adenovirus-based vectors are weU known to those with ordinary skiU in the art.
  • Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adeno viral vectors are described in U.S. Patent No.
  • Adadenovirus vectors for gene therapy hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
  • a herpes-based, gene therapy dehvery system is used to dehver polynucleotides encoding REMAP to target ceUs which have one or more genetic abnormahties with respect to the expression of REMAP.
  • the use of herpes simplex virus (HSV)-based vectors maybe especiaUy valuable for introducing REMAP to ceUs of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are weU known to those with ordinary skiU in the art.
  • a rephcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to dehver a reporter gene to the eyes of primates (Liu, X.
  • HSV-1 virus vector has also been disclosed in detail in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference.
  • the manipulation of cloned herpesvirus sequences, the generation of recombinant virus foUowing the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of ceUs with herpesvirus are techniques weU known to those of ordinary skiU in the art.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to dehver polynucleotides encoding REMAP to target ceUs.
  • SFV Senihki Forest Virus
  • This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for REMAP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of REMAP-coding RNAs and the synthesis of high levels of REMAP in vector transduced ceUs.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abihty to estabhsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic rephcation of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses wiU aU ow the introduction of REMAP into a variety of ceU types.
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are weU known to those with ordinary skiU in the art.
  • Ohgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple hehx base-pairing methodology.
  • Triple hehx pairing is useful because it causes inhibition of the abihty of the double hehx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Pubhshing, Mt. Kisco NY, pp. 163-177.)
  • a complementary sequence or antisense molecule may also be designed to block translation of RNA 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 REMAP.
  • RNA sequences of between 15 and 20 ribonucleotides may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitabihty of candidate targets may also be evaluated by testing accessibihty to hybridization with complementary ohgonucleotides 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 ohgonucleotides such as sohd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding REMAP. Such DNA sequences may be incorporated 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 lines, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stabihty and half-hfe. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding REMAP.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, ohgonucleotides, antisense ohgonucleotides, triple hehx-foiming ohgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encoding REMAP may be therapeuticaUy useful, and in the treatment of disorders associated with decreased REMAP expression or activity, a compound which specificaUy promotes expression of the polynucleotide encoding REMAP may be therapeuticaUy useful.
  • At least one, and up to a plurality, of test compounds maybe screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary hbrary 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 hbrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding REMAP is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabilized ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding REMAP 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 REMAP.
  • 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 po be 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 line 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 hbrary of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) 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 ceUs or tissues are available and equaUy suitable for use in vivo, in vitro, and ex vivo.
  • ohgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides
  • vectors For ex vivo therapy, vectors maybe introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by liposome 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 maybe apphed 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 generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, cehuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Pubhshing, Easton PA).
  • Such compositions may consist of REMAP, antibodies to REMAP, and mimetics, agonists, antagonists, or inhibitors of REMAP.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generaUy aerosohzed immediately prior to inhalation by the patient.
  • aerosol dehvery of fast- acting formulations is weU-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary dehvery 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 purpose.
  • the determination of an effective dose is weU within the capabihty of those skiUed in the art.
  • Speciahzed forms of compositions may be prepared for direct intraceUular dehvery of macromolecules comprising REMAP or fragments thereof.
  • liposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular dehvery of the macromolecule.
  • REMAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HJN 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 adniinistration. 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 REMAP or fragments thereof, antibodies of REMAP, and agonists, antagonists or inhibitors of REMAP, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity maybe 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 httle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • the exact dosage wiU be determined by the practitioner, in hght of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combinations), reaction sensitivities, and response to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-hfe and clearance rate of the particular formulation. Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • antibodies which specificaUy bind REMAP maybe used for the diagnosis of disorders characterized by expression of REMAP, or in assays to monitor patients being treated with REMAP or agonists, antagonists, or inhibitors of REMAP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for REMAP include methods which utilize the antibody and a label to detect REMAP 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.
  • a variety of protocols for measuring REMAP including ELISAs, RTAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of REMAP expression.
  • Normal or standard values for REMAP expression are estabhshed by combining body fluids or ceU extracts taken from normal mammahan subjects, for example, human subjects, with antibodies to REMAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of REMAP expressed in subject, control, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.
  • the polynucleotides encoding REMAP may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides maybe used to detect and quantify gene expression in biopsied tissues in which expression of REMAP maybe correlated with disease.
  • the diagnostic assay maybe used to determine absence, presence, and excess expression of REMAP, and to monitor regulation of REMAP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding REMAP or closely related molecules maybe used to identify nucleic acid sequences which encode REMAP.
  • the specificity of the probe determine whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amphfication wiU determine whether the probe identifies only naturaUy occurring sequences encoding REMAP, aUehc 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 REMAP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and maybe derived from the sequence of SEQ ID NO:44-86 or from genomic sequences including promoters, enhancers, and introns of the REMAP gene.
  • Means for producing specific hybridization probes for DNAs encoding REMAP include the cloning of polynucleotide sequences encoding REMAP or REMAP derivatives into vectors for the production of mRNA probes.
  • 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 maybe labeled by a variety of reporter groups, for example, by radionuchdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding REMAP may be used for the diagnosis of disorders associated with expression of REMAP.
  • disorders include, but are not limited to, a cardiovascular disorder including blood vessel disorders such as arteriovenous fistula, atherosclerosis, hypertension, vascuhtis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and p ebothrombosis, vascular tumors, and comphcations of thrombolysis, baUoon angioplasty, vascular replacement, and coronary artery bypass graft surgery, heart disorders such as congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitaUy bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rhe
  • the polynucleotide sequences encoding REMAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered REMAP expression. Such qualitative or quantitative methods are weU known in the art.
  • the nucleotide sequences encoding REMAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding REMAP maybe labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
  • 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 REMAP in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of
  • REMAP a normal or standard profile for expression is estabhshed. This may be accomphshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding REMAP, under conditions suitable for hybridization or amphfication. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner maybe compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabhsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • ohgonucleotides designed from the sequences encoding REMAP may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
  • Ohgomers wiU preferably contain a fragment of a polynucleotide encoding REMAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding REMAP, and wiUbe employed under optimized conditions for identification of a specific gene or condition.
  • Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oligonucleotide primers derived from the polynucleotide sequences encoding REMAP may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from the polynucleotide sequences encoding REMAP are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oligonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in gh-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in sihco SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs maybe detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insuhn-dependent diabetes meUitus. SNPs are also useful for exarruning differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as hfe-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in chminished clinical response to treatment with an anti-asthma drug that targets the 5-hpoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations.
  • Methods which may also be used to quantify the expression of REMAP include radiolabehng or biotinylating nucleotides, coamphfication of a control nucleic acid, and mterpolating results from standard curves.
  • radiolabehng or biotinylating nucleotides coamphfication of a control nucleic acid
  • mterpolating results from standard curves See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
  • the speed of quantitation of multiple samples maybe accelerated by nning the assay in a Mgh-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • ohgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms.
  • This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects maybe selected for a patient based on his/her pharmacogenomic profile.
  • REMAP REMAP
  • the microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, expressly incorporated by reference herein.)
  • a transcript image maybe generated by hybridizing the polynucleotides of the present invention or their complements to the totahty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in Mgh-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU lines, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU line.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental compounds.
  • AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • the toxicity of a test compound 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 maybe quantified.
  • the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or ceU type.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU type. Each protein component of a proteome can be subjected rndividuaUy to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a 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 (Sterner and Anderson, supra).
  • the proteins are visuahzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for REMAP to quantify the levels of REMAP expression.
  • the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788).
  • Detection maybe performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or arnino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
  • There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling maybe more reliable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
  • the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays maybe prepared, used, and analyzed using methods known in the art.
  • nucleic acid sequences encoding REMAP maybe used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
  • Either coding or noncoding sequences maybe used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may 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 constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial Pl constructions, or single chromosome cDNA hbraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • BACs bacterial artificial chromosomes
  • the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP).
  • RFLP restriction fragment length polymorphism
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendehan Inheritance in Man (OMJM) World Wide Web site. Correlation between the location of the gene encoding REMAP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • OMJM Online Mendehan Inheritance in Man
  • nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • REMAP in another embodiment, REMAP, its catalytic or immunogenic fragments, or ohgopeptides thereof can be used for screening hbraries 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 sohd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between REMAP and the agent being tested maybe 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 sohd substrate. The test compounds are reacted with REMAP, or fragments thereof, and washed. Bound REMAP is then detected by methods weU known in the art. Purified REMAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobihze it on a sohd support.
  • nucleotide sequences which encode REMAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pah interactions.
  • Incyte cDNAs were derived from cDNA hbraries described in the LJFESEQ GOLD database (Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A)+ RNA was isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • Stratagene was provided with RNA and constructed the corresponding cDNA hbraries. Otherwise, cDNA was synthesized and cDNA hbraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using ohgo d(T) or random primers. Synthetic ohgonucleotide adapters were hgated 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 S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were hgated into compatible restriction enzyme sites of the polyhnker of a suitable plasmid, e.g., PBLUESCRJPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE (Incyte Genomics), or pJNCY (Incyte Genomics), or derivatives thereof.
  • Recombinant plasmids were transformed into competent E. coli ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DHl 0B from Life Technologies.
  • Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E. A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophihzation, at 4°C
  • plasmid DNA was amphfied from host ceU lysates using direct link PCR in a Mgh-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of amphfied plasmid DNA was quantified fluorometeicaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN U fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Incyte cDNA recovered in plasmids as described in Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supphed in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Apphed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VJJI.
  • the polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic prograrnming, and dinucleotide nearest neighbor analysis.
  • Incyte cDNA sequences or translations thereof were then queried against a selection of pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae.
  • pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae.
  • HMM hidden Markov model
  • PFAM hidden Markov model
  • HMM-based protein domain databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabihstic approach which analyzes consensus primary structures of gene famihes. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol.
  • 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. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • 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, bidden Markov model (HMM)-based protein fa ⁇ hly databases such as PFAM; and HMM-based protein domain databases such as SMART.
  • 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 ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnrnent program (DNASTAR), which also calculates the percent identity between aligned sequences. Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of
  • Incyte cDNA and fuU length sequences and provides apphcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where apphcable, the scores, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabihty value, the greater the identity between two sequences).
  • Genscan gene identification program against pubhc genomic sequence databases e.g., gbpri and gbhtg.
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354).
  • the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • the output of Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • Genscan The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode receptors and membrane-associated proteins, the encoded polypeptides were analyzed by querying against PFAM models for receptors and membrane- associated proteins. Potential receptors and membrane-associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as receptors and membrane-associated proteins. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubhc 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 pubhc 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 assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example UI. Alternatively, fuU length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial DNA sequences were extended to fuU length with an algorithm based on BLAST analysis.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pairs
  • GenBank protein homolog The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of REMAP Encoding Polynucleotides The sequences which were used to assemble SEQ ID NO:44-86 were compared with sequences from the Incyte LTFESEQ database and pubhc domain databases using BLAST and other implementations of the Smith- Waterman algorithm.
  • pubhc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of aU sequences of that cluster, including its particular SEQ JD NO:, to that map location.
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normalized value between 0 and 100, and is calculated as foUows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pah (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 pah with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quahty in a BLAST ahgnrnent. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding REMAP 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 HI).
  • Each cDNA sequence is derived from a cDNA hbrary 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; genitaha, female; genitaha, male; germ ceUs; hernic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflamniation, neurological, trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding REMAP.
  • cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA). In this manner, SEQ JD NO:44 was mapped to chromosome 3 within the interval from 30.4 to
  • SEQ ID NO:68 was mapped to chromosome 3 within the interval from 60.0 to
  • FuU length polynucleotide sequences were also produced by extension of an appropriate fragment of the fuU length molecule using ohgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other prirner was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • Selected human cDNA hbraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to dete ⁇ riine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coh ceUs. Transformed ceUs were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384- weU plates in LB/2x carb liquid media.
  • SNPs Single nucleotide polymorphisms
  • LIFESEQ database Incyte Genomics
  • Prehminary filters removed the majority of basecaU errors by requiring a minimum Phred quahty score of 15, and removed sequence ahgnrnent errors and errors resulting from improper trimming of vector sequences, chimeras, and sphce variants.
  • An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP.
  • Clone error filters used statisticaUy generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering error filters used statisticaUy generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences.
  • a final set of filters removed duphcates and SNPs found in immunoglobulins or T-ceU receptors.
  • Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three deciualan, and two Amish individuals.
  • the African population comprised 194 individuals (97 male, 97 female), aU African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
  • AUele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no aUehc variance in this population were not further tested in the other three populations.
  • Hybridization probes derived from SEQ ID NO.-44-86 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pahs, is specificaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
  • An ahquot containing IO 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl U, Eco RL Pst I, Xba I, or Pvu U (DuPont NEN).
  • the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytean 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 saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuahzed using autoradiography or an alternative imaging means and compared.
  • microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photohthography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical n ⁇ crospotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena (1999), supra).
  • Suggested substrates include sihcon, sihca, glass slides, glass chips, and sihcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using available methods and machines weU known to those of ordinary skill 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; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microarray. Fragments or ohgomers suitable for hybridization can be selected using software weU known in the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the ohgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l ohgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham 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 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in l4 l 5X SSC/0.2% SDS. Microarray Preparation
  • Sequences of the present invention are used to generate array elements.
  • Each array element is amplified from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amphfied in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
  • Purified array elements are immobihzed on polymer-coated glass shdes.
  • Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments.
  • Glass shdes are etched in 4% hydrofluoric acid (VWR
  • Array elements are apphed to the coated glass substrate using a procedure described in U.S. Patent No. 5,807,522, incorporated herein by reference.
  • 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, 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 shde.
  • Microarrays are UV-crosslinked using a STRATALINKER UV-crosshnker (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 saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2%
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 covershp.
  • the arrays are transferred to a waterproof chamber having a cavity just shghtly larger than a microscope shde.
  • the chamber is kept at 100% humidity internaUy by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
  • the chamber containing the arrays is incubated for about 6.5 hours at 60°C
  • the arrays are washed for 10 in at 45°C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C in a second wash buffer (0.1X SSC), and dried. Detection
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser hght is focused on the array using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the shde 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 hght is spht, based on wavelength, into two photomultipher tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipher tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each array is 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 calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the cahbration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipher tube is digitized using a 12-bit RTT-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 linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). XII. Complementary Polynucleotides
  • Sequences complementary to the REMAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring REMAP.
  • ohgonucleotides comprising from about 15 to 30 base pans is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate ohgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of REMAP.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary ohgonucleotide is designed to prevent ribosomal binding to the REMAP-encoding transcript.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express REMAP upon induction with isopropyl beta-D- thiogalactopyranoside (JPTG).
  • JPTG isopropyl beta-D- thiogalactopyranoside
  • Expression of REMAP in eukaryotic ceUs is achieved by infecting insect or mammalian ceU lines with recombinant Autographica cahfornica nuclear polyhedrosis virus (AcMNPV), commonly, known as baculoviras.
  • AcMNPV Autographica cahfornica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculoviras is replaced with cDNA encoding REMAP 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.
  • baculoviras Recombinant baculoviras is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requhes additional genetic modifications to baculoviras. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
  • REMAP is synthesized as a fusion protein with, e.g., glutathione S-teansferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST glutathione S-teansferase
  • FLAG or 6-His peptide epitope tag
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobihzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
  • the GST moiety can be proteolyticaUy cleaved from REMAP 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
  • REMAP function is assessed by expressing the sequences encoding REMAP at physiologicaUy elevated levels in mammalian ceU culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endothelial or hematopoietic ceU line, using either hposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs from nontransfected ceUs and is a rehable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward hght scatter and 90 degree side hght scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York NY.
  • the influence of REMAP on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding REMAP 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 inrmunoglobulin 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 REMAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • REMAP substantiaUy purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
  • PAGE polyacrylamide gel electrophoresis
  • REMAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high i munogenicity, and a corresponding oligopeptide 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 hydrophihc regions are weU described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleinhdobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MFS N-maleinhdobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the ohgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-REMAP activity by, for example, binding the peptide or REMAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Media containing REMAP are passed over the innnunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of REMAP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/REMAP binding (e. g. , a buffer of pH 2 to pH 3 , or a high concentration of a chaotiope, such as urea or thiocyanate ion), and REMAP is coUected.
  • REMAP or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
  • Bolton-Hunter reagent See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
  • Candidate molecules previously arrayed in the weUs of a multi- weU plate are incubated with the labeled REMAP, washed, and any weUs with labeled REMAP complex are assayed. Data obtained using different concentrations of REMAP are used to calculate values for the number, affinity, and association of REMAP with the candidate molecules.
  • molecules interacting with REMAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • REMAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a higli-throughput manner to determine ah interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • PATHCALLING process CuraGen Corp., New Haven CT
  • yeast two-hybrid system in a higli-throughput manner to determine ah interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • XVIII Demonstration of REMAP Activity Gap Junction Activity of REMAP
  • Gap junction activity of REMAP is demonstrated as the abihty to induce the formation of interceUular channels between paired Xenopus laevis oocytes injected with REMAP cRNA
  • REMAP cRNA antisense ohgonucleotide to REMAP to reduce background.
  • REMAP cRNA-injected oocytes are incubated overnight, stripped of viteUine membranes, and paired for recording of junctional currents by dual ceU voltage clamp. The measured conductances are proportional to gap junction activity of REMAP.
  • an assay for REMAP activity measures the ion channel activity of REMAP using an electrophysiological assay for ion conductance.
  • REMAP can be expressed by transforming a mammalian ceU line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding REMAP. Eukaryotic expression vectors are commerciaUy available, and the techniques to introduce them into ceUs are weU known to those skilled in the art.
  • a second plasmid which expresses any one of a number of marker genes, such as ⁇ -galactosidase, is co-transformed into the ceUs to aUow rapid identification of those ceUs which have taken up and expressed the foreign DNA.
  • ceUs are incubated for 48-72 hours after transformation under conditions appropriate for the ceU line to aUow expression and accumulation of REMAP and ⁇ -galactosidase.
  • Transformed ceUs expressing ⁇ -galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are weU known in the art. Stained ceUs are tested for differences in membrane conductance by electrophysiological techniques that are weU known in the art.
  • Untransformed ceUs, and/or ceUs transformed with either vector sequences alone or ⁇ -galactosidase sequences alone, are used as controls and tested in paraUel.
  • CeUs expressing REMAP wiU have higher anion or cation conductance relative to control ceUs.
  • REMAP Transmembrane Protein Activity
  • An assay for REMAP activity measures the expression of REMAP on the ceU surface.
  • cDNA encoding REMAP is transfected into an appropriate mammalian ceU line.
  • CeU surface proteins are labeled with biotin as described (de la Fuente, M. A. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed using REMAP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled irnmunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of REMAP expressed on the ceU surface.
  • An alternative assay for REMAP activity is based on a prototypical assay for ligand/receptor- mediated modulation of ceU prohferation. This assay measures the amount of newly synthesized DNA in Swiss mouse 3T3 ceUs expressing REMAP.
  • An appropriate mammalian expression vector containing cDNA encoding REMAP is added to quiescent 3T3 cultured ceUs using transfection methods weU known in the art. The transfected ceUs are incubated in the presence of [ 3 H]thymidine and varying amounts of REMAP ligand.
  • Incorporation of [ 3 H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a tritium radioisotope counter, and the amount incorporated is directly proportional to the amount of newly synthesized DNA.
  • a linear dose-response curve over at least a hundred-fold REMAP ligand concentration range is indicative of receptor activity.
  • One unit of activity per miUihter is defined as the concentration of REMAP producing a 50% response level, where 100% represents maximal incorporation of [ 3 H thymidine into acid-precipitable DNA (McKay, I. and Leigh, I., eds. (1993) Growth Factors: A Practical Approach, Oxford University ' Press, New York, NY, p. 73).
  • An assay for REMAP activity measures the expression of REMAP on the ceU surface.
  • cDNA encoding REMAP is transfected into an appropriate mammalian ceU line.
  • CeU surface proteins are labeled with biotin as described (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed using REMAP-specific antibodies, and immunoprecipitated samples are analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of REMAP expressed on the ceU surface.
  • an assay for REMAP activity is based on a prototypical assay for hgand/receptor-mediated modulation of ceU prohferation.
  • This assay measures the rate of DNA synthesis in Swiss mouse 3T3 ceUs.
  • a plasmid containing polynucleotides encoding REMAP is added to quiescent 3T3 cultured ceUs using transfection methods weU known in the art.
  • the transiently transfected ceUs are then incubated in the presence of [ 3 H]thymidine, a radioactive DNA precursor molecule. Varying amounts of REMAP ligand are then added to the cultured ceUs.
  • the assay for REMAP activity is based upon the abihty of GPCR family proteins to modulate G protein-activated second messenger signal transduction pathways (e.g., cAMP; Gaudin, P. et al. (1998) J. Biol. Chem. 273:4990-4996).
  • a plasmid encoding fuU length REMAP is transfected into a mammalian ceU line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) ceU lines) using methods weU-known in the art.
  • Transfected ceUs are grown in 12-weU trays in culture medium for 48 hours, then the culture medium is discarded, and the attached ceUs are gently washed with PBS. The ceUs are then incubated in culture medium with or without ligand for 30 minutes, then the medium is removed and ceUs lysed by treatment with 1 M perchloric acid. The cAMP levels in the lysate are measured by radioimmunoassay using methods weU-known in the art. Changes in the levels of cAMP in the lysate from ceUs exposed to ligand compared to those without ligand are proportional to the amount of REMAP present in the transfected ceUs.
  • the ceUs are grown in 24-weU plates containing lxlO 5 ceUs/weU and incubated with inositol-free media and [ 3 H]myoinositol, 2 mCi/weU, for 48 hr.
  • the culture medium is removed, and the ceUs washed with buffer containing 10 M LiCl foUowed by addition of ligand.
  • the reaction is stopped by addition of perchloric acid.
  • Inositol phosphates are extracted and separated on Dowex AG1-X8 (Bio-Rad) anion exchange resin, and the total labeled inositol phosphates counted by liquid scintiUation. Changes in the levels of labeled inositol phosphate from ceUs exposed to ligand compared to those without ligand are proportional to the amount of REMAP present in the transfected ceUs.
  • REMAP is expressed by transforming a mammalian ceU line such as
  • COS7, HeLa or CHO with a eukaryotic expression vector encoding REMAP Eukaryotic expression vectors are commerciaUy available, and the techniques to introduce them into ceUs are weU known to those skiUed in the art.
  • the ceUs are incubated for 48-72 hours after transformation under conditions appropriate for the ceU line to aUow expression and accumulation of REMAP and b-galactosidase.
  • Transformed ceUs expressing b- galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are weU known in the art. Stained ceUs are tested for differences in membrane conductance due to various ions by electrophysiological techniques that are weU known in the art. Untransformed ceUs, and/or ceUs transformed with either vector sequences alone or b-galactosidase sequences alone, are used as controls and tested in paraUel.
  • the contribution of REMAP to cation or anion conductance can be shown by incubating the cells using antibodies specific for either REMAP. The respective antibodies wiUbind to the extraceUular side of REMAP, thereby blocking the pore in the ion channel, and the associated conductance.
  • REMAP transport activity is assayed by measuring uptake of labeled substrates into Xenopus laevis oocytes.
  • Oocytes at stages V and VI are injected with REMAP mRNA (10 ng per oocyte) and incubated for 3 days at 18 °C in OR2 medium (82.5 mM NaCl, 2.5 mM KC1, 1 mM CaCL , , 1 mM MgCl j , 1 mM Na 2 HPO 4 , 5 mM Hepes, 3.8 mM NaOH , 50 ⁇ g/ml gentamycin, pH 7.8) to aUow expression of REMAP protein.
  • OR2 medium 82.5 mM NaCl, 2.5 mM KC1, 1 mM CaCL , , 1 mM MgCl j , 1 mM Na 2 HPO 4 , 5 mM Hepes, 3.8 mM NaOH , 50 ⁇ g/ml gentamycin, pH 7.8
  • Oocytes are then transferred to standard uptake medium (100 mM NaCl, 2 mM KC1, 1 mM CaCl j , 1 mM MgCl 2 , 10 mM Hepes/Tris pH 7.5).
  • uptake of various substrates e.g., amino acids, sugars, drugs, and neurotransmitters
  • substrates e.g., amino acids, sugars, drugs, and neurotransmitters
  • uptake is terminated by washing the oocytes three times in Na + -free medium, measuring the incorporated 3 H, and comparing with controls.
  • REMAP activity is proportional to the level of internalized 3 H substrate.
  • REMAP protein kinase (PK) activity is measured by phosphorylation of a protein substrate using gamma-labeled [ 3 P]-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter.
  • REMAP is incubated with the protein substrate, t 32 P]-ATP, and an appropriate kinase buffer.
  • the 3 P incorporated into the product is separated from free [ 32 P]-ATP by electrophoresis and the incorporated 32 P is counted.
  • the amount of 32 P recovered is proportional to the PK activity of REMAP in the assay.
  • a determination of the specific a ino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
  • adenylyl cylcase activity of REMAP is demonstrated by the abihty to convert ATP to cAMP (Mittal, C.K. (1986) Methods Enzymol. 132:422-428).
  • REMAP is incubated with the substrate [ ⁇ - 32 P]ATP, foUowing which the excess substrate is separated from the product cychc [ 32 P] AMP.
  • REMAP activity is determined in 12 x 75 mm disposable culture tubes containing 5 ⁇ l of 0.6 M Tris-HCl, pH 7.5, 5 ⁇ l of 0.2 M MgCl 2 , 5 ⁇ l of 150 mM creatine phosphate containing 3 units of creatine phosphokinase, 5 ⁇ l of 4.0 mM l-me yl-3-isobutylxanthine, 5 ⁇ l of 20 mM cAMP, 5 ⁇ l 20 mM ⁇ tMothreitol, 5 ⁇ l of 10 mM ATP, 10 ⁇ l [ ⁇ - 32 P]ATP (2-4 x IO 6 cpm), and water in a total volume of 100 ⁇ l.
  • the reaction mixture is prewarmed to 30 °C.
  • the reaction is initiated by adding REMAP to the prewarmed reaction mixture. After 10-15 minutes of incubation at 30°C, the reaction is terminated by adding 25 ⁇ l of 30% ice-cold trichloroacetic acid (TCA). Zero-time incubations and reactions incubated in the absence of REMAP are used as negative controls. Products are separated by ion exchange chromatography, and cyclic [ 32 P] AMP is quantified using a ⁇ -radioisotope counter. The REMAP activity is proportional to the amount of cychc [ 32 P] AMP formed in the reaction.

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WO2006000753A2 (en) * 2004-06-25 2006-01-05 Celltech R & D Limited Use of flj40787, a protein involved in colon, colorectal, ovarian, lung and/or liver cancer
JP2007020403A (ja) * 2005-07-12 2007-02-01 National Institute Of Advanced Industrial & Technology 新規糖鎖認識蛋白質及びその遺伝子

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EP1402051A2 (en) * 2001-06-05 2004-03-31 Exelixis, Inc. Lces as modifiers of the p53 pathway and methods of use
EP1402051A4 (en) * 2001-06-05 2005-08-17 Exelixis Inc LCE AS MODIFIERS OF THE P53 PATHWAY AND METHOD OF USE
WO2006000753A2 (en) * 2004-06-25 2006-01-05 Celltech R & D Limited Use of flj40787, a protein involved in colon, colorectal, ovarian, lung and/or liver cancer
WO2006000753A3 (en) * 2004-06-25 2006-04-06 Celltech R&D Ltd Use of flj40787, a protein involved in colon, colorectal, ovarian, lung and/or liver cancer
JP2007020403A (ja) * 2005-07-12 2007-02-01 National Institute Of Advanced Industrial & Technology 新規糖鎖認識蛋白質及びその遺伝子

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