# Patent

## Title
CD40-related receptor that binds CD40L

## Abstract

Various embodiments of the invention provide human receptors and membrane-associated proteins (REMAP) and Q polynucleotides which identify and encode REMAP. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of REMAP.

## Background
TECHNICAL FIELD
The invention relates to novel nucleic acids, receptors and membrane-associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and receptors and membrane-associated proteins.
BACKGROUND OF THE INVENTION
Signal transduction is the general process by which cells respond to extracellular signals. Signal transduction across the plasma membrane begins with the binding of a signal molecule, e.g., a hormone, neurotransmitter, or growth factor, to a cell membrane receptor. The receptor, thus activated, triggers an intracellular biochemical cascade that ends with the activation of an intracellular target molecule, such as a transcription factor. This process of signal transduction regulates all types of cell functions including cell proliferation, differentiation, and gene transcription.
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
Plasma membrane proteins (MPs) are divided into two groups based upon methods of protein extraction from the membrane. Extrinsic or peripheral membrane proteins can be released using extremes of ionic strength or pH, urea, or other disruptors of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent.
The majority of known integral membrane proteins are transmembrane proteins (TM) which are characterized by an extracellular, a transmembrane, and an intracellular domain. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an α-helical conformation. TM proteins are classified as bitopic (Types I and II) and polytopic (Types III and IV) (Singer, S. J. (1990) Annu. Rev. Cell Biol. 6:247-296). Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. TM proteins carry out a variety of important cellular functions, including acting as cell-surface receptor proteins involved in signal transduction. These functions are represented by 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 may be vesicle organelle-forming molecules, such as caveolins, or cell recognition molecules, such as cluster of differentiation (CD) antigens, glycoproteins, and mucins.
Many MPs contain amino acid sequence motifs that serve to localize proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, ROD, NGR, and GSL sequence motifs, von Willebrand 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). Furthermore, MPs may also contain amino acid sequence motifs that serve to interact with extracellular or intracellular molecules, such as carbohydrate recognition domains (CRD).
Chemical modification of amino acid residue side chains alters the manner in which MPs interact with other molecules, for example, phospholipid membranes. Examples of such chemical modifications to amino acid residue side chains are covalent bond formation 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 amino 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.
Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines; peptide hormones; growth and differentiation factors; small peptide factors such as thyrotropin-releasing hormone; galanin, somatostatin, and tachykinins; and circulatory system-borne signaling molecules. Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell. This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transferrin, glucose- or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (Lodish, H. et al. (1995) Molecular Cell Biology, Scientific American Books, New York N.Y., p. 723; Mikhailenko, I. et al. (1997) J. Biol. Chem. 272:6784-6791).
Receptor Protein Kinases
Many growth factor receptors, including receptors for epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator α-thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor, it triggers the autophosphorylation of a serine, threonine, or tyrosine residue on the receptor. These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in signaling pathways that eventually link the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule. In the case of tyrosine residue autophosphorylation, signaling proteins can bind these motifs using several common domains, for example Src homology-2 (SH2) domains, phosphotyrosine-binding (PTB) domains, and forkhead-associated (FHA) domains. These domains, alone or in combination, are found in many signaling proteins, such as phospholipase C-γ (PLC-γ), the p85 regulatory subunit of PI-3 kinase, pp60c-crc,Ras-GTPase activating protein, Chk2, AF-6, insulin receptor substrate-i (IRS-1), and Shc (Li, J. et al. (2000) J. Cell Sci. 113:4143-4149; Guy, G. R. et al. (2002) Cell Signal. 14:11-20; Vidal, M. et al. (2001) Crit. Rev. Oncol. Hematol. 40:175-186; Lowenstein, E. J. et al. (1992) Cell 70:431-442). The cytoline family of receptors share a different common binding domain and include transmembrane receptors for growth hormone (GH), interleukins, erythropoietin, and prolactin.
Other receptors and second messenger-binding proteins have intrinsic serine/threonine protein kinase activity. These include activin/TGF-β/BMP-superfamily receptors, calcium- and diacylglycerol-activated/phospholipid-dependant protein kinase (PK-C), and RNA-dependant protein kinase (PK-R). In addition, other serine/threonine protein kinases, including nematode Twitchin, have fibronectin-like, immunoglobulin C2-like domains.
G-Protein Coupled Receptors
The G-protein coupled receptors (GPCRs), encoded by one of the largest families of genes yet identified, play a central role in the transduction of extracellular signals across the plasma membrane. GPCRs have a proven history of being successful therapeutic targets.
GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha (α) helices. GPCRs range in size from under 400 to over 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem. 196:1-10; Cougllin, S. R. (1994) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of a helices forms a ligand-binding pocket The extracellular N-terminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins. (See, e.g., Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press,. San Diego Calif., pp. 2-6; Bolander, F. F. (1994) Molecular Endocrinology, Academic Press, San Diego Calif., pp. 162-176; Baldwin, J. M. (1994) Curr. Opin. Cell Biol. 6:180-190.)
GPCRs include receptors for sensory signal mediators (e.g., light and olfactory stimulatory molecules); adenosine, γ-aminobutyric acid (GABA), hepatocyte growth factor, melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin, tachykinins, vasoactive intestinal polypeptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine and norepinephrine, histamine, glutamate (metabotropic effect), acetylcholine (muscarinic effect), and serotonin); chemokines; lipid mediators of inflammation (e.g., prostaglandins and prostanoids, platelet activating factor, and leukotrienes); and peptide hormones (e.g., bombesin, bradykinin, calcitonin, C5a anaphylatoxin, endothelin, follicle-stimulating hormone (FSH), gonadotropic-releasing hormone (GnRH), neurokinin, and thyrotropin-releasing hormone (TRH), and oxytocin). GPCRs which act as receptors for stimuli that have yet to be identified are known as orphan receptors.
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Furthermore, somatic activating mutations in the thyrotropin receptor have been reported to cause hyperfunctioning thyroid adenomas, suggesting that certain GPCRs susceptible to constitutive activation may behave as protooncogenes (Parma, J. et al. (1993) Nature 365:649-651). GPCR receptors for the following ligands also contain mutations associated with human disease: luteinizing hormone (precocious puberty); vasopressin V2 (X-linked nephrogenic diabetes); glucagon (diabetes and hypertension); calcium (hyperparathyroidism, hypocalcuria, hypercalcemia); parathyroid hormone (short limbed dwarfism); β3-adrenoceptor (obesity, non-insulin-dependent diabetes mellitus); growth hormone releasing hormone (dwarfism); and adrenocorticotropin (glucocorticoid deficiency) (Wilson, S. et al. (1998) Br. J. Pharmocol. 125:1387-1392; Stadel, J. M. et al. (1997) Trends Pharmacol. Sci. 18:430-437). GPCRs are also involved in depression, schizophrenia, sleeplessness, hypertension, anxiety, stress, renal failure, and several cardiovascular disorders (Horn, F. and G. Vriend (1998) J. Mol. Med. 76:464468).
In addition, within the past 20 years several hundred new drugs have been recognized that are directed towards activating or inhibiting GPCRs. The therapeutic targets of these drugs span a wide range of diseases and disorders, including cardiovascular, gastrointestinal, and central nervous system disorders as well as cancer, osteoporosis and endometriosis (Wilson et al., supra; Stadel et al., supra). For example, the dopamine agonist L-dopa is used to treat Parkinson's disease, while a dopamine antagonist is used to treat schizophrenia and the early stages of Huntington's disease. Agonists and antagonists of adrenoceptors have been used for the treatment of asthma, high blood pressure, other cardiovascular disorders, and anxiety; muscarinic agonists are used in the treatment of glaucoma and tachycardia; serotonin 5HT1D antagonists are used against migraine; and histamine Hi antagonists are used against allergic and anaphylactic reactions, hay fever, itching, and motion sickness (Horn et al., supra).
Nuclear Receptors
Nuclear receptors bind small molecules such as hormones or second messengers, leading to increased receptor-binding affinity to specific chromosomal DNA elements. In addition the affinity for other nuclear proteins may also be altered. Such binding and protein-protein interactions may regulate and modulate gene expression. Examples of such receptors include the steroid hormone receptors family, the retinoic acid receptors family, and the thyroid hormone receptors family.
Ligand-Gated Receptor Ion Channels
Ligand-gated receptor ion channels fall into two categories. The first category, extracellular ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-binding events into electrical signals, such as fast synaptic neurotransmission. ELG function is regulated by post-translational modification. The second category, intracellular ligand-gated receptor ion channels (ILGs), are activated by many intracellular second messengers and do not require post-translational modification(s) to effect a channel-opening response.
ELGs depolarize excitable cells to the threshold of action potential generation. In non-excitable cells, ELGs permit a limited calcium ion-influx during the presence of agonist ELGs include channels directly gated by neurotransmitters such as acetylcholine, L-glutamate, glycine, ATP, serotonin, GABA, and histamine. ELG genes encode proteins having strong structural and functional similarities. ELGs are encoded by distinct and unrelated gene families and include receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of arachidonic acid.
Macrophage Scavenger Receptors
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-terminal cysteine-rich domain. The extracellular domain contains a short spacer domain, an α-helical coiled-coil domain, and a triple helical collagenous domain. These receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:9133-9137; Elomaa, O. et al. (1995) Cell 80:603-609). The 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.
T-Cell Receptors
T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells. Although a population of T cells can recognize a wide range of different antigens, an individual T cell can only recognize a single antigen and only when it is presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T cells consists of immunoglobulin-like integral membrane glycoproteins containing two polypeptide subunits, a and p, of similar molecular weight. Both TCR subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. (1984) Nature 309:757-762). The genes for the TCR subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A. (1991) Annu. Rev. Genet. 25:487-510). Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg, A. C. et al. (1985) N. Engl. J. Med. 313:529-533; Weiss, supra).
Netrin Receptors
The netrins are a family of molecules that function as diffusible attractants and repellants to guide migrating cells and axons to their targets within the developing nervous system. The netrin receptors include the C. elegans protein UNC-5, as well as homologues recently identified in vertebrates (Leonardo, E. D. et al. (1997) Nature 386:833-838). These receptors are members of the immunoglobulin superfamily, and also contain a characteristic domain called the ZU5 domain. Mutations in the mouse member of the netrin receptor family, Rcm (rostral cerebellar malformation) result in cerebellar and midbrain defects as an apparent result of abnormal neuronal migration (Ackerman, S. L. et al. (1997) Nature 386:838-842).
VPS10 Domain Containing Receptors
The members of the VPS10 domain containing receptor family all contain a domain with homology to the yeast vacuolar sorting protein 10 (VPS10) receptor. This family includes the mosaic receptor SorLA, the neurotensin receptor sortilin, and SorCS, which is expressed during mouse embryonal and early postnatal nervous system development (Hermey, G. et al. (1999) Biochem. Biophys. Res. Commun. 266:347-351; Hermey, G. et al. (2001) Neuroreport 12:29-32). A recently identified member of this family, SorCS2, is highly expressed in the developing and mature mouse central nervous system. Its main site of expression is the floor plate, and high levels are also detected transiently in brain regions including the dopaminergic brain nuclei and the dorsal thalamus (Rezgaoui, M. (2001) Mech. Dev. 100:335-338).
Membrane-Associated Proteins
Tetraspan Family Proteins
The transmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene family encoding type mi integral membrane proteins (Wright, M. D. and M. G. Tomlinson (1994) Immunol. Today 15:588-594). The 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. A number of 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 may be altered when cells are growing or activated.
Tumor Antigens
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. For example, chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithelial membranes. 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 phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion channel activity in cells. Inappropriate phosphorylation of proteins in cells has been linked to 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.
Cerebellar granule neurons possess a non-inactivating potassium current which modulates firing frequency upon receptor stimulation by neurotransmitters and controls the resting membrane potential. Potassium channels that exhibit non-inactivating currents include the ether a go-go (EAG) channel. A membrane protein designated KCR1 specifically binds to rat EAG by means of its C-terminal region and regulates the cerebellar non-inactivating potassium current. KCR1 is predicted to contain 12 transmembrane domains, with intracellular amino and carboxyl termini. Structural characteristics of these transmembrane regions appear to be similar to those of the transporter superfamily, but no homology between KCR1 and known transporters was found, suggesting that KCR1 belongs to a novel class of transporters. KCR1 appears to be the regulatory component of non-inactivating potassium channels (Hoshi, N. et al. (1998) J. Biol. Chem. 273:23080-23085).
ABC Transporters
ATP-binding cassette (ABC) transporters, also called the “traffic ATPases”, are a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8:67-113). ABC proteins share a similar overall structure and significant sequence homology. All 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 II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-linked adrenoleukodystrophy, multidrug resistance, celiac disease, and cystic fibrosis.
Semaphorins and Neuropilins
Semaphorins are a large group of axonal guidance molecules consisting of at least 30 different members and are found in vertebrates, invertebrates, and even certain viruses. All semaphorins contain the sema domain which is approximately 500 amino acids in length. Neuropilin, a semaphorin receptor, has been shown to promote neurite outgrowth in vitro. The extracellular region of neuropilins consists of three different domains: CUB, discoidin, and MAM domains. The CUB and the MAM motifs of neuropilin have been suggested to have roles in protein-protein interactions and are thought to be involved in the binding of semaphorins through the sema and the C-terminal domains (reviewed in Raper, J. A. (2000) Curr. Opin. Neurobiol. 10:88-94).
Membrane Proteins Associated with Intercellular Communication
Intercellular communication is essential for the development and survival of multicellular organisms. Cells communicate with one another through the secretion and uptake of protein signaling molecules. The uptake of proteins into the cell 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 cell is achieved by exocytosis, in which molecules inside of the cell 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 extracellular 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.
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-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66, facilitating potential recovery from CNS damage (Fournier, A. E. et al. (2001) Nature 409:341-346).
The slit proteins are extracellular matrix proteins expressed by cells 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) Cell 96:795-806).
Lysosomes are the site of degradation of intracellular material during autophagy and of extracellular molecules following endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the trans-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 organelles and other intracellular components occurs.
Protein sorting by transport vesicles, such as the endosome, has important consequences for a variety of physiological processes including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled secretion of hormones and neurotransmitters (Rothman, J. E. and F. T. Wieland (1996) Science 272:227-234). In particular, 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).
Three classes of molecular motors—kinesins, dyneins and myosins—are involved in a variety of biological movements, such as mitosis, axoplasmic transport and secretion. Structurally, motor proteins consist of two functional parts: a motor domain that reversibly binds to the cytoskeleton and converts chemical energy into motion; and the rest of the molecule, often referred to as the tail, that interacts with cargo directly or through accessory light chains. These movements are required for the spatial organization of cytoplasm and, as a consequence, are crucial for cell division, embryonic development, and the formation of specialized areas of cytoplasm such as cilia and flagella. The ability of these proteins to transport a wide array of cargo is due, in part, to the fact that the tail domains are quite divergent from one another. This has allowed them to evolve into adaptors, linking themselves to cargo through interactions with receptor proteins on the cargo surface (Karcher, R. L. et al. (2002) TRENDS in Cell Biology Vol. 12 No. 1).
Kinesin is the most abundant motor in many cell types and is responsible for movement of a variety of different cargoes. The best characterized of kinesin receptors is kinectin, a receptor isolated as an endoplasmic-reticulum specific protein (Kumar, J. et al. (1995) Science 267, 1834-1837). Kinesin exists as a tetramer of two heavy chains, which contain the N-terminal motor domain and C-terminal tail, as well as two light chains, which bind to the heavy chain tail. Kinectin binds to the heavy chain of kinesin and is considered an ER-specific receptor for this motor protein. Interactions between motor proteins and corresponding receptors may be verified using a yeast two-hybrid system or co-immunoprecipitation assays.
Peroxisomes are organelles independent from the secretory pathway. They are the site of many peroxide-generating oxidative reactions in the cell. Peroxisomes are unique among eukaryotic organelles in that their size, number, and enzyme content vary depending upon organism, cell type, and metabolic needs (Waterham, H. R. and J. M. Cregg (1997) BioEssays 19:57-66). Genetic defects in peroxisome proteins which result in peroxisomal deficiencies have been linked to a number of human pathologies, including Zellweger syndrome, rhizomelic chonrodysplasia punctata, X-linked adrenoleukodystrophy, acyl-CoA oxidase deficiency, bifunctional enzyme deficiency, classical Refsum's disease, DHAP alkyl transferase deficiency, and acatalasemia (Moser, H. W. and A. B. Moser (1996) Ann. NY Acad. Sci. 804:427-441). In addition, Gartner, J. et al. (1991; Pediatr. Res. 29:141-146) found a 22 kDa integral membrane protein associated with lower density peroxisome-like subcellular fractions in patients with Zellweger syndrome.
Normal embryonic development and control of germ cell maturation is modulated by a number of secretory proteins which interact with their respective membrane-bound receptors. Cell fate during embryonic development is determined by members of the activin/TGF-β superfamily, cadherins, IGF-2, and other morphogens. In addition, proliferation, maturation, and redifferentiation of germ cell and reproductive tissues are regulated, for example, by IGF-2, inhibins, activins, and follistatins (Petraglia, F. (1997) Placenta 18:3-8; Mather, J. P. et al. (1997) Proc. Soc. Exp. Biol. Med. 215:209-222). Transforming growth factor beta (TGFβ) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFβ receptor and (TβR-II) phosphorylating type I TGFβ receptor (TβR-I). Signaling is initiated when the ligand binds to the TβR-II which is followed by recruitment of TβR-I into a heteromeric complex. Within the complex, TβR-II transphosphorylates and activates TβR-I kinase, which phosphorylates and activates downstream signaling components of the pathway. TβR-I-associated protein-1 (TRECAP-1), which distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor, has been associated with TGFβ signaling (Charng, M. J. et al. (1998) J. Biol. Chem. 273:9365-9368).
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).
The μ-opioid receptor (MOR) mediates the actions of analgesic agents including morphine, codeine, methadone, and fentanyl as well as heroin. MOR is functionally coupled to a G-protein-activated potassium channel (Mestek A. et al. (1995) J. Neurosci. 15:2396-2406). A variety of 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-hydroxytryptamine4 and 5-hydroxytryptamine7 (Pan, Y. X. et al. (1999) Mol. Pharm. 56:396403).
Peripheral and Anchored Membrane Proteins
Some membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins. Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups. Membrane localization 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.
Expression Profiling
Microarrays are analytical tools used in bioanalysis. A microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support. Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
One area in particular in which microarrays find use is in gene expression analysis. Array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
Breast Cancer
Breast cancer is the most frequently diagnosed type of cancer in American women and the second most frequent cause of cancer death. There are more than 180,000 new cases of breast cancer diagnosed each year, and the mortality rate for breast cancer approaches 10% of all deaths in females between the ages of 45-54 (K. Gish (1999) AWIS Magazine 28:7-10). The lifetime risk of an American woman developing breast cancer is 1 in 8, and one-third of women diagnosed with breast cancer die of the disease. However the survival rate based on early diagnosis of localized breast cancer is extremely high (97%), compared with the advanced stage of the disease in which the tumor has spread beyond the breast (22%). A number of risk factors have been identified, including hormonal and genetic factors. Current procedures for clinical breast examination are lacking in sensitivity and specificity, and efforts are underway to develop comprehensive gene expression profiles for breast cancer that may be used in conjunction with conventional screening methods to improve diagnosis and prognosis of this disease (Perou C M et al. (2000) Nature 406:747-752).
Breast cancer evolves through a multi-step process whereby premalignant mammary epithelial cells undergo a relatively defined sequence of events leading to tumor formation. An early event in tumor development is ductal hyperplasia. Cells undergoing rapid neoplastic growth gradually progress to invasive carcinoma and become metastatic to the lung, bone, and potentially other organs. Variables that may influence the process of tumor progression and malignant transformation include genetic factors, environmental factors, growth factors, and hormones.
Breast cancer is a genetic disease commonly caused by mutations in cellular disease. One genetic defect associated with breast cancer results in a loss of heterozygosity (LOH) at multiple loci such as p53, Rb, BRCA1, and BRCA2. Another genetic defect is gene amplification involving genes such as c-myc and c-erbB2 (Her2-neu gene). Steroid and growth factor pathways are also altered in breast cancer, notably the estrogen, progesterone, and epidermal growth factor (EGF) pathways. Mutations in two genes, BRCA1 and BRCA2, are known to greatly predispose a woman to breast cancer and may be passed on from parents to children (Gish, supra). However, this type of hereditary breast cancer accounts for only about 5% to 9% of breast cancers, while the vast majority of breast cancer is due to noninherited mutations that occur in breast epithelial cells.
A good deal is already known about the expression of specific genes associated with breast cancer. For example, the relationship between expression of epidermal growth factor (EGF) and its receptor, EGFR, to human mammary carcinoma has been particularly well studied. (See Khazaie et al., supra, and references cited therein for a review of this area.) Overexpression of EGFR, particularly coupled with down-regulation of the estrogen receptor, is a marker of poor prognosis in breast cancer patients. In addition, EGFR expression in breast tumor metastases is frequently elevated relative to the primary tumor, suggesting that EGFR is involved in tumor progression and metastasis. This is supported by accumulating evidence that EGF has effects on cell functions related to metastatic potential, such as cell motility, chemotaxis, secretion and differentiation. Changes in expression of other members of the erbB receptor family, of which EGFR is one, have also been implicated in breast cancer. The abundance of erbB receptors, such as HER-2/neu, HER-3, and HER-4, and their ligands in breast cancer points to their functional importance in the pathogenesis of the disease, and may therefore provide targets for therapy of the disease (Bacus, S S et al. (1994) Am J Clin Pathol 102:S13S24). Other known markers of breast cancer include a human secreted frizzled protein mRNA that is downregulated in breast tumors; the matrix G1a protein which is overexpressed is human breast carcinoma cells; Drg1 or RTP, a gene whose expression is diminished in colon, breast, and prostate tumors; maspin, a tumor suppressor gene downregulated in invasive breast carcinomas; and CaN19, a member of the S100 protein family, all of which are down regulated in mammary carcinoma cells relative to normal mammary epithelial cells (Zhou Z et al. (1998) Int J Cancer 78:95-99; Chen, L et al. (1990) Oncogene 5:1391-1395; Ulrix W et al (1999) FEBS Lett 455:23-26; Sager, R et al. (1996) Curr Top Microbiol Immunol 213:51-64; and Lee, S W et al. (1992) Proc Natl Acad Sci USA 89:2504-2508).
Cell lines derived from human mammary epithelial cells at various stages of breast cancer provide a useful model to study the process of malignant transformation and tumor progression as it has been shown that these cell lines retain many of the properties of their parental tumors for lengthy culture periods (Wistuba II et al. (1998) Clin Cancer Res 4:2931-2938). Such a model is particularly useful for comparing phenotypic and molecular characteristics of human mammary epithelial cells at various stages of malignant transformation.
Prostate Cancer
As with most tumors, prostate cancer develops through a multistage progression ultimately resulting in an aggressive tumor phenotype. The initial step in tumor progression involves the hyperproliferation of normal luminal and/or basal epithelial cells. Androgen responsive cells become hyperplastic and evolve into early-stage tumors. Although early-stage tumors are often androgen sensitive and respond to androgen ablation, a population of androgen independent cells evolve from the hyperplastic population. These cells represent a more advanced form of prostate tumor that may become invasive and potentially become metastatic to the bone, brain, or lung. A variety of genes may be differentially expressed during tumor progression. For example, loss of heterozygosity (LOH) is frequently observed on chromosome 8p in prostate cancer. Fluorescence in situ hybridization (FISH) revealed a deletion for at least 1 locus on 8p in 29 (69%) tumors, with a significantly higher frequency of the deletion on 8p21.2-p21.1 in advanced prostate cancer than in localized prostate cancer, implying that deletions on 8p22-p21.3 play an important role in tumor differentiation, while 8p21.2-p21.1 deletion plays a role in progression of prostate cancer (Oba, K. et al. (2001) Cancer Genet. Cytogenet. 124: 20-26). As with breast cancer, there is a need for diagnostic and therapeutic agents that will improve treatment options for prostate cancer patients that can be fulfilled by the use of microarray expression analysis.
Colon Cancer
While soft tissue sarcomas are relatively rare, more than 50% of new patients diagnosed with the disease will die from it. The molecular pathways leading to the development of sarcomas are relatively unknown, due to the rarity of the disease and variation in pathology. Colon cancer evolves through a multi-step process whereby pre-malignant colonocytes undergo a relatively defined sequence of events leading to tumor formation. Several factors participate in the process of tumor progression and malignant transformation including genetic factors, mutations, and selection.
To understand the nature of gene alterations in colorectal cancer, a number of studies have focused on the inherited syndromes. The first, Familial Adenomatous Polyposis (FAP), is caused by mutations in the Adenomatous Polyposis Coli gene (APC), resulting in truncated or inactive forms of the protein. This tumor suppressor gene has been mapped to chromosome 5q. The second known inherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC), which is caused by mutations in mismatch repair genes.
Although hereditary colon cancer syndromes occur in a small percentage of the population, and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be applied broadly. For instance, somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in disease progression. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer. Less is understood about downstream targets of these mutations and the role they may play in cancer development and progression.
Lung Cancer
Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred. Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and liver. The decision to treat with surgery, radiation therapy, or chemotherapy is made on the basis of tumor histology, response to growth factors or hormones, and sensitivity to inhibitors or drugs. With current treatments, most patients die within one year of diagnosis. Earlier diagnosis and a systematic approach to identification, staging, and treatment of lung cancer could positively affect patient outcome.
Lung cancers progress through a series of morphologically distinct stages from hyperplasia to invasive carcinoma. Malignant lung cancers are divided into two groups comprising four histopathological classes. The Non Small Cell Lung Carcinoma (NSCLC) group includes squamous cell carcinomas, adenocarcinomas, and large cell carcinomas and accounts for about 70% of all lung cancer cases. Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands. Squamous cell carcinomas typically arise in proximal airways. The histogenesis of squamous cell carcinomas may be related to chronic inflammation and injury to the bronchial epithelium, leading to squamous metaplasia. The Small Cell Lung Carcinoma (SCLC) group accounts for about 20% of lung cancer cases. SCLCs typically arise in proximal airways and exhibit a number of paraneoplastic syndromes including inappropriate production of adrenocorticotropin and anti-diuretic hormone.
Lung cancer cells accumulate numerous genetic lesions, many of which are associated with cytologically visible chromosomal aberrations. The high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one of the earliest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common. Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2.
Genes differentially regulated in lung cancer have been identified by a variety of methods. Using mRNA differential display technology, Manda et al. (1999; Genomics 51:5-14) identified five genes differentially expressed in lung cancer cell lines compared to normal bronchial epithelial cells. Among the known genes, pulmonary surfactant apoprotein A and alpha 2 macroglobulin were down regulated whereas nm23H1 was upregulated. Petersen et al. (2000; Int J. Cancer, 86:512-517) used suppression subtractive hybridization to identify 552 clones differentially expressed in lung tumor derived cell lines, 205 of which represented known genes. Among the known genes, thrombospondin-1, fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentially expressed in lung cancers. Wang et al. (2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentially overexpresssed in squamous cell carcinoma compared with normal lung epithelium. Among the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 and cytokeratin 13.
Ovarian Cancer
Ovarian cancer is the leading cause of death from a gynecologic cancer. The majority of ovarian cancers are derived from epithelial cells, and 70% of patients with epithelial ovarian cancers present with late-stage disease. As a result, the long-term survival rates for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate. Genetic variations involved in ovarian cancer development include mutation of p53 and microsatellite instability. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.
Immune Response
Tumor cells stimulate the formation of stroma that secretes various mediators, such as growth factors, cytokines, and proteases, all of which are pivotal for tumor growth. One such cytokine, interferon gamma (IFN-γ) induces growth arrest in normal human mammary epithelial cells by establishing a block during mid-G1 phase. EFN-γ inhibits the kinase activities of cdk2, cdk4 and cdk6 within 24 h of treatment. IFN-γ-mediated growth inhibition requires signal transducers and activators of transcrip-tion (STAT)-1 activation and may require induction of the cyclin-dependent kinase inhibitor p21. IFN-γ, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.
IFN-γ, also known as Type U interferon or immune interferon, is produced primarily by T-lympho-cytes and natural killer cells. IFN-γ was originally characterized based on its antiviral characteristics. The protein exhibits antiproliferative, immunoregulatory and proinflammatory activities and is thus important in host defense mechanisms. IFN-γ induces the production of cytokines, upregulates the expression of class I and II MHC antigens, Fc receptor, and leukocyte adhesion molecules. It modulates macrophage effector functions, influences isotype switching and potentiates the secretion of immunoglobulins by B cells. IFN-γ also augments TH1 cell expansion and may be required for TH1 cell differentiation. The IFN-γ receptor has been cloned and characterized, and is structurally related to the recently cloned IL-10 receptor. It is present on almost all cell types except mature erythrocytes.
Human Peripheral Blood Mononuclear Cells (PBMCs)
Human peripheral blood mononuclear cells (PBMCs) represent the major cellular components of the immune system. PBMCs contain about 52% lymphocytes (12% B lymphocytes, 40% T lymphocytes {25% CD4+ and 15% CD8+}), 20% NK cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells. The proportions, as well as the biology of these cellular components tend to vary slightly between healthy individuals, depending on factors such as age, gender, past medical history, and genetic background. These cells are responsible for immune responses and fighting infections, and thus represent a crucial system designed to maintain human health. Understanding the factors that activate and maintain this system requires analysis of cellular responses to stimuli, examining differences in the gene expression patterns of the various cell types, and determination of potential therapeutic targets that could be exploited for bolstering the immune response in individuals with deficiencies in this system. Microarray expression analysis can play an important role in achieving these goals.
Leukocytes comprise lymphocytes, granulocytes, and monocytes. Lymphocytes include T and B cells, which specifically recognize and respond to foreign pathogens. T cells fight viral infections and activate other leukocytes, while B cells secrete antibodies that neutralize bacteria and other microbes. Lymphoblast cell lines can be used to study signaling in human B cells and identify factors produced by those cells. An example is the RPMI 6666 B cell lymphoblast cell line derived from the peripheral blood of a male donor with Hodgkin's disease, which produces immunoglobulins and presents cell-associated Epstein-Barr virus (EBV) particles. Granulocytes and monocytes are primarily migratory, phagocytic cells that exit the bloodstream to fight infection in tissues. Monocytes, which are derived from immature promonocytes, further differentiate into macrophages that engulf and digest microorganisms and damaged or dead cells. Monocytes and macrophages modulate the immune response by secreting signaling molecules such as growth factors and cytokines. Tumor necrosis factor-α (TNF-α), for example, is a macrophage-secreted protein with anti-tumor and anti-viral activity. In addition, monocytes and macrophages are recruited to sites of infection and inflammation by signaling proteins secreted by other leukocytes. The differentiation of the monocyte blood cell lineage can be studied in vitro using cultured cell lines. For example, THP-1 is a human promonocyte cell line that can be activated by treatment with both phorbol ester such as phorbol myristate acetate (PMA) and ionomycin, a calcium ionophore that permits the entry of calcium in the cell, which increases the intracellular concentration of calcium. PMA is a broad activator of the protein kinase C-dependent pathways. The combination of PMA and ionomycin activates two of the major signaling pathways used by mammalian cells to interact with their environment. In T cells, the combination of PMA and ionomycin mimics the type of secondary signaling events elicited during optimal B cell activation. THP-1 can also be activated by treatment with both phorbol ester such as phorbol myristate acetate (PMA), and lipopolysaccharide (LPS). In another example, K-562 is a myeloid precursor cell line derived from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia. The K-562 cell line has been extensively used to study differentiation of the erythrocytic, granulocytic, and monocytic lineage in humans. In addition, the K-562 cell line is widely used as an extremely sensitive target to the cytolytic activity of human natural killer cells in vitro. Another cell line, Jurkat, is an acute T cell leukemia cell line that grows actively in the absence of external stimuli and has been extensively used to study signaling in human T cells. In T cells, the combination of PMA and ionomycin mimics the type of secondary signaling events elicited during optimal B cell activation.
Monocytes are involved in the initiation and maintenance of inflammatory immune responses. The outer membrane of gram-negative bacteria expresses lipopolysaccharide (LPS) complexes called endotoxins. Toxicity is associated with the lipid component (Lipid A) of LPS, and immunogenicity is associated with the polysaccharide components of LPS. LPS elicits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections. For the most part, endotoxins remain associated with the cell wall until the bacteria disintegrate. LPS released into the bloodstream by lysing gram-negative bacteria is first bound by certain plasma proteins identified as LPS-binding proteins. The LPS-binding protein complex interacts with CD14 receptors on monocytes, macrophages, B cells, and other types of receptors on endothelial cells. Activation of human B cells with LPS results in mitogenesis as well as immunoglobulin synthesis. In monocytes and macrophages three types of events are triggered during their interaction with LPS: 1) Production of cytokines, including IL-1, IL6, IL-8, TNF-α, and platelet-activating factor, which stimulate production of prostaglandins and leukotrienes that mediate inflammation and septic shock; 2) Activation of the complement cascade; and 3) Activation of the coagulation cascade. Thus, LPS stimulation of lymphocytic cells can be used to examine changes in gene expression that occur in response to infectious stimuli, and can be analyzed by microarray expression analysis.
Functional interaction of the cell types involved in immune responses involves transfer of signals via soluble messenger molecules known as cytokines. Both hematopoietic cells and non-hematopoietic cells produce cytokines, which stimulate the activation, differentiation and proliferation of T cells, B cells, macrophages, and granulocytes during an active immune response. Cytokines bind to specific receptors expressed on cellular membranes and transduce a signal through the cell. Depending on the type of cytokine and the cell to which it binds, this signal initiates activation, differentiation, growth, and/or apoptosis. IL-10 is a pleiotrophic cytoline that can exert either immunostimulatory or immunosupressive effects on a variety of cell types. IL-10 suppresses the accessory cell function of macrophages and dendritic cells in part by downregulating class II MHC expression, preventing antigen presentation. IL-10 directly suppresses macrophage and monocyte production of inflammatory molecules such as tumor necrosis factor alpha (TNF-α), EL-1α, and IL6, while maintaining production of transforming growth factor beta (TGF-β) which curbs Th1 responses. In contrast to its suppressive activities on T cells and macrophages, IL-10 boosts proliferation and differentiation of activated B cells into plasma cells.
Staphylococcal exotoxins specifically activate human T cells, expressing an appropriate TCR-Vbeta chain. Although polyclonal in nature, T cells activated by Staphylococcal exotoxins require antigen presenting cells (APCs) to present the exotoxin molecules to the T cells and deliver the costimulatory signals required for optimum T cell activation. Although, Staphylococcal exotoxins must be presented to T cells by APCs, these molecules are not required to be processed by APC. Indeed, Staphylococcal exotoxins directly bind to a non-polymorphic portion of the human MHC class II molecules, bypassing the need for capture, cleavage, and binding of the peptides to the polymorphic antigenic groove of the MHC class II molecules.
There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders.
SUMMARY OF THE INVENTION
The present invention relates to novel receptors and membrane-associated proteins, referred to collectively as “REMAPs.” The present invention relates to isolated polypeptides encoding REMAPs, REMAP variants, REMAP fragments, REMAP fusion polypeptides, polynucleotides encoding such polypeptides and methods of producing such polypeptides. One exemplary REMAP, termed REMAP-22, corresponds to SEQ ID NO: 22.
In some embodiments, an isolated REMAP polypeptide may be biologically active. For example, an isolated REMAP polypeptide may be capable of binding a ligand, such as CD40L. Such an isolated polypeptide may include the amino acid of SEQ ID NO: 22 or an amino acid sequence that is 90% identical to SEQ ID NO: 22. In some embodiments, the isolated polypeptide may share one or more antigenic determinants with a polypeptide encoded by SEQ ID NO: 22. In still other embodiments, the isolated polypeptide may be biologically active, e.g., may bind CD40L, and share one or more antigenic determinants with the polypeptide encoded by SEQ ID NO: 22.
In further embodiments, the isolated polypeptide may be encoded by (i) a polynucleotide such as SEQ ID NO: 60, (ii) an RNA equivalent of SEQ ID NO: 60, or (iii) a polynucleotide which varies from (i) or (ii) due to the genetic code. In some embodiments, the polynucleotide may be operably linked to a regulatory or expression sequence, such as a promoter sequence.
In further embodiments, an isolated polypeptide may include a REMAP fragment or a biologically active REMAP fragment. In still other embodiments, the isolated polypeptide may comprise a fusion protein that includes a portion of a REMAP polypeptide. For example, in some embodiments, an isolated polypeptide may include amino acids 188-237 of SEQ ID NO:22. In other embodiments, an isolated peptide may include an amino acid sequence of SEQ ID NO: 22 fused to a heterologous fusion polypeptide.
The polypeptides of the present invention may be isolated and produced by any number of methods known in the art. For example, such polypeptides may be produced recombinantly. In some embodiments, such polypeptides may be produced by culturing cells under conditions suitable for expression of the polypeptide, and recovering the polypeptide so expressed. In some embodiments, cells may be transformed with a polynucleotide including, for example, SEQ ID NO: 60, an RNA equivalent of SEQ ID NO: 60 or a polynucleotide which varies from SEQ ID NO: 60 due to the genetic code; in some embodiments, the polynucleotides may be operably linked to a promoter sequence.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention. The probability scores for the matches between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
Table 5 shows representative cDNA libraries for polynucleotide embodiments.
Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.
Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with allele frequencies in different human populations.


DESCRIPTION OF THE INVENTION
Before the present proteins, nucleic acids, and methods are described, it is understood that embodiments of the invention are not limited to the particular machines, instruments, materials, and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with various embodiments of the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Definitions
“REMAP” refers to the amino acid sequences of substantially 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.
The term “agonist” refers to a molecule which intensifies or mimics the biological activity of REMAP. Agonists may include proteins, nucleic acids, carbohydrates, small 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 “allelic variant” is an alternative form of the gene encoding REMAP Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
“Altered” nucleic acid sequences encoding 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 allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide 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 functionally equivalent REMAP. Deliberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of REMAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms “amino acid” and “amino acid sequence” can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
“Amplification” relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies well known in the art.
The term “antagonist” refers to a molecule which inhibits or attenuates the biological activity of REMAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small 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.
The term “antibody” refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab′)2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind REMAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term “antigenic determinant” refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term “aptamer” refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by EXponential Enrichment), described in U.S. Pat. No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups (e.g., the 2′-OH group of a ribonucleotide may be replaced by 2′-F or 2′-NH2), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).
The term “intramer” refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
The term “antisense” refers to any composition capable of base-pairing with the “sense” (coding) strand of a polynucleotide having a specific nucleic acid sequence. Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation “negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
The term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, “immunologically active” or “immunogenic” refers to the capability of the natural, recombinant, or synthetic REMAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
“Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5′-AGT-3′ pairs with its complement, 3′-TCA-5′.
A “composition comprising a given polynucleotide” and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotides encoding REMAP or fragments of REMAP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
“Consensus sequence” refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.) in the 5′ and/or the 3′ direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (Accelrys,. Burlington Mass.) or Phrap (University of Washington, Seattle Wash.). Some sequences have been both extended and assembled to produce the consensus sequence.
“Conservative amino acid substitutions” are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.














Original
Conservative



Residue
Substitution









Ala
Gly, Ser



Arg
His, Lys



Asn
Asp, Gln, His



Asp
Asn, Glu



Cys
Ala, Ser



Gln
Asn, Glu, His



Glu
Asp, Gln, His



Gly
Ala



His
Asn, Arg, Gln, Glu



Ile
Leu, Val



Leu
Ile, Val



Lys
Arg, Gln, Glu



Met
Leu, Ile



Phe
His, Met, Leu, Trp, Tyr



Ser
Cys, Thr



Thr
Ser, Val



Trp
Phe, Tyr



Tyr
His, Phe, Trp



Val
Ile, Leu, Thr










Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
“Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
“Exon shuffling” refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
A “fragment” is a unique portion of REMAP or a polynucleotide encoding REMAP which can be identical in sequence to, but shorter in length than, the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID NO:39-76 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:39-76, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:39-76 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:39-76 from related polynucleotides. The precise length of a fragment of SEQ ID NO:39-76 and the region of SEQ ID NO:39-76 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:1-38 is encoded by a fragment of SEQ ID NO:39-76. A fragment of SEQ ID NO:1-38 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-38. For example, a fragment of SEQ ID NO:1-38 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-38. The precise length of a fragment of SEQ ID NO:1-38 and the region of SEQ ID NO:1-38 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
A “full length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
“Homology” refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described in Higgins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins, D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The “weighted” residue weight table is selected as the default.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, Md., and on the NCBI World Wide Web site available on the Internet. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively on the Internet via the NCBI World Wide Web site as well. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: −2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases “percent similarity” and “% similarity,” as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix is selected as the default residue weight table.
Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties
Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
“Human artificial chromosomes” (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
“Hybridization” refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the “washing” step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68° C. in the presence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/ml sheared, denaturedsalmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm 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 Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D. W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor N.Y., ch. 9).
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68° C. in the presence of about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSC concentration may be varied from about 0.1 to 2×SSC, with SDS being present at about 0.1%. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term “hybridization complex” refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C0t or R0t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words “insertion” and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
“Immune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An “immunogenic fragment” is a polypeptide or oligopeptide fragment of REMAP which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of REMAP which is useful in any of the antibody production methods disclosed herein or known in the art.
The term “microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
The terms “element” and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microarray.
The term “modulate” refers to a change in the activity of 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.
The phrases “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
“Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
“Peptide nucleic acid” (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
“Post-translational modification” of an REMAP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of REMAP.
“Probe” refers to nucleic acids encoding REMAP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acids. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. “Primers” are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
Methods for preparing and using probes and primers are described in, for example, Sambrook, J. and D. W. Russell (2001; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor N.Y.), Ausubel, F. M. et al. (1999; Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, New York N.Y.), and Innis, M. et al. (1990; PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego Calif.). PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge Mass.).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas Tex.) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge Mass.) allows the user to input a “mispriming library,” in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A “recombinant nucleic acid” is a nucleic acid that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and Russell (supra). The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
“Reporter molecules” are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
An “RNA equivalent,” in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term “sample” is used in its broadest sense. A sample suspected of containing REMAP, nucleic acids encoding REMAP, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms “specific binding” and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope “A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term “substantially purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturally associated.
A “substitution” refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
“Substrate” refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
“Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A “transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. In another embodiment, the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872). The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
A “variant” of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an “allelic” (as defined above), “splice,” “species,” or “polymorphic” variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotides that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May 7, 1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%,.at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.
The Invention
Various embodiments of the invention include new human receptors and membrane-associated proteins (REMAP), the polynucleotides encoding REMAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders.
Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown. Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention. The full length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability scores for the matches between each polypeptide and its homolog(s). Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, all of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accelrys, Burlington Mass.). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are receptors and membrane-associated proteins. For example, SEQ ID NO:6 is 100% identical, from residue M1 to residue S208, to human tumor necrosis factor receptor 1 (GenBank ID g339750) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 4.5e-119, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:6 also has homology to proteins that are localized to the plasma membrane, function as receptors, and are tumor necrosis factor receptors, type 1, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:6 also contains a TNF-receptor internal cysteine rich domain, a TNFR/NGFR cysteine-rich region domain, and a tumor necrosis factor receptor/nerve domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM, INCY, and SMART databases of conserved protein family domains. (See Table 3.) Data from BUMPS, MOTIFS, and other BLAST analyses provide further corroborative evidence that SEQ ID NO:6 is a type 1 tumor necrosis factor receptor. In another example, SEQ ID NO:8 is 99% identical, from residue M1 to residue A272, to human gastrin receptor (GenBank ID g406076) as determined by the Basic Local Alignment Search Tool (BLAST). The BLAST probability score is 3.2e-206, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:8 also has homology to cholecystolcnin B (gastrin) receptors that are localized to the basolateral plasma membrane, as determined by BLAST analysis using the PROTEOME database. These receptors are G protein-coupled receptors. They are involved in stimulating phospholipase C and intracellular calcium flux, regulating digestion, gastric mucosal cell proliferation, and opioidergic and dopaminergic signaling. The human CCKBR variant is associated with colorectal cancer. SEQ ID NO:8 also contains a 7 transmembrane receptor (rhodopsin family) domain as determined by searching for statistically significant matches in the hidden Markov model (HM)-based PFAM database of conserved protein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFHLESCAN analyses provide further corroborative evidence that SEQ ID NO:8 is a G-protein coupled gastrin receptor. In another example, SEQ ID NO:22 is 99% identical, from residue Ml to residue G187, to human CDw40 (GenBank ID g29851) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.7e-107, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:22 is also a member of the tumor necrosis factor receptor superfamily, binds the ligand CD40L, and is expressed specifically in B lymphocytes. It also has a role in B lymphocyte maturation, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:22 also contains a TNF-receptor internal cysteine rich, Tumor necrosis factor receptor/nerve, and TNFR/NGFR cysteine-rich region domains as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM, SMART, and INCY databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:22 is a CDw40. In another example, SEQ ID NO:27 is 100% identical, from residue M1 to residue M224, to Homo sapiens ocular melanoma-associated antigen (GenBank ID g246539) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.8e-115, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:27 also has homology to proteins that are members of the tetraspanning superfamily and specifically CD63 antigen, form complexes with integrins and MHC class II molecules, and act to limit the invasion and progression of melanoma, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:27 also contains a tetraspanin family domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:27 is a transmembrane 4 family or tetraspanning family member. In another example, SEQ ID NO:31 is 85% identical, from residue F6 to residue I786, to human CD97 (GenBank ID g1685051) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:31 also has homology to proteins that are localized to the plasma membrane and are members of the EGF TM7 family of class II seven-span transmembrane receptors, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:31 also contains a 7 transmembrane receptor (secretin family) domain, an EGF-like domain, a G-protein coupled receptor proteolytic site domain, and a latrophilin/CL-1-like GPS domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and TMHMMER analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:31 is a CD97 antigen. In another example, SEQ ID NO:38 is 99% identical, from residue M1 to residue K792, to H. sapiens CD97 (GenBank ID g1685051) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:38 also has homology to proteins that are localized to the plasma membrane, are receptors for the complement cascade regulator, CD55 (Daf1), may play a role in lymphocyte activation, and are CD97 antigens as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:38 also contains a 7 transmembrane receptor domain, an EGF-like domain, and a Latrophilin/CL-1-like GPS domain, as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:38 is a CD97 antigen. SEQ ID NO:1-5, SEQ ID NO:7, SEQ ID NO:9-21, SEQ ID NO:23-26, SEQ ID NO:28-30, and SEQ ID NO:32-37 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-38 are described in Table 7.
As shown in Table 4, the full length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start (5′) and stop (3′) positions of the cDNA and/or genomic sequences used to assemble the full length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO:39-76 or that distinguish between SEQ ID NO:39-76 and related polynucleotides.
The polynucleotide fragments described in Column 2 of Table 4 may refer specifically, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries. Alternatively, the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides. In addition, the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation “ENST”). Alternatively, the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence. Records Database (i.e., those sequences including the designation “NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation “NP”). Alternatively, the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an “exon stitching” algorithm. For example, a polynucleotide sequence identified as FL_XXXXXX_N1—N2—YYYYY_N3—N4 represents a “stitched” sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N1,2,3 . . . , if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an “exon-stretching” algorithm. For example, a polynucleotide sequence identified as FL_XXXXXX_gAAAAA_gBBBBB—1_N is a “stretched” sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the “exon-stretching” algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the “exon-stretching” algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V).











Prefix
Type of analysis and/or examples of programs







GNN, GFG,
Exon prediction from genomic sequences using, for


ENST
example, GENSCAN (Stanford University, CA, USA)



or FGENES (Computer Genomics Group, The Sanger



Centre, Cambridge, UK).


GBI
Hand-edited analysis of genomic sequences.


FL
Stitched or stretched genomic sequences (see Example V).


INCY
Full length transcript and exon prediction from mapping of



EST sequences to the genome. Genomic location



and EST composition data are combined to predict



the exons and resulting transcript.









In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length polynucleotides which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with allele frequencies in different human populations. Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the corresponding Incyte project identification number (PID) for polynucleotides of the invention. Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID). Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full-length polynucleotide sequence (CB1 SNP). Column 7 shows the allele found in the EST sequence. Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST. Columns 11-14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of allele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.
The invention also encompasses REMAP variants. Various embodiments of REMAP variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the REMAP amino acid sequence, and can contain at least one functional or structural characteristic of REMAP.
Various embodiments also encompass polynucleotides which encode REMAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:39-76, which encodes REMAP. The polynucleotide sequences of SEQ ID NO:39-76, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses variants of a polynucleotide encoding REMAP. In particular, such a variant polynucleotide will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding REMAP. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:39-76 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:39-76. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of REMAP.
In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding REMAP. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding REMAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing. A splice variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding REMAP over its entire length; however, portions of the splice variant will have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding REMAP. For example, a polynucleotide comprising a sequence of SEQ ID NO:69 and a polynucleotide comprising a sequence of SEQ ID NO:76 are splice variants of each other. Any one of the splice variants described above can encode a polypeptide which contains at least one functional or structural characteristic of REMAP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding REMAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring REMAP, and all such variations are to be considered as being specifically disclosed.
Although polynucleotides which encode REMAP and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring REMAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding REMAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding REMAP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of polynucleotides which encode REMAP and REMAP derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic polynucleotide may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a polynucleotide encoding REMAP or any fragment thereof.
Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO:39-76 and fragments thereof, under various conditions of stringency (Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in “Definitions.”
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway N.J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad Calif.). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853).
The nucleic acids encoding REMAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68° C. to 72° C.
When screening for full length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5′ regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotides or fragments thereof which encode REMAP may be cloned in recombinant DNA molecules that direct expression of REMAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or a functionally equivalent polypeptides may be produced and used to express REMAP.
The polynucleotides of the invention can be engineered using methods generally 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 oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of REMAP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through “artificial” breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, polynucleotides encoding REMAP may be synthesized, in whole or in part, using one or more chemical methods well known in the art (Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232). Alternatively, REMAP itself or a fragment thereof may be synthesized using chemical methods known in the art. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH Freeman, New York N.Y., pp. 55-60; Roberge, J. Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of 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 naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
In order to express a biologically active REMAP, the polynucleotides 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 polynucleotides encoding REMAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding REMAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding REMAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
Methods which are well known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding REMAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and Russell, supra, ch. 14, and 8; Ausubel et al., supra, ch. 1, 3, and 15).
A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding 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 cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook and Russell, supra; Ausubel et al., supra; Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technology l (1992) McGraw Hill, New York N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355). Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Buller, R. M. et al. (1985) Nature 317:813-815; McGregor, D. P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I. M. and N. Somia (1997) Nature 389:239-242). The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding REMAP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding REMAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding REMAP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509). When large quantities of 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 may be 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. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G. A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C. A. et al. (1994) Bio/Technology 12:181-184).
Plant systems may also be used for expression of REMAP. Transcription of polynucleotides 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. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191-196).
In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, polynucleotides encoding REMAP may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses REMAP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355).
For long term production of recombinant proteins in mammalian systems, stable expression of REMAP in cell lines is preferred. For example, polynucleotides encoding REMAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr cells, respectively (Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites (Hartan, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β-glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131).
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding REMAP is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding REMAP can be identified by the absence of marker gene function. Alternatively, 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 usually indicates expression of the tandem gene as well.
In general, host cells that contain the polynucleotide 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 amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of 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 cell sorting (FACS). 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. These and other assays are well known in the art (Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York N.Y.; Pound, J. D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).
A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding REMAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, polynucleotides encoding REMAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes i t vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Biosciences, Promega (Madison Wis.), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
Host cells transformed with polynucleotides encoding REMAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode REMAP may be designed to contain signal sequences which direct secretion of REMAP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding REMAP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric REMAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of REMAP activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-nyc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the 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 et al. (supra, ch. 10 and 16). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In another embodiment, synthesis of radiolabeled REMAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
REMAP, fragments of REMAP, or variants of REMAP may be used to screen for compounds that specifically bind to REMAP. One or more test compounds may be screened for specific binding to REMAP. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to REMAP. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
In related embodiments, variants of REMAP can be used to screen for binding of test compounds, such as antibodies, to REMAP, a variant of REMAP, or a combination of REMAP and/or one or more variants REMAP. In an embodiment, a variant of REMAP can be used to screen for compounds that bind to a variant of REMAP, but not to REMAP having the exact sequence of a sequence of SEQ ID NO:1-38. REMAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to REMAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
In an embodiment, a compound identified in a screen for specific binding to REMAP can be 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 (Coligan, J. E. et al. (1991) Current Protocols in Immunology 1(2):Chapter 5). In another embodiment, the compound thus identified can be a natural ligand of a receptor REMAP (Howard, A. D. et al. (2001) Trends Pharmacol. Sci. 22: 132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
In other embodiments, a compound identified in a screen for specific binding to REMAP can be closely related to the natural receptor to which REMAP binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the ligand binding site or binding pocket For example, the compound may be a receptor for REMAP which is capable of propagating a signal, or a decoy receptor for REMAP which is not capable of propagating a signal (Ashkenazi, A. and V. M. Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336). The compound can be rationally designed using known techniques. Examples of such techniques include those used to construct the compound etanercept (ENBREL; Amgen Inc., Thousand Oaks Calif.), which is efficacious for treating rheumatoid arthritis in humans. Etanercept is an engineered p75 tumor necrosis factor (CNE) receptor dimer linked to the Fc portion of human IgG1 (Taylor, P. C. et al. (2001) Curr. Opin. Immunol. 13:611-616).
In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to REMAP, fragments of REMAP, or variants of REMAP. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of REMAP. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of REMAP. In another embodiment, an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of REMAP.
In an embodiment, anticalins can be screened for specific binding to REMAP, fragments of REMAP, or variants of REMAP. Anticalins are,ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit REMAP involves producing appropriate cells which express REMAP, either as a secreted protein or on the cell membrane. Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing REMAP or cell 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. For example, the assay may comprise the steps of combining at least one test compound with REMAP, either in solution or affixed to a solid support, and detecting the binding of REMAP to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors. Examples of such assays include radio-labeling assays such as those described in U.S. Pat. No. 5,914,236 and U.S. Pat. No. 6,372,724. In a related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural ligands (Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1:25-30). In another related embodiment, one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B. C. and J. A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H. B. et al. (1991) J. Biol. Chem. 266:10982-10988).
REMAP, fragments of REMAP, or variants of REMAP may be used to screen for compounds that modulate the activity of REMAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, 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. Alternatively, a test compound is combined with an in vitro or cell-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 plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding REMAP or their mammalian homologs may be “knocked out” in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J. D. (1996) Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding REMAP may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).
Polynucleotides encoding REMAP can also be used to create “knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding REMAP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress 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
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of REMAP and receptors and membrane-associated proteins. In addition, examples of tissues expressing REMAP can be found in Table 6 and can also be found in Example XI. Therefore, REMAP appears to play a role in cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders. In the treatment of disorders associated with increased REMAP expression or activity, it is desirable to decrease the expression or activity of REMAP. In the treatment of disorders associated with decreased REMAP expression or activity, it is desirable to increase the expression or activity of REMAP.
Therefore, in one embodiment, 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. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCrD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis, phenylketonuria, pseudovitamin D-deficiency rickets, disorders of carbohydrate metabolism such as congenital type II dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism, disorders of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's disease, hyperlipidemia, hyperlipemia, and lipid myopathies, and disorders of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX diabetes; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; and an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma, a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism, a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer' s disease, a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia), a pancreatic disorder such as Type I or Type II diabetes mellitus and associated complications, a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 α-reductase, and gynecomastia.
In another embodiment, a vector capable of expressing 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 including, but not limited to, those described above.
In a further embodiment, a composition comprising a substantially 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.
In still another embodiment, 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 listed above.
In a further embodiment, 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 cell proliferative, autoimmune/inflammatory, neurological, metabolic, developmental, and endocrine disorders described above. In one aspect, an antibody which specifically binds REMAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express REMAP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding REMAP may be 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.
In other embodiments, any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of REMAP may be produced using methods which are generally known in the art. In particular, purified REMAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind REMAP. Antibodies to REMAP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeutically. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have application in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with REMAP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to REMAP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are substantially identical to a portion of the amino acid sequence of the natural protein. Short stretches of REMAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to REMAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120).
In addition, techniques developed for the production of “chimeric antibodies,” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce REMAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
Antibody fragments which contain specific binding sites for REMAP may also be generated. For example, such 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. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W. D. et al. (1989) Science 246:1275-1281).
Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between REMAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering REMAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for REMAP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of REMAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple REMAP epitopes, represents the average affinity, or avidity, of the antibodies for REMAP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific fdr a particular REMAP epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 1012 L/mole are preferred for use in immunoassays in which the REMAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 107 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 D.C.; Liddell, J. E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York N.Y.).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of REMAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (Catty, supra; Coligan et al., supra).
In another embodiment of the invention, polynucleotides encoding REMAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding REMAP. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding REMAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa N.J.).
In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J. E. et al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, K. J. et al. (1995) 9:1288-1296). Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (Miller, A. D. (1990) Blood 76:271; Ausubel et al., supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art (Rossi, J. J. (1995) Br. Med. Bull. 51:217-225; Boado, R. J. et al. (1998) J. Pharm. Sci. 87:1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res. 25:2730-2736).
In another embodiment of the invention, polynucleotides encoding 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)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410; Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HI) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in REMAP expression or regulation causes disease, the expression of REMAP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in REMAP are treated by constructing mammalian expression vectors encoding REMAP and introducing these vectors by mechanical means into REMAP-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445450).
Expression vectors that may be effective for the expression of REMAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). REMAP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V. and H. M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding REMAP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSPECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F. L. and A. J. Eb (1973) Virology 52:456467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to 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 (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, L. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol. 61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 to Rigg (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”) discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding REMAP to cells which have one or more genetic abnormalities with respect to the expression of REMAP. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M. E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano (“Adenovirus vectors for gene therapy”), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P. A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I. M. and N. Somia (1997; Nature 18:389:239-242).
In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding REMAP to target cells which have one or more genetic abnormalities with respect to the expression of REMAP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing REMAP to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Pat. No. 5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”), which is hereby incorporated by reference. U.S. Pat. No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W. F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding REMAP to target cells. The biology of the prototypic alphavirus, Senilti Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for 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 cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S. A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of REMAP into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177). A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding REMAP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding REMAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
In other embodiments of the invention, the expression of one or more selected polynucleotides of the present invention can be altered, inhibited, decreased, or silenced using RNA interference (RNAi) or post-transcriptional gene silencing (PTGS) methods known in the art. RNAi is a post-transcriptional mode of gene silencing in which double-stranded RNA (dsRNA) introduced into a targeted cell specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces the expression of the targeted gene. PTGS can also be accomplished by use of DNA or DNA fragments as well. RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T. (2000; Nature 404:804-808). PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
RNAi can be induced in mammalian cells by the use of small interfering RNA also known as siRNA. SiRNA are shorter segments of dsRNA (typically about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease. SiRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNA& with 2 nucleotide 3′ overhangs. The use of siRNA for inducing RNAi in mammalian cells is described by Elbashir, S. M. et al. (2001; Nature 411:494-498).
SiRNA can either be generated indirectly by introduction of dsRNA into the targeted cell, or directly by mammalian transfection methods and agents described herein or known in the art. (such as liposome-mediated transfection, viral vector methods, or other polynucleotide delivery/introductory methods). Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occurrence of each nucleotide and the 3′ adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred. Regions to be avoided for target siRNA sites include the 5′ and 3′ untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex. The selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be eliminated from consideration. The selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commercially available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin Tex.).
In alternative embodiments, long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA. This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T. R. et al. (2002) Science 296:550-553; and Paddison, P. J. et al. (2002) Genes Dev. 16:948-958). In these and related embodiments, shRNAs can be delivered to target cells using expression vectors known in the art. An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to the target tissue, shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene-specific silencing.
In various embodiments, the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis. Expression levels of the mRNA of a targeted gene, can be determined by northern analysis methods using, for example, the NORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein. Expression levels of the protein encoded by the targeted gene can be determined by Western analysis using standard techniques known in the art.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding REMAP. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased REMAP expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding REMAP may be therapeutically useful, and in the treatment of disorders associated with decreased REMAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding REMAP may be therapeutically useful.
In various embodiments, one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding REMAP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding REMAP are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding 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. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. et al. (2000) U.S. Pat. No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C. K. et al. (1997) Nat. Biotechnol. 15:462-466).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa.). Such compositions may consist of REMAP, antibodies to REMAP, and mimetics, agonists, antagonists, or inhibitors of REMAP.
In various embodiments, the compositions described herein, such as pharmaceutical compositions, may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No. 5,997,848). Pulmonary delivery allows administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising REMAP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, REMAP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S. R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example REMAP or fragments thereof, antibodies of REMAP, and agonists, antagonists or inhibitors of REMAP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED50 (the dose therapeutically effective in 50% of the population) or LD50 (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD50/ED50 ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
Diagnostics
In another embodiment, antibodies which specifically bind REMAP may be 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 cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring REMAP, including ELISAs, RIAs, 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 established by combining body fluids or cell extracts taken from normal mammalian 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 from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, polynucleotides encoding REMAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of REMAP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of REMAP, and to monitor regulation of REMAP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding REMAP or closely related molecules may be used to identify nucleic acid sequences which encode REMAP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding REMAP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the REMAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:39-76 or from genomic sequences including promoters, enhancers, and introns of the REMAP gene.
Means for producing specific hybridization probes for polynucleotides encoding REMAP include the cloning of polynucleotides encoding REMAP or REMAP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotides encoding REMAP may be used for the diagnosis of disorders associated with expression of REMAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kurui, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; a metabolic disorder such as Addison's disease, cerebrotendinous xanthomatosis, congenital adrenal hyperplasia, coumarin resistance, cystic fibrosis, fatty hepatocirrhosis, fructose-1,6-diphosphatase deficiency, galactosemia, goiter, glucagonoma, glycogen storage diseases, hereditary fructose intolerance, hyperadrenalism, hypoadrenalism, hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, hyperthyroidism, hypoglycemia, hypothyroidism, hyperlipidemia, hyperlipemia, lipid myopathies, lipodystrophies, lysosomal storage diseases, mannosidosis, neuraminidase deficiency, obesity, osteoporosis, phenylketonuria, pseudovitamin D-deficiency rickets, disorders of carbohydrate metabolism such as congenital type II dyserythropoietic anemia, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria, pentosuria, and inherited abnormalities of pyruvate metabolism, disorders of lipid metabolism such as fatty liver, cholestasis, primary biliary cirrhosis, carnitine deficiency, carnitine palmitoyltransferase deficiency, myoadenylate deaminase deficiency, hypertriglyceridemia, lipid storage disorders such Fabry's disease, Gaucher's disease, Niemann-Pick's disease, metachromatic leukodystrophy, adrenoleukodystrophy, GM2 gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease, hyperlipoproteinemia, lipodystrophy, lipomatoses, acute panniculitis, disseminated fat necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal change disease, lipomas, atherosclerosis, hypercholesterolemia, hypercholesterolemia with hypertriglyceridemia, primary hypoalphalipoproteinemia, hypothyroidism, renal disease, liver disease, lecithin:cholesterol acyltransferase deficiency, cerebrotendinous xanthomatosis, sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoffs disease, hyperlipidemia, hyperlipemia, and lipid myopathies, and disorders of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type DX diabetes; a developmental disorder such as renal tubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, hydrocephalus, a seizure disorder such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; and an endocrine disorder such as a disorder of the hypothalamus and/or pituitary resulting from lesions such as a primary brain tumor, adenoma, infarction associated with pregnancy, hypophysectomy, aneurysm, vascular malformation, thrombosis, infection, immunological disorder, and complication due to head trauma, a disorder associated with hypopituitarism including hypogonadism, Sheehan syndrome, diabetes insipidus, Kallman's disease, Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism, a disorder associated with hyperpituitarism including acromegaly, giantism, and syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) often caused by benign adenoma, a disorder associated with hypothyroidism including goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism, a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease, a disorder associated with hyperparathyroidism including Conn disease (chronic hypercalemia), a pancreatic disorder such as Type I or Type II diabetes mellitus and associated complications, a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease, a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteoporosis, and, in men, Leydig cell deficiency, male climacteric phase, and germinal cell aplasia, a hypergonadal disorder associated with Leydig cell tumors, androgen resistance associated with absence of androgen receptors, syndrome of 5 α-reductase, and gynecomastia. Polynucleotides 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 well known in the art.
In a particular embodiment, polynucleotides encoding REMAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding REMAP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding REMAP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of REMAP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding REMAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding REMAP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding REMAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding REMAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from polynucleotides encoding 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. In SSCP, oligonucleotide primers derived from polynucleotides 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. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego Calif.).
SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes mellitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle cell anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity. For example, a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway. Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as well as for tracing the origins of populations and their migrations (Taylor, J. G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin. Neurobiol. 11:637-641).
Methods which may also be used to quantify the expression of REMAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or calorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, REMAP, fragments of REMAP, or antibodies specific for REMAP may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat. No. 5,840,484; hereby expressly incorporated by reference herein). Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (see, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released Feb. 29, 2000, available on the Internet at the National Institute of Environmental Health Sciences (“NIEHS”) government World Wide Web site. Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.
In an embodiment, the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
Another embodiment relates to the use of the polypeptides disclosed herein to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for REMAP to quantify the levels of REMAP expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/25116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662). Various types of microarrays are well known and thoroughly described in Schena, M., ed. (1999; DNA Microarrays: A Practical Approach, Oxford University Press, London).
In another embodiment of the invention, nucleic acid sequences encoding REMAP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; Trask, B. J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acid sequences may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E. S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding 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.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R. A. et al. (1988) Nature 336:577-580). The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, REMAP, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between REMAP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT application W084/03564). In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with REMAP, or fragments thereof, and washed. Bound REMAP is then detected by methods well 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 immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding REMAP specifically compete with a test compound for binding REMAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with REMAP.
In additional embodiments, the 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 pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, including U.S. Ser. No. 60/358,279, U.S. Ser. No. 60/364,338, U.S. Ser. No. 60/375,657, U.S. Ser. No. 60/376,669, U.S. Ser. No. 60/379,837, and U.S. Ser. No. 60/379,853, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.). Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CLAB column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad Calif.), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto Calif.), pRARE (Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Invitrogen.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V. B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.
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 programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto Calif.); hidden Markov model WMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HHM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full 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. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HM)-based protein family databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda Calif.) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignments 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 full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO:39-76. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative receptors and membrane-associated proteins were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once 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 public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
“Stitched” Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified were then “stitched” together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.
“Stretched” Sequences
Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. 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. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public 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:39-76 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID NO:39-76 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public 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 all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
Map locations are represented by ranges, or intervals, of human chromosomes. The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI “GeneMap'99” World Wide Web site, can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook and Russell, supra, ch. 7; Ausubel et al., supra, ch. 4).
Analogous computer techniques applying BLAST were used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:




  
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    Score
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    ⁢
    
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          length
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The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and −4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.

Alternatively, polynucleotides encoding REMAP are analyzed with respect to the tissue sources from which they were derived. For example, some fill length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding REMAP. cDNA sequences and cDNA library/tissue information are found in the LIESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).
VIII. Extension of REMAP Encoding Polynucleotides
Full length polynucleotides are produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5′ extension of the known fragment, and the other primer was synthesized to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)2SO4, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C.
The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1×TE and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsysterns Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Biosciences). For shotgun sequencing, the digested nucleotides 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 religated using T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37OC in 384-well plates in LB/2×carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above. Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotides are verified using the above procedure or are used to obtain 5′ regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.
IX. Identification of Single Nucleotide Polymorphisms in REMAP Encoding Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPS) were identified in SEQ ID NO:39-76 using the LIFESEQ database (Incyte Genomics). Sequences from the same gene were clustered together and assembled as described in Example III, allowing the identification of all sequence variants in the gene. An algorithm consisting of a series of filters was used to distinguish SNPs from other sequence variants. Preliminary filters removed the majority of basecall errors by requiring a minimum Phred quality score of 15, and removed sequence alignment errors and errors resulting from improper trimming of vector sequences, chimeras, and splice variants. An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP. Clone error filters used statistically generated algorithms to identify errors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation. Clustering error filters used statistically generated algorithms to identify errors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed duplicates and SNPs found in immunoglobulinsor T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations. The Caucasian population comprised 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezualan, and two Amish individuals. The African population comprised 194 individuals (97 male, 97 female), all African Americans. The Hispanic population comprised 324 individuals (162 male, 162 female), all 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. Allele frequencies were first analyzed in the Caucasian population; in some cases those SNPs which showed no allelic variance in this population were not further tested in the other three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO:39-76 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 μCi of [γ-32P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston Mass.). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: AseI, BglII, EcoRI, PstI, XbaI, or PvuII (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.). Hybridization is carried out for 16 hours at 40° C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena, M., ed. (1999) DNA Microarrays: A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection. After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element. Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM DATP, 500 μM dGTP, 500 μM dTTP, 40 ηM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte Genomics). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C. for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech, Palo Alto Calif.) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Amplified array elements are then purified using SEPHACRYL400 (Amersham Biosciences).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester Pa.), washed extensively in distilled water, and coated with 0.05% aininopropyl silane (Sigma-Aldrich, St. Louis Mo.) in 95% ethanol. Coated slides are cured in a 110° C. oven.
Array elements are applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522, incorporated herein by reference. 1 μl of the array element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALSKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.
Hybridization
Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65° C. for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5×SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20× microscope objective (Nikon, Inc., Melville N.Y.). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm×1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood Mass.) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Genomics). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentially expressed.
Expression
For example, T-47D is a breast carcinoma cell line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast. T-47D cells were treated with interferon gamma for from one hour to three days and then compared to untreated T-47D cells. Expression of SEQ ID NO:39 was decreased from 2- to 5.8-fold in treated T-47D cells when compared to untreated T-47D cells. Therefore, SEQ ID NO:39 is useful as a diagnostic marker or as a potential therapeutic target for breast cancer and inflammatory and immune diseases.
In another example, SEQ ID NO:65 demonstrated differential expression in a number of breast cancer and prostate cancer cell lines, as determined by microarray expression analysis. Normal breast cancer cells were represented by the HMEC (human mammary epithelial cells) cell line, and the fibrocystic cell line MCF-10A, derived from a donor with fibrocystic breast disease, was also used as a control, non-cancerous cell line. SEQ ID NO:65 showed at least a 2-fold decrease in expression in Sk-Br-3 cells, a Her2-positive cell line derived from a malignant adenocarcinoma of the breast, when compared to expression levels in either HMEC or MCF-10A cells. In addition, the BT-20 cell line, a cell line that forms stage II adenocarcinomas in mice derived from a donor with malignant adenocarcinoma of the breast, had at least a 2-fold decrease in SEQ ID NO:65 gene expression levels when compared to MCF-10A expression levels. Interestingly, the MCF-10A cell line showed at least a 2-fold decrease in expression of SEQ ID NO:65 when compared to the expression profile in HMEC normal epithelial cell line.
In another example, expression levels of SEQ ID NO:65 were compared in prostate cancer cell lines and in the normal prostate epithelial cell line PrEC. SEQ ID NO:9 showed a 2-fold increase in expression in PC3 cells (a adenocarcinoma cell line isolated from a bone metastasis of a donor with grade IV prostate cancer) when compared to starved PrEC cells. In other experiments, there was a 2-fold decrease in expression in DU 145 cells (derived from a brain metastasis of a donor with metastatic prostatic carcinoma), and a 2-fold decrease in expression in LNCaP cells (derived from a metastatic site in the lymph node of a prostate cancer donor), when compared to gene expression levels in PrEC cells grown in defined media LNCaP cells also showed at least a 2-fold decrease in SEQ ID NO:65 gene expression levels when compared to levels in another control prostate cell line, PZ-HPV-7. Additionally, treatment of LNCaP cells with PMA and ionomycin, activating PKC and calcium influx into the cells, lead to a time-dependent increase in expression of SEQ ID NO:65 (at least 2-fold after 4 hours, and at least 3-fold after 8 hours) when compared to untreated cells. Therefore, SEQ ID NO:65 is useful for staging of, monitoring treatment of, and diagnostic assays for breast and prostate cancer.
In another example, SEQ ID NO:62 and SEQ ID NO:65 were shown to have differential expression patterns in a number of lymphocyte cell models upon treatment with various stimuli, as determined by microarray expression analysis. Human peripheral blood mononuclear cells (PBMCs) were treated with PMA and ionomycin, to activate PKC- and calcium-dependent signaling pathways, and SEQ ID NO:62 expression levels were compared to levels in untreated cells. SEQ ID NO:62 showed a time-dependent increase in expression, at least 2.5-fold above untreated cell levels at 1 hour, peaking at 4.8-fold after 2 hours, then declining back to at least 2.5-fold at the 4 hour time point. Also, PBMCs from a number of different donors were treated with LPS for 4 to 24 hours, and these cells showed a general decrease in expression of SEQ ID NO:65 of between 2- and 4.5-fold when compared to untreated cells. In addition, RPMI 6666 cells (B cells derived from a donor with Hodgkin's disease) showed at least a 2-fold decrease in expression of SEQ ID NO:65 upon LPS treatment for 8 hours, when compared to expression levels in untreated RPMI 6666 cells. Treatment of donor PBMCs with SEB (staphylococcal endotoxin), however, resulted in a 2- to 4-fold increase in expression after 24 to 72 hours of SEQ ID NO:65, when compared to untreated cells.
In another example, THP-1 cells, a monocytic cell line, demonstrated differential expression of SEQ ID NO:62 and SEQ ID NO:65 upon differentiation into macrophage-like cells or foam cells, as determined by microarray expression analysis. Stimulation of THP-1 cells with PMA induces differentiation into a macrophage-like cell that displays many characteristics of peripheral human macrophages. The gene expression levels of SEQ ID NO:65 were shown to increase in PMA-treated cells from 2- to 6-fold, when compared to untreated THP-1 cells. Further treatment of THP-1 cells with oxidized LDL (oxLDL) induces differentiation into foam cells. Upon LPS treatment of macrophage-like or foam cells, the expression of SEQ ID NO:62 increased at least 2-fold when compared to untreated cells. Therefore, SEQ ID NO:62 and SEQ ID NO:65 are useful for study of activated immune system cells, and for monitoring treatment of and diagnostic assays for diseases of the immune system.
In another example, SEQ ID NO:70 and SEQ ID NO:73 showed differential expression in association with breast cancer, as determined by microarray analysis. Gene expression profiles were obtained by comparing the results of competitive hybridization experiments. The gene expression profile of cells isolated from a tumor in the right breast was compared to the gene expression profile of cells originating from grossly uninvolved breast tissue from the same donor, a 43-year-old female diagnosed with invasive lobular carcinoma (Huntsman Cancer Institute, Salt Lake City, Utah). The tumor was described as well differentiated and metastatic to 2 out of 13 lymph nodes. SEQ ID NO:73 showed decreased gene expression by at least two-fold in the tumorous tissue sample as compared to the uninvolved tissue sample from the same donor. In another example, the gene expression profile of a breast carcinoma cell line treated with interferon gamma (IFN-γ) was compared to the gene expression profile of untreated cells from the same line. T-47D is a breast carcinoma cell line isolated from a pleural effusion obtained from a 54-year-old female with an infiltrating ductal carcinoma of the breast. T-47D cells were treated with IFN-γ for 1, 4, 8, 24, 48 hours and 3 days. The expression of SEQ ID NO:70 was decreased by at least two-fold in the treated breast carcinoma cell lines as compared to the untreated T-47D population. Thus, SEQ ID NO:70 and SEQ ID NO:73 are useful as diagnostic markers for breast cancer, as well as for monitoring the progression and treatment of breast cancer.
In another example, SEQ ID NO:73 and SEQ ID NO:76 showed differential expression in association with colon cancer, as determined by microarray analysis. Gene expression profiles were obtained by comparing the results of competitive hybridization experiments between normal colon tissue and tumorous rectal tissue from the same donor. Different pieces of normal tissue were also compared against a pool of normal tissue from the same donor to determine gene expression variation in normal colon tissue. The expression of SEQ ID NO:73 was decreased by at least two-fold in tumorous rectal tissue as compared to normal rectal tissue from the same donor. In addition, the gene expression profiles of 6 different colon cancer tissues were analyzed by comparing one individual sample to 5 others, keeping one element in common between the various pairs of comparisons. The reference tissue sample is a metastatic adenocarcinoma of ovarian origin, which distinguishes this sample from the others and may be of special interest. The other five samples include tumorous colon tissue collected from an 85-year-old male, an 81-year-old male, an 83-year-old female, as well as a mucinous adenocarcinoma from a 58-year-old female, and a poorly differentiated metastatic adenocarcinoma from a 56-year-old female. The gene expression of SEQ ID NO:76 was decreased by two-fold in the tumorous rectal tissue samples as compared to the reference tissue. Therefore, SEQ ID NO:73 and SEQ ID NO:76 are useful as diagnostic markers for colon cancer, as well as for monitoring the progression and treatment of colon cancer.
In another example, SEQ ID NO:73 showed differential expression in association with lung cancer. Gene expression profiles were obtained by comparing the results of competitive hybridization experiments. Messenger RNA isolated from grossly uninvolved lung tissue with no visible abnormalities, from a 73-year-old male, was compared to lung squamous cell adenocarcinoma tissue from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). The expression of SEQ ID NO:73 was decreased by at least two-fold in tumorous lung tissue as compared to normal lung tissue from the same donor. Therefore, SEQ ID NO:73 is useful as a diagnostic marker for lung cancer, as well as for monitoring the progression and treatment of lung cancer.
In another example, SEQ ID NO:73 showed differential expression in association with inflammatory and immune responses, as determined by microarray analysis. Gene expression profiles were obtained by comparing the results of competitive hybridization experiments. Human peripheral blood mononuclelar cells (PBMCs) from seven healthy donors were stimulated in vitro with Staphylococal extoxin B (SEB) for 24 and 72 hours. The SEB treated PBMCs from each donor were compared to PBMCs from the same donor, kept in culture for 24 hours, in the absence of SEB. The gene expression of SEQ ID NO:73 was decreased by at least two-fold in SEB treated PBMCs as compared to untreated PBMCs from the same donors. In another example, SEQ ID NO:73 showed differential expression in treated versus untreated cells in a promonocyte cell line. THP-1 was isolated from the peripheral blood of a 1-year-old male with acute monocytic leukemia. PMA is a broad activator of the protein kinase C-dependent pathways. Upon stimulation with PMA, THP-1 differentiates into a macrophagelike cell that displays many characteristics of peripheral human macrophages. Promonocytes and monocytes to LPS, PMA-activated THP-1 cells (monocytic) and untreated THP-1 cells (promonocytic) were stimulated in vitro with LPS for 4 hours. LPS-treated THP-1 cells were compared to untreated THP-1 cells. In addition, PMA-activated THP-1 cells were compared to untreated THP-1 cells. The expression of SEQ ID NO:73 was decreased by at least two-fold in treated cells as compared to untreated cells. Therefore, SEQ ID NO:73 is useful as a diagnostic marker for inflammatory and immune response diseases, as well as for monitoring the progression and treatment of inflammatory and immune response diseases.
XII. Complementary Polynucleotides
Sequences complementary to the REMAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring REMAP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of REMAP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the REMAP-encoding transcript.
XIII. Expression of REMAP
Expression and purification of REMAP is achieved using bacterial or virus-based expression systems. For expression of REMAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express REMAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of REMAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding 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. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945).
In most expression systems, REMAP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences). Following purification, the GST moiety can be proteolytically cleaved from REMAP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). Purified REMAP obtained by these methods can be used directly in the assays shown in Examples XVII, XVIII, and XIX, where applicable.
XIV. Functional Assays
REMAP function is assessed by expressing the sequences encoding REMAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad Calif.) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994; Flow Cytometry, Oxford, New York N.Y.).
The influence of REMAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding REMAP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding REMAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XV. Production of REMAP Specific Antibodies
REMAP substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.
Alternatively, the REMAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (Ausubel et al., supra, ch. 11).
Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-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.
XVI. Purification of Naturally Occurring REMAP Using Specific Antibodies
Naturally occurring or recombinant REMAP is substantially purified by immunoaffinity chromatography using antibodies specific for REMAP. An immunoaffinity column is constructed by covalently coupling anti-REMAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing REMAP are passed over the immunoaffinity column, and the column is washed under conditions that allow 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 chaotrope, such as urea or thiocyanate ion), and REMAP is collected.
XVII. Identification of Molecules which Interact with REMAP
REMAP, or biologically active fragments thereof, are labeled with 125I Bolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J. 133:529-539). Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled REMAP, washed, and any wells 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.
Alternatively, 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 commercially 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 Conn.) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Pat. No. 6,057,101).
XVIII. Demonstration of REMAP Activity
An assay for REMAP activity measures the expression of REMAP on the cell surface. cDNA encoding REMAP is transfected into an appropriate mammalian cell line. Cell 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 cell surface.
In the alternative, an assay for REMAP activity is based on a prototypical assay for ligand/receptor-mediated modulation of cell proliferation. This assay measures the rate of DNA synthesis in Swiss mouse 3T3 cells. A plasmid containing polynucleotides encoding REMAP is added to quiescent 3T3 cultured cells using transfection methods well known in the art. The transiently transfected cells are then incubated in the presence of [3H]thymidine, a radioactive DNA precursor molecule. Varying amounts of REMAP ligand are then added to the cultured cells. Incorporation of [3H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a 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 milliliter is defined as the concentration of REMAP producing a 50% response level, where 100% represents maximal incorporation of [3H]thymidine into acid-precipitable DNA (McKay, L. and I. Leigh, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York NY, p. 73.)
In a further alternative, the assay for REMAP activity is based upon the ability 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 full length REMAP is transfected into a mammalian cell line (e.g., Chinese hamster ovary (CHO) or human embryonic kidney (HEK-293) cell lines) using methods well-known in the art. Transfected cells are grown in 12-well trays in culture medium for 48 hours, then the culture medium is discarded, and the attached cells are gently washed with PBS. The cells are then incubated in culture medium with or without ligand for 30 minutes, then the medium is removed and cells lysed by treatment with 1 M perchloric acid. The cAMP levels in the lysate are measured by radioimmunoassay using methods well-known in the art. Changes in the levels of cAMP in the lysate from cells exposed to ligand compared to those without ligand are proportional to the amount of REMAP present in the transfected cells.
To measure changes in inositol phosphate levels, the cells are grown in 24-well plates containing 1×105 cells/well and incubated with inositol-free media and [3H]myoinositol, 2 μCi/well, for 48 hr. The culture medium is removed, and the cells washed with buffer containing 10 mM LiCl followed 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 scintillation. Changes in the levels of labeled inositol phosphate from cells exposed to ligand compared to those without ligand are proportional to the amount of REMAP present in the transfected cells.
In a further alternative, the ion conductance capacity of REMAP is demonstrated using an electrophysiological assay. REMAP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding REMAP. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A small amount of a second plasmid, which expresses any one of a number of marker genes such as β-galactosidase, is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of REMAP and β-galactosidase. Transformed cells expressing β-galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are well known in the art. Stained cells are tested for differences in membrane conductance due to various ions by electrophysiological techniques that are well known in the art. Untransformed cells, and/or cells transformed with either vector sequences alone or β-galactosidase sequences alone, are used as controls and tested in parallel. 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 will bind to the extracellular side of REMAP, thereby blocking the pore in the ion channel, and the associated conductance.
In a further alternative, 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 KCl, 1 mM CaCd2, 1 mM MgCl2, 1 mM Na2OPO4, 5 mM Hepes, 3.8 mM NaOH, 50 μg/ml gentamycin, pH 7.8) to allow expression of REMAP protein. Oocytes are then transferred to standard uptake medium (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Hepes/Tris pH 7.5). Uptake of various substrates (e.g., amino acids, sugars, drugs, and neurotransmitters) is initiated by adding a 3H substrate to the oocytes. After incubating for 30 minutes, uptake is terminated by washing the oocytes three times in Na+-free medium, measuring the incorporated 3H, and comparing with controls. REMAP activity is proportional to the level of internalized 3H substrate.
In a further alternative, REMAP protein kinase (PK) activity is measured by phosphorylation of a protein substrate using gamma-labeled [32P]-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. REMAP is incubated with the protein substrate, [32P]-ATP, and an appropriate kinase buffer. The 32P incorporated into the product is separated from free [32P]-ATP by electrophoresis and the incorporated 32P is counted. The amount of 32P recovered is proportional to the PK activity of REMAP in the assay. A determination of the specific amino acid residue phosphorylated is made by phosphoamino acid analysis of the hydrolyzed protein.
XIX. Identification of REMAP Ligands
REMAP is expressed in a eukaryotic cell line such as CHO (Chinese Hamster Ovary) or BEK (Human Embryonic Kidney) 293 which have a good history of GPCR expression and which contain a wide range of G-proteins allowing for functional coupling of the expressed REMAP to downstream effectors. The transformed cells are assayed for activation of the expressed receptors in the presence of candidate ligands. Activity is measured by changes in intracellular second messengers, such as cyclic AMP or Ca2+. These may be measured directly using standard methods well known in the art, or by the use of reporter gene assays in which a luminescent protein (e.g. firefly luciferase or green fluorescent protein) is under the transcriptional control of a promoter responsive to the stimulation of protein kinase C by the activated receptor (Milligan, G. et al. (1996) Trends Pharmacol. Sci. 17:235-237). Assay technologies are available for both of these second messenger systems to allow high throughput readout in multi-well plate format, such as the adenylyl cyclase activation FlashPlate Assay (NEN Life Sciences Products), or fluorescent Ca2+ indicators such as Fluo-4 AM (Molecular Probes) in combination with the FLIPR fluorimetric plate reading system (Molecular Devices). In cases where the physiologically relevant second messenger pathway is not known, REMAP may be coexpressed with the G-proteins Gα15/16 which have been demonstrated to couple to a wide range of G-proteins (Offermanns, S. and M. I. Simon (1995) J. Biol. Chem. 270:15175-15180), in order to funnel the signal transduction of the REMAP through a pathway involving phospholipase C and Ca2+ mobilization. Alternatively, REMAP may be expressed in engineered yeast systems which lack endogenous GPCRs, thus providing the advantage of a null background for REMAP activation screening. These yeast systems substitute a human GPCR and Gα protein for the corresponding components of the endogenous yeast pheromone receptor pathway. Downstream signaling pathways are also modified so that the normal yeast response to the signal is converted to positive growth on selective media or to reporter gene expression (Broach, J. R. and J. Thorner (1996) Nature 384 (supp.): 14-16). The receptors are screened against putative ligands including known GPCR ligands and other naturally occurring bioactive molecules. Biological extracts from tissues, biological fluids and cell supernatants are also screened.
XX. Other Embodiments
Various embodiments of the invention provide purified polypeptides, receptors and membrane-associated proteins, referred to collectively as ‘REMAP’ and individually as ‘REMAP-1,’ ‘REMAP-2,’ ‘REMAP-3,’ ‘REMAP4,’ ‘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,’ and ‘REMAP-38,’ and methods for using these proteins and their encoding polynucleotides for the detection, diagnosis, and treatment of diseases and medical conditions. Embodiments also provide methods for utilizing the purified receptors and membrane-associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology. Related embodiments provide methods for utilizing the purified receptors and membrane-associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-38.
Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. In another embodiment, the polynucleotide encode& a polypeptide selected from the group consisting of SEQ ID NO:1-38. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:39-76.
Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.
Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38.
Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In other embodiments, the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex. In a related embodiment, the method can include detecting the amount of the hybridization complex. In still other embodiments, the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
Still another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof. In a related embodiment, the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
Another embodiment provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and a pharmaceutically acceptable excipient. In one embodiment, the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
Still another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional REMAP, comprising administering to a patient in need of such treatment the composition.
Another embodiment provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-38. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
Still another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NQ:39-76, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:39-76, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.












TABLE 1









Incyte




Polypeptide
Incyte
Polynucleotide
Polynucleotide


Incyte Project ID
SEQ ID NO:
Polypeptide ID
SEQ ID NO:
ID
Incyte Full Length Clones




















3048626
1
3048626CD1
39
3048626CB1



2684425
2
2684425CD1
40
2684425CB1


7505960
3
7505960CD1
41
7505960CB1


7507021
4
7507021CD1
42
7507021CB1
90122224CA2


7509099
5
7509099CD1
43
7509099CB1
90137914CA2


7509361
6
7509361CD1
44
7509361CB1
90134650CA2


7506815
7
7506815CD1
45
7506815CB1
90115637CA2


7506814
8
7506814CD1
46
7506814CB1
90115621CA2


7506852
9
7506852CD1
47
7506852CB1
90123356CA2, 90123372CA2, 90123380CA2, 90123571CA2


7503782
10
7503782CD1
48
7503782CB1


7504647
11
7504647CD1
49
7504647CB1
6352669CA2, 90036485CA2, 90036561CA2, 95121338CA2,







95121529CA2, 95121605CA2, 95121637CA2, 95121653CA2,







95121693CA2, 95121745CA2, 95121761CA2, 95121812CA2,







95121868CA2, 95121884CA2, 95121905CA2


7500424
12
7500424CD1
50
7500424CB1
90030465CA2, 90030473CA2, 90030573CA2, 90030581CA2


7500449
13
7500449CD1
51
7500449CB1


7503281
14
7503281CD1
52
7503281CB1
90041241CA2, 90041301CA2, 90041317CA2, 90041341CA2


7503292
15
7503292CD1
53
7503292CB1


7503311
16
7503311CD1
54
7503311CB1


7510384
17
7510384CD1
55
7510384CB1


7509976
18
7509976CD1
56
7509976CB1


7510454
19
7510454CD1
57
7510454CB1
55062756CA2, 90005113CA2, 90005121CA2, 90005137CA2,







90005145CA2, 90005205CA2, 90005213CA2, 90005221CA2,







90005237CA2, 90082826CA2, 90208706CA2, 90208714CA2,







90208785CA2, 90208793CA2


8017335
20
8017335CD1
58
8017335CB1


7510197
21
7510197CD1
59
7510197CB1
3833001CA2


7510055
22
7510055CD1
60
7510055CB1
95110475CA2


7501754
23
7501754CD1
61
7501754CB1
3576444CA2


7510517
24
7510517CD1
62
7510517CB1


7511014
25
7511014CD1
63
7511014CB1
90115446CA2


7506687
26
7506687CD1
64
7506687CB1


7510621
27
7510621CD1
65
7510621CB1


7505533
28
7505533CD1
66
7505533CB1
95136216CA2, 95136264CA2


7511220
29
7511220CD1
67
7511220CB1


7510967
30
7510967CD1
68
7510967CB1


7511298
31
7511298CD1
69
7511298CB1
90171160CA2


7510937
32
7510937CD1
70
7510937CB1
90051283CA2


7511852
33
7511852CD1
71
7511852CB1
95001926CA2


7511077
34
7511077CD1
72
7511077CB1
1929803CA2


7511576
35
7511576CD1
73
7511576CB1


7511492
36
7511492CD1
74
7511492CB1


7511141
37
7511141CD1
75
7511141CB1
2776443CA2, 95021920CA2


7511300
38
7511300CD1
76
7511300CB1




















TABLE 2





Poly-






pep-


tide


SEQ
Incyte
GenBank ID NO:




ID
Polypeptide
or PROTEOME
Probability


NO:
ID
ID NO:
Score
Annotation



















1
3048626CD1
g6523391
 8.1E−210
[Mus musculus] phtf protein






Manuel, A. et al. (2000) Molecular characterization of a novel gene family (PHTF)






conserved from drosophila to mammals. Genomics 64: 216-220




587245|Phtf
 6.8E−211
[Mus musculus][Transcription factor; DNA-binding protein] Putative homeodomain






transcription factor; expressed in testis






Manuel, A. et al. (2000) Molecular characterization of a novel gene family (PHTF)






conserved from drosophila to mammals. Genomics 64: 216-220




432628|
 2.1E−209
[Homo sapiens][Transcription factor; DNA-binding protein] Putative homeodomain




PHTF1

transcription factor; may play role in development






Raich, N. et al. (1999) PHTF, A novel atypical homeobox gene on chromosome 1p13, is






evolutionarily conserved. Genomics 59: 108-109


2
2684425CD1
g8439531
0.0
[Homo sapiens] transmembrane molecule with thrombospondin module




599850|
0.0
[Homo sapiens] Protein containing a type 1 thrombospondin domain




LOC55901


3
7505960CD1
g4529890
0.0
[Homo sapiens] NG22




692052|NG22
0.0
[Homo sapiens] Protein of unknown function, has strong similarity to uncharacterized






mouse 2210409B01Rik




664607|2210409
 2.3E−294
[Mus musculus] RIKEN cDNA 2210409B01 gene




B01Rik


4
7507021CD1
g2338292
 1.4E−78
[Homo sapiens] proline-rich Gla protein 2






Kulman, J. D. et al. (1997) Primary structure and tissue distribution of two novel proline-






rich gamma-carboxyglutamic acid proteins. Proc. Natl. Acad. Sci. U.S.A. 94: 9058-9062


5
7509099CD1
g307046
 2.4E−274
[Homo sapiens] interleukin 1 receptor precursor




336000|
 2.0E−275
[Homo sapiens][Receptor (signalling)][Plasma membrane] Type I interleukin-1 receptor, a




IL1R1

member of the IL1R like protein family regulated by IL1R associated kinase IRAK1,






involve in immune and inflammatory responses, involved in leukemia, atherosclerosis,






sepsis and growth of solid tumors






Chen, G. et al. (2000) Selection of insulinoma cell lines with resistance to interleukin-1beta-






and gamma-interferon-induced cytotoxicity. Diabetes 49: 562-570




583367|Il1r1
 2.4E−193
[Mus musculus][Receptor (signalling)][Plasma membrane] Type I interleukin-1 receptor, a






member of the IL1R like protein family regulated by IL1R associated kinase IRAK (Il1rak),






involved in immune and inflammatory resposes and signal transduction






Parnet, P. et al. (1994) Expression of type I and type II interleukin-1 receptors in mouse






brain. Brain Res. Mol. Brain Res. 27: 63-70


6
7509361CD1
g339750
 4.5E−119
[Homo sapiens] tumor necrosis factor receptor 1






Fuchs, P. et al. (1992) Structure of the human TNF receptor 1 (p60) gene (TNFR1) and






localization to chromosome 12p13 [corrected] [published erratum appears in (1992)






Genomics 13: 1384] Genomics 13: 219-224




338586|
 3.8E−120
[Homo sapiens][Receptor (signalling)][Plasma membrane] FPF Type I tumor necrosis




TNFRSF1A

factor receptor, mediates proinflammatory cellular responses, juxtamembrane domain






interacts with phosphatidylinositol-4-phosphate 5-kinase






Baranzini, S. E. et al. (2000) Transcriptional analysis of multiple sclerosis brain lesions






reveals a complex pattern of cytokine expression. J. Immunol. 165: 6576-6582




723062|1ext_A
 3.0E−95
[Protein Data Bank] Tumor Necrosis Factor Receptor




590719|
 1.1E−85
[Rattus norvegicus][Receptor (signalling)] Type I tumor necrosis factor receptor, a




Tnfrsf1a

glycoprotein that mediates proinflammatory cellular responses, contains an extracellular






domain that is proteolytically cleaved to yield a tumor necrosis factor binding protein






Laabich, A. et al. (2001) Characterization of apoptosis-genes associated with NMDA






mediated cell death in the adult rat retina. Brain Res. Mol. Brain Res. 91: 34-42


7
7506815CD1
g12653895
 1.1E−194
[Homo sapiens] cholecystokinin B receptor




334486|
 9.0E−196
[Homo sapiens][Regulatory subunit; Receptor (signalling)] [Basolateral plasma membrane;




CCKBR

Cytoplasmic; Plasma membrane] Cholecystokinin B (gastrin) receptor, G protein-coupled






receptor stimulating phospholipase C and intracellular calcium flux, associated with anxiety






and likely digestion and dopamine signaling, constitutively active form is expressed in






colorectal cancers






Smith, A. M. and Watson, S. A. (2000) Gastrin and gastrin receptor activation: an early






event in the adenoma-carcinoma sequence. Gut 4: 820-824




589913|Cckbr
 1.3E−170
[Rattus norvegicus][Receptor (signalling)] [Nuclear; Cytoplasmic; Plasma membrane]






Cholecystokinin B (gastrin) receptor, G protein-coupled receptor stimulating phospholipase






C and intracellular calcium flux, associated with digestion and opioidergic and






dopaminergic signaling; a human CCKBR variant is associated with colorectal cancer






Coudore-Civiale, M. A. et al. (2000) Spinal effect of the cholecystokinin-B receptor






antagonist CI-988 on hyperalgesia, allodynia and morphine-induced analgesia in diabetic






and mononeuropathic rats. Pain 88: 15-22


8
7506814CD1
g12653895
3.20E−206
[Homo sapiens] cholecystokinin B receptor



7506814CD1
334486|
2.70E−207
[Homo sapiens][Regulatory subunit; Receptor (signalling)][Basolateral plasma membrane;




CCKBR

Cytoplasmic; Plasma membrane] Cholecystokinin B (gastrin) receptor, G protein-coupled






receptor stimulating phospholipase C and intracellular calcium flux, associated with anxiety






and likely digestion and dopamine signaling, constitutively active form is expressed in






colorectal cancers (Desbois, C. et al. (1999) Eur J Biochem 266, 1003-10)



7506814CD1
589913|Cckbr
3.50E−194
[Rattus norvegicus][Receptor (signalling)][Nuclear; Cytoplasmic; Plasma membrane]






Cholecystokinin B (gastrin) receptor, G protein-coupled receptor stimulating phospholipase






C and intracellular calcium flux, associated with digestion and opioidergic and






dopaminergic signaling; a human CCKBR variant is associated with colorectal cancer






(Wank, S. A. et al. (1992) Proc Natl Acad Sci USA 89, 8691-5)


9
7506852CD1
g400450
1.80E−53
[Homo sapiens] A1 adenosine receptor



7506852CD1
334066|
1.50E−54
[Homo sapiens][Receptor (signalling)][Cytoplasmic; Plasma membrane] Adenosine A1




ADORA1

receptor, a glycoprotein and G protein-coupled receptor that selectively binds adenosine;






stimulates cell death of thymocytes and phagocytosis; density is reduced in hippocampus






from Alzheimer's disease patients; may play a role in obesity (Libert, F. et al. (1992)






Biochem Biophys Res Commun 187, 919-26)



7506852CD1
590847|
6.40E−54
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Adenosine A1 receptor, a G




Adoral

protein-coupled receptor that selectively binds adenosine; modulates adenosine effects in






neural and endocrine systems; may play a role in inherited obesity (Mahan, L. C. et al.






(1991) Mol Pharmacol 40, 1-7)


10
7503782CD1
g7209574
4.20E−19
[Homo sapiens] LAK-4p


11
7504647CD1
g533184
3.60E−23
[Homo sapiens] 50 kD dystrophin-associated glycoprotein (McNally, E. et al. (1994) Proc.






Natl. Acad. Sci. U.S.A. 11; 91(21): 9690-4)



7504647CD1
337978|SGCA
3.00E−24
[Homo sapiens][Anchor Protein][Extracellular matrix (cuticle and basement membrane);






Basement membrane (extracellular matrix); Plasma membrane] Alpha-sarcoglycan






(adhalin), a dystrophin-associated glycoprotein required for normal striated muscle






development, protects against contraction-induced sarcolemmal damage; mutations in the






corresponding gene cause limb girdle muscular dystrophy type 2D (Barresi, R. et al. (2000)






J Biol Chem 275, 38554-60)



7504647CD1
581329|Sgca
5.80E−15
[Mus musculus][Structural protein][Extracellular matrix (cuticle and basement membrane);






Basement membrane (extracellular matrix); Cytoplasmic; Plasma membrane] Alpha-






sarcoglycan (adhalin), a dystrophin-associated glycoprotein required for normal striated






muscle development, protects against contraction-induced sarcolemmal damage; mutations






in the human SGCA gene cause limb girdle muscular dystrophy type 2D (Coral-Vazquez, R.






et al. (1999) Cell 98, 465-74).


12
7500424CD1
g14250620
4.50E−66
[Homo sapiens] G protein-coupled receptor 56



7500424CD1
342484|
3.70E−67
[Homo sapiens][Receptor (signalling)][Plasma membrane] G protein-coupled receptor 56, a




GPR56

putative G protein-coupled receptor that may function in cell adhesion, cell-cell signaling,






and is differentially expressed during metastatic progression of melanomas (Zendman, A. J.






et al. (1999) FEBS Lett 446, 292-8)



7500424CD1
732759|
6.10E−38
[Mus musculus][Receptor (signalling)][Plasma membrane] G protein-coupled receptor 56




Gpr56


13
7500449CD1
g456353
1.50E-131
[Homo sapiens] intestinal VIP receptor related protein (Couvineau, A. et al. (1994)






Biochem. Biophys. Res. Commun. 200, 769-776)



7500449CD1
749162|
4.80E−97
[Homo sapiens][Receptor (signalling)][Plasma membrane] Vasoactive intestinal activating




VIPR1

polypeptide receptor 1, a stimulatory G protein coupled receptor; mediates gastrointestinal,






nervous system, pulmonary, vascular and immune functions, inhibits inflammation






(Sreedharan, S. P. et al. (1995) Proc Natl Acad Sci USA 92, 2939-43)



7500449CD1
590753|Vipr1
3.50E−78
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Vasoactive intestinal






activating polypeptide receptor 1, a stimulatory G protein coupled receptor; inhibits






inflammatory responses and may mediate central and peripheral nervous system functions






(Ishihara, T. et al (1992). Neuron 8, 811-9)


14
7503281CD1
g178198
1.40E−112
[Homo sapiens] alpha-2-adrenergic receptor (alpha-2 C2) old gene name ‘ADRA2RL1’






(Lomasney, J. W. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5094-5098)



7503281CD1
343936|
1.10E−113
[Homo sapiens][Receptor (signalling)][Plasma membrane] Adrenergic alpha-2B receptor, a




ADRA2B

G protein-coupled receptor that binds epinephrine and norepinephrine, signals through






regulation of adenylyl cyclase and MAPK pathways to mediate cell-cell signaling, may have






a role in fat metabolism (Smith, M. S. et al. (1995) Brain Res Mol Brain Res 34, 109-17)



7503281CD1
429618|
3.90E−102
[Mus musculus][Receptor (signalling)][Endosome/Endosomal vesicles; Cytoplasmic;




Adra2b

Plasma membrane] Adrenergic receptor alpha 2b, a G protein-coupled receptor that binds






epinephrine and norepinephrine, signals through regulation of adenylyl cyclase activity,






involved in blood pressure regulation, sensory perception, synaptic transmission, and






analgesia (Link, R. E. et al. (1996) Science 273, 803-5)


15
7503292CD1
g3901028
1.70E−144
[Homo sapiens] neurotensin receptor 2 (Vita, N. et al. (1998) Eur. J. Pharmacol. 360, 265-272)



7503292CD1
428880|
1.40E−145
[Homo sapiens][Receptor (signalling)][Plasma membrane] Levocabastine-sensitive




NTSR2

neurotensin receptor, a low affinity putative G protein-coupled receptor that binds, but is






not activated by, neurotensin; activation by SR48692 and SR142948A stimulates IP






formation, Ca2+ mobilization, and arachidonic acid release (Mazella, J. et al. (1996) J






Neurosci 16, 5613-20)



7503292CD1
659258|Ntsr2
7.10E−119
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Levocabastine-sensitive






neurotensin receptor, a G protein-coupled receptor that binds neurotensin, and the H1






antihistaminic drug levocabastine, activation by SR48692 induces Ca2+ mobilization, may






help modulate neuronal osmosensitivity (Botto, J. M. et al. (1998) Biochem Biophys Res






Commun 243, 585-90)


16
7503311CD1
g1785516
3.50E−146
[Homo sapiens] gastric inhibitory polypeptide receptor (Yamada, Y. et al. (1995) Genomics






29, 773-776)



7503311CD1
335524|GIPR
2.80E−147
[Homo sapiens][Receptor (signalling)][Plasma membrane] Gastric inhibitory polypeptide






receptor, a G protein-coupled receptor that increases intracellular cAMP levels and MAPK






kinase activity, may be associated with Gushing's syndrome (Lacroix, A. et al. (1998)






Endocr Res 24, 835-43)



7503311CD1
590141|Gipr
5.70E−117
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Gastric inhibitory






polypeptide receptor, a G protein-coupled receptor that increases intracellular cAMP and






calcium levels, mediates effects of glucose-dependent insulinotropic polypeptide (GIP) on






insulin secretion, may be associated with type 2 diabetes (Usdin, T. B. et al. (1993)






Endocrinology 133, 2861-70)


17
7510384CD1
g3242762
1.10E−110
[Homo sapiens] growth hormone-releasing hormone receptor



7510384CD1
335522|
3.00E−111
[Homo sapiens][Receptor (signalling)][Plasma membrane] Growth hormone releasing




GHRHR

hormone receptor, a G protein-coupled receptor that regulates pituitary growth hormone






synthesis and secretion, may act through increasing intracellular cAMP levels; deficiency is






a cause of dwarfism (Wajnrajch, M. P. et al. (1996) Nat Genet 12, 88-90)



7510384CD1
590139|Ghrhr
3.10E−86
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Growth hormone releasing






hormone receptor, a member of the G protein-coupled receptor family expressed primarily






in pituitary, has probable roles in regulating growth; has strong similarity to human






GHRHR, deficiency of which is associated with dwarfism (Zeitler, P. et al. (1998) J Mol






Endocrinol 21, 363-71)


18
7509976CD1
g1200235
0
[Homo sapiens] SEX protein



7509976CD1
599756|
0
[Homo sapiens][Receptor (signalling)][Plasma membrane] Protein with strong similarity to




HSSEXGENE

murine Plxn3, which is a member of the plexin family of semaphorin receptors involved in






cell guidance (Kameyama, T. et al. (1996) Biochem Biophys Res Commun 226, 396-402)



7509976CD1
582527|Plxn3
0
[Mus musculus][Receptor (signalling)][Plasma membrane] Plexin 3, a member of the plexin






family of semaphorin receptors, may play a role in the regulation of neuronal development






(Kameyama, T. et al. (supra))


19
7510454CD1
g17481324
1.10E−21
[Mus musculus] vomeronasal receptor 1 E9



7510454CD1
613285|
2.50E−11
[Homo sapiens][Receptor (signalling)] V1R-like 1, a predicted member of the G-protein




V1RL1

coupled receptor family and a putative olfactory mucosal pheromone receptor (Rodriguez, I.






et al. (2000) Nat Genet 26, 18-9)


20
8017335CD1
g15082375
 6.7E−81
[Homo sapiens] Similar to transmembrane 7 superfamily member 1 (upregulated in kidney)



8017335CD1
338556|
 5.5E−82
[Homo sapiens][Plasma membrane] Transmembrane 7 superfamily member 1, may be a




TM7SF1

member of the G protein-coupled receptor family, contains seven alpha helical






transmembrane domains; expression is upregulated during kidney development






Spangenberg, C. et al.






Cloning and characterization of a novel gene (TM7SF1) encoding a putative seven-pass






transmembrane protein that is upregulated during kidney development.






Genomics 48, 178-85 (1998).



8017335CD1
746563|
  1E−80
[Mus musculus] Transmembrane 7 superfamily member 1, may be a member of the G




Tm7sf1

protein-coupled receptor family, contains seven alpha helical transmembrane domains;






expression is upregulated during kidney development


22
7510055CD1
g29851
 2.7E−107
[Homo sapiens] CDw40






Stamenkovic, I. et al.






A B-lymphocyte activation molecule related to the nerve growth factor receptor and






induced by cytokines in carcinomas






EMBO J. 8, 1403-1410 (1989)



7510055CD1
338592|
 2.2E−108
[Homo sapiens][Receptor (signalling)][Plasma membrane] Member of the tumor necrosis




TNFRSF5

factor receptor superfamily, binds the ligand CD40L and is expressed specifically in B






lymphocytes, has a role in B lymphocyte maturation






Stamenkovic, I. et al.






A B-lymphocyte activation molecule related to the nerve growth factor receptor and






induced by cytokines in carcinomas.






Embo Journal 8, 1403-10 (1989).






Mach, F. et al.






Reduction of atherosclerosis in mice by inhibition of CD40 signalling.






Nature 394, 200-3 (1998).



7510055CD1
586037|
 4.1E−68
[Mus musculus][Receptor (signalling)][Plasma membrane] Member of the tumor necrosis




Tnfrsf5

factor receptor superfamily, binds the ligand CD40L and is expressed specifically in B






lymphocytes, has a role in B lymphocyte maturation






Torres, R. M. et al.






Differential increase of an alternatively polyadenylated mRNA species of murine CD40






upon B lymphocyte activation.






J Immunol 148, 620-6 (1992).


23
7501754CD1
g9944291
 2.5E−223
[Homo sapiens] TTYH1






Campbell, H. D. et al.






Human and mouse homologues of the drosophila melanogaster tweety (tty) gene: A novel






gene family encoding predicted transmembrane proteins






Genomics 68, 89-92 (2000)



7501754CD1
613379|
  2E−224
[Homo sapiens][Active transporter, secondary; Transporter] Tweety homolog 1




TTYH1

(Drosophila), a member of a family of putative membrane proteins with five potential






transmembrane domains






Campbell, H. D. et al.






Human and mouse homologues of the drosophila melanogaster tweety (tty) gene; A novel






gene family encoding predicted transmembrane proteins






Genomics 68, 89-92 (2000).



7501754CD1
618612|
  4E−203
[Mus musculus] Tweety homolog 1 (Drosophila), a member of a family of putative




Ttyh1

membrane proteins with five potential transmembrane domains






Campbell, H. D. et al. (supra)


24
7510517CD1
g1359731
 1.0E−98
[Homo sapiens] EP4 prostaglandin receptor






Foord, S. M. et al. (1996) The structure of the prostaglandin EP4 receptor gene and related






pseudogenes. Genomics 35: 182-188.




337370|
 8.6E−100
[Homo sapiens][Receptor (signaling)][Plasma membrane] Prostaglandin E receptor 4, a G




PTGER4

protein-coupled receptor that signals through stimulatory G-protein, mediates a variety of






physiological effects including inflammatory response and cell motility, may increase






invasive growth of colorectal carcinoma cells






Bastien, L. et al. (1994) Cloning, functional expression and characterization of the human






Prostaglandin E2 receptor EP2 subtype. J. Biol. Chem. 269: 11873-11877.






An, S. et al. (1993) Cloning and expression of the EP2 subtype of human receptors for






prostaglandin E2. Biochem. Biophys. Res. Commun. 197: 263-270.






Dumais, N. et al. (1998) Prostaglandin E2 up-regulates HIV-1 long terminal repeat-driven






gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways. J.






Biol. Chem. 273: 27306-27314.






Pai, R. et al. (2002) Prostaglandin E2 transactivates EGF receptor: a novel mechanism for






promoting colon cancer growth and gastrointestinal hypertrophy. Nat. Med. 8: 289-293.






Mutoh, M. et al. (2002) Involvement of prostaglandin E receptor subtype EP(4) in colon






carcinogenesis. Cancer Res. 62: 28-32.






Sheng, H. et al. (2001) Prostaglandin E2 increases growth and motility of colorectal






carcinoma cells. J. Biol. Chem. 276: 18075-18081.




582643|
 8.8E−91
[Mus musculus][Receptor (signaling)][Plasma membrane] Prostaglandin E receptor 4, a G




Ptger4

protein-coupled receptor that signals through a stimulatory G-protein, mediates a variety of






physiological and pathophysiological effects including immune and inflammatory responses






and heart and skeletal development






Honda, A. et al. (1993) Cloning and expression of a cDNA for mouse prostaglandin E






receptor EP2 subtype. J. Biol. Chem. 268: 7759-7762.






Suzawa, T. et al. (2000) The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and






EP4) in bone resorption: an analysis using specific agonists for the respective Eps.






Endocrinology 14: 1554-1559.






Miyaura, C. et al. (2000) Impaired bone resorption to prostaglandin E2 in prostaglandin E






receptor EP4-knockout mice. J. Biol. Chem. 275: 19819-19823.


25
7511014CD1
g456564
 9.9E−140
[Homo sapiens] prostanoid FP receptor






Abramovitz, M. et al. (1994) Cloning and expression of a cDNA for the human prostanoid






FP receptor. J. Biol. Chem. 269: 2632-2636.




337372|
 8.5E−141
[Homo sapiens][Receptor (signaling)][Plasma membrane] Prostanoid FP receptor




PTGFR

(prostaglandin F2-alpha receptor), activation induces calcium flux, regulates smooth muscle






contraction, and predicted to be necessary for luteolysis; mutations in the corresponding






gene are associated with breast cancer






Sossey-Alaoui, K. et al. (2001) Fine mapping of the PTGFR gene to 1p31 region and






mutation analysis in human breast cancer. Int. J. Mol. Med 7: 543-546.






Sugimoto, Y. et al. (1997) Failure of parturition in mice lacking the prostaglandin F






receptor. Science 277: 681-683.




582645|Ptgfr
 4.3E−130
[Mus musculus][Receptor (signaling)][Plasma membrane] Prostanoid FP receptor






(prostaglandin F2-alpha receptor), a G protein-coupled receptor that mediates intracellular






signaling, necessary for luteolysis; mutations in human PTGFR gene are associated with






breast cancer






Sugimoto, Y. et al. (1994) Cloning and expression of a cDNA for mouse prostaglandin F






receptor. J. Biol. Chem. 269: 1356-1360.


26
7506687CD1
g6010211
0.0
[Homo sapiens] semaphorin receptor






Tamagnone, L. et al. (1999) Plexins are a large family of receptors for transmembrane,






secreted, and GPI-anchored semaphorins in vertebrates. Cell 99: 71-80.




568412|
0.0
[Homo sapiens][Receptor (signaling)][Plasma membrane] Plexin 5, member of the plexin




PLXNB1

family of semaphorin receptors involved in mediating cell guidance, expressed in the brain






Maestrini, E. et al. (1996) A family of transmembrane proteins with homology to the MET-






hepatocyte growth factor receptor. Proc. Natl. Acad. Sci. USA 93: 674-678.




608600|Plxn6
 2.5E−149
[Mus musculus] Protein containing a plexin repeat, a Sema domain, and three IPT/TIG






domains, all of which are found in receptors


27
7510621CD1
g246539
 1.8E−115
[Homo sapiens] ocular melanoma-associated antigen; OMA81H






Wang, M. X. et al. (1992) An ocular melanoma-associated antigen. Molecular






characterization. Arch. Ophthalmol. 110: 399-404.




344036|CD63
 1.6E−116
[Homo sapiens][Lysosome/vacuole; Cytoplasmic; Plasma membrane] Melanoma 1 antigen,






a member of the tetraspanning superfamily (TM4SF), forms multicomponent complexes






with beta 1 integrins, associates with peptide-loaded MHC class II molecules; acts to limit






the invasion and progression of melanoma






Metzelaar, M. J. et al. (1991) CD63 antigen. A novel lysosomal membrane glycoprotein,






cloned by a screening procedure for intracellular antigens in eukaryotic cells. J. Biol. Chem.






266: 3239-3245.






Gwynn, B. et al. (1996) Genetic localization of Cd63, a member of the transmembrane 4






superfamily, reveals two distinct loci in the mouse genome. Genomics 35: 389-391.






Radford, K. J. et al. (1996) CD63 associates with transmembrane 4 superfamily members,






CD9 and CD81, and with beta 1 integrins in human melanoma. Biochem. Biophys. Res.






Commun. 222: 13-18.






Smith, D. A. et al. (1995) Antibodies against human CD63 activate transfected rat






basophilic leukemia (RBL-2H3) cells. Mol. Immunol. 32: 1339-1344.




583753|Cd63
 2.3E−92
[Mus musculus][Plasma membrane] Melanoma 1 antigen, a member of the tetraspanning






superfamily (TM4SF), may play a role in maintaining normal renal function, highly






expressed in activated macrophages






Miyamoto, H. et al. (1994) Molecular cloning of the murine homologue of CD63/ME491






and detection of its strong expression in the kidney and activated macrophages. Biochim.






Biophys. Acta 1217: 312-316.


28
7505533CD1
g7768496
 6.9E−13
[Schizosaccharomyces pombe] putative ER-derived vesicles protein similar to yeast erv14




569856|
 8.9E−43
[Homo sapiens] Protein of unknown function, has moderate similarity to S. cerevisiae




HSPC163

Erv14p, which is a protein of ER-derived vesicles that is required for efficient degradation






of soluble ER quality control substrates




6677|ERV14
 4.0E−15
[Saccharomyces cerevisiae][Vesicle coat protein; Docking protein][Endoplasmic reticulum;






Other vesicles of the secretory/endocytic pathways]Protein of ER-derived vesicles that is






required for efficient degradation of soluble ER quality control substrates, has similarity to






Drosophila melanogaster cni protein






Powers, J. et al. (1998) Transport of Ax12p depends on Erv14p, an ER-vesicle protein






related to the Drosophila cornichon gene product. J. Cell Biol. 142: 1209-1222.


29
7511220CD1
g7259234
 1.0E−75
[Mus musculus] contains transmembrane (TM) region






Inoue, S. et al.






Growth suppression of Escherichia coli by induction of expression of mammalian genes






with transmembrane or ATPase domains






Biochem. Biophys. Res. Commun. 268, 553-561 (2000)


30
7510967CD1
g14091952
0.0
[Rattus norvegicus] KIDINS220






Iglesias, T. et al.






Identification and cloning of Kidins220, a novel neuronal substrate of protein kinase D






J. Biol. Chem. 275, 40048-40056 (2000)



7510967CD1
735217|
0.0
[Homo sapiens] Protein containing eleven ankyrin (Ank) repeats, which may mediate




KIDINS220

protein-protein interactions, has a region of low similarity to a region of ankyrin 1 (human






ANK1), which is a a cytoskeletal anchor protein and is associated with hereditary






spherocytosis



7510967CD1
244565|
 9.6E−181
[Caenorhabditis elegans] Ankyrin repeat-containing protein with similarity to C. elegans




F36H1.2

UNC-44 and human and D. melanogaster ankyrins






Iglesias, T. et al. (supra)


31
7511298CD1
g1685051
0.0
[Homo sapiens] CD97






Gray, J. X. et al.






CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with






inflammation






J Immunol 157, 5438-47 (1996).



7511298CD1
762597|CD97
0.0
[Homo sapiens][Receptor (signalling)][Plasma membrane] CD97 antigen, a leukocyte






activation antigen that binds CD55 (DAF), may be involved in cell-cell signaling, cell






adhesion, immune and inflammatory responses, expressed in thyroid and gastrointestinal






tract cancer






Zendman, A. J. et al.






TM7XN1, a novel human EGF-TM7-like cDNA, detected with mRNA differential display






using human melanoma cell lines with different metastatic potential.






FEBS Lett 446, 292-8 (1999).






Aust, G. et al.






CD97: a dedifferentiation marker in human thyroid carcinomas.






Cancer Res 57, 1798-806 (1997).



7511298CD1
584465|Cd97
 5.4E−213
[Mus musculus][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] CD97






antigen, a member of the EGF TM7 family that is a group of class II seven-span






transmembrane receptors, receptor for the complement cascade regulator, CD55(Daf1),






plays a role in cell adhesion, may play a role in lymphocyte activation






Qian, Y. M. et al.






Structural characterization of mouse CD97 and study of its specific interaction with the






murine decay-accelerating factor (DAF, CD55).






Immunology 98, 303-11 (1999).


32
7510937CD1
g3766232
0.0
[Vulpes vulpes] kinectin




341688|
0.0
[Homo sapiens][Anchor Protein; Activator][Endoplasmic reticulum; Cytoplasmic] Kinectin,




KTN1

functions as a receptor for the microtubule-motor protein kinesin and plays a role in






intracellular movement of organelles; mutations in the corresponding gene are associated






with childhood papillary thyroid carcinoma.






Salassidis, K. et al. Translocation t(10; 14)(q11.2:q22.1) fusing the kinetin to the RET gene






creates a novel rearranged form (PTC8) of the RET proto-oncogene in radiation-induced






childhood papillary thyroid carcinoma. Cancer Res 60, 2786-9. (2000).




581915|Ktn1
0.0
[Mus musculus][Anchor Protein][Endoplasmic reticulum; Cytoplasmic; Plasma membrane]






Kinectin, functions as a receptor for the microtubule-motor protein kinesin and plays a role






in intracellular movement of organelles; mutations in the human KTN1 gene are associated






with childhood papillary thyroid carcinoma.






Leung, E. et al. Cloning of novel kinectin splice variants with alternative C-termini:






structure, distribution and evolution of mouse kinectin. Immunol Cell Biol 74, 421-33






(1996).


33
7511852CD1
g189186
 8.1E−149
[Homo sapiens] tumor necrosis factor receptor






Smith, C. A. et al. A receptor for tumor necrosis factor defines an unusual family of cellular






and viral proteins. Science 248, 1019-1023 (1990).




338588|
 6.5E−150
[Homo sapiens][Receptor (signalling)][Plasma membrane] Tumor necrosis factor receptor




TNFRSF1B

1b, a receptor for tumor necrosis factor (TNF), mediates proinflammatory responses






associated with wounding and immunity; mutation in gene is associated familial combined






hyperlipidemia and narcolepsy.






Chan, F. K. et al. A domain in TNF receptors that mediates ligand-independent receptor






assembly and signaling. Science 288, 2351-4 (2000).




586035|
 1.5E−81
[Mus musculus][Receptor (signalling)][Extracellular (excluding cell wall); Plasma




Tnfrsf1b

membrane] Tumor necrosis factor receptor 1b, a receptor for tumor necrosis factor (TNF),






mediates proinflammatory responses; mutation in human TNFRSF1B gene is associated






familial combined hyperlipidemia and narcolepsy.






Kurrelmeyer, K. M., Michael, L. H., Baumgarten, G., Taffet, G. E., Peschon, J. J.,






Sivasubramanian, N., Entman, M. L., and Mann, D. L. Endogenous tumor necrosis factor






protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of






acute myocardial infarction. Proc Natl Acad Sci U S A 97, 5456-61 (2000).






Azuma, Y., Kaji, K., Katogi, R., Takeshita, S., and Kudo, A. Tumor necrosis factor-alpha






induces differentiation of and bone resorption by osteoclasts. J Biol Chem 275, 4858-64.






(2000).


34
7511077CD1
g15079236
 3.3E−81
[Mus musculus] Similar to tumor differentially expressed 1




585979|Tde1
 3.0E−81
[Mus musculus] Tumor differentially expressed 1, a putative membrane protein that is






overexpressed in testicular tumor cells.






Bossolasco, M. et al. The human TDE gene homologue: localization to 20q13.1-13.3 and






variable expression in human tumor cell lines and tissue. Mol Carcinog 26, 189-200 (1999).




428528|
 3.7E−76
[Homo sapiens] Tumor differentially expressed 1, a putative membrane protein that is




TDE1

overexpressed in lung tumors and colorectal tumor cells.






Bossolasco, M. et al. (supra)






Nimmrich, I. et al. Seven genes that are differentially transcribed in colorectal tumor cell






lines. Cancer Lett 160, 37-43 (2000).


35
7511576CD1
g13661645
 1.3E−77
[Homo sapiens] MS4A6A-polymorph






Liang, Y. et al. Identification of a CD2O-, FcepsilonRIbeta-, and HTm4-related gene family:






sixteen new MS4a family members expressed in human and mouse. Genomics 72, 119-127






(2001).




697394|
 4.1E−61
[Homo sapiens][Plasma membrane]Member 6 of the membrane-spanning four-domains,




MS4A6A

subfamily A group of proteins, has similarity to CD20, HTm4 (CD20L), and high affinity






IgE receptor beta chain (FCER1B).






Ishibashi, K. et al., Identification of a new multigene four-transmembrane family (MS4A)






related to CD20, HTm4 and beta subunit of the high-affinity IgE receptor., Gene 264, 87-93.






(2001).


36
7511492CD1
g506861
 1.1E−46
[Homo sapiens] BST-2






Ishikawa, J. et al., Molecular cloning and chromosomal mapping of a bone marrow stromal






cell surface gene, BST2, that may be involved in pre-B-cell growth, Genomics 26, 527-534






(1995).




340100|BST2
 9.3E−48
[Homo sapiens][Plasma membrane] Bone marrow stromal antigen 2, a cell surface antigen






that may play a role in proliferation and cell-cell communication, likely to be involved in






humoral defense; elevated levels are associated with myeloma.






Ohtomo, T. et al. Molecular cloning and characterization of a surface antigen preferentially






overexpressed on multiple myeloma cells. Biochem Biophys Res Commun 258, 583-91.






(1999).


37
7511141CD1
g974282
 2.9E−73
[Homo sapiens] secretin receptor






Chow, B. K. Molecular cloning and functional characterization of a human secretin






receptor. Biochem. Biophys. Res. Commun. 212, 204-211 (1995).




337902|SCTR
 2.3E−74
[Homo sapiens][Receptor (signalling)][Plasma membrane] Secretin receptor, a class II G






protein-coupled receptor that can couple the cAMP and phosphatisylinositol intracellular






signaling pathways and is involved in the control of water, bicarbonate and enzyme






secretion in pancreas, gall bladder and stomach.






Shetzline, M. A. et al. A role for receptor kinases in the regulation of class II G protein-






coupled receptors. Phosphorylation and desensitization of the secretin receptor. J Biol






Chem 273, 6756-62 (1998).




705026|Sctr
 5.9E−46
[Rattus norvegicus][Receptor (signalling)][Plasma membrane] Secretin receptor, a class II






G protein-coupled receptor that couples to a stimulatory G protein, activates the cAMP






signaling pathway and is involved in the control of water, bicarbonate and enzyme secretion






in pancreas, gall bladder and stomach.






Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E., and Miller, L. J. Identification






of an interaction between residue 6 of the natural peptide ligand and a distinct residue






within the amino-terminal tail of the secretin receptor. J Biol Chem 274, 19161-7 (1999).


38
7511300CD1
g1685051
0.0
[Homo sapiens] CD97






Gray, J. X. et al. CD97 is a processed, seven-transmembrane, heterodimeric receptor






associated with inflammation. J. Immunol. 157(12): 5438-47 (1996).




762597|CD97
0.0
[Homo sapiens][Receptor (signalling)][Plasma membrane] CD97 antigen, a leukocyte






activation antigen that binds CD55 (DAF), may be involved in cell-cell signaling, cell






adhesion, immune and inflammatory responses, expressed in thyroid and gastrointestinal






tract cancer.






Gray, J. X. et al. (supra)






Aust, G. et al. CD97: a dedifferentiation marker in human thyroid carcinomas. Cancer Res






57, 1798-806 (1997).




584465|Cd97
 2.1E−242
[Mus musculus][Adhesin/agglutinin; Receptor (signalling)][Plasma membrane] CD97






antigen, a member of the EGF TM7 family that is a group of class II seven-span






transmembrane receptors, receptor for the complement cascade regulator, CD55 (Daf1),






plays a role in cell adhesion, may play a role in lymphocyte activation.






Caminschi, I., Lucas, K. M., O'Keeffe, M. A., Hochrein, H., Laabi, Y., Kontgen, F., Lew,






A. M., Shortman, K., and Wright, M. D. Molecular cloning of F4/80-like-receptor, a seven-






span membrane protein expressed differentially by dendritic cell and monocyte-macrophage






subpopulations. J Immunol 167, 3570-6. (2001).






















TABLE 3





SEQ
Incyte

Potential
Potential




ID
Polypeptide
Amino Acid
Phosphorylation
Glycosylation

Analytical Methods


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





















1
3048626CD1
747
S150 S184 S222
N291 N659 N718
Cytosolic domains: M1-K97, S150-I456,
TMHMMER





S250 S297 S307

C534-D595, T648-P714





S345 S350 S351

Transmembrane domains: V98-F117,





S357 S362 S384

I132-V149, P457-F479, V511-L533,





S440 S494 S505

V596-H618, Y628-V647, L715-G737





S555 S580 S654

Non-cytosolic domains: C118-V131, R480-I510,





S658 T74 T95

V619-H627, F738-S747





T153 T233 T247





T264 T276 T285





T386 T485 T539





T653







Class IA and IB cytochrome C signature
BLIMPS_PRINTS







PR00604: H374-S381







Cytochrome c family heme-binding site signature:
MOTIFS







C375-S380


2
2684425CD1
799
S137 S174 S182
N39 N53 N58
signal_cleavage: M1-A24
SPSCAN





S191 S254 S322
N69 N80 N135





S328 S405 S410
N304 N557 N761





S424 S435 S458





S476 S523 S545





S552 S587 S602





S629 S713 S718





S722 S726 S782





T55 T73 T85





T190 T306 T444





T495 T509 T621





T752 T767 Y779







Signal Peptide: M1-G22
HMMER







Signal Peptide: M1-A24
HMMER







Cytosolic domain: M1-N360
TMHMMER







Transmembrane domain: I361-W383







Non-cytosolic domain: R384-I799


3
7505960CD1
663
S22 S31 S102
N29 N69 N155
signal_cleavage: M1-G52
SPSCAN





S119 S218 S304
N197 N298 N393





S430 S526 S572
N405 N416 N631





T135 T447 Y13







Cytosolic domains: M1-V35, R251-R251,
TMHMMER







R331-M355, E473-D549, S610-K663







Transmembrane domains:







I36-Y58, S228-L250, L252-Y274,







T308-L330, F356-Y378, Y450-L472,







L550-S572, L587-F609







Non-cytosolic domains: G59-Q227, Y275-E307,







L379-R449, G573-H586







Leucine zipper pattern: L245-L266
MOTIFS


4
7507021CD1
150
S21 S73 T16 T26

signal_cleavage: M1-D19
SPSCAN





T85 T87 Y96







Signal Peptide: M1-D19
HMMER







Signal Peptide: M1-P22
HMMER







Signal Peptide: M1-E25
HMMER







Signal Peptide: M1-S23
HMMER







Signal Peptide: M1-T20
HMMER







Vitamin K-dependent carboxylation/gamma-carb:
HMMER_PFAM







L55-Y96







Cytosolic domain: R133-L150
TMHMMER







Transmembrane domain: L110-L132







Non-cytosolic domain: M1-S109







Vitamin K-dependent carboxylation domain:
PROFILESCAN







V30-A111







Coagulation factor GLA domain signature
BLIMPS_PRINTS







PR00001: D54-C67, L68-F81, E82-Y96







PROLINERICH GLA PROTEIN 2 PD059428:
BLAST_PRODOM







M1-D54







PROLINERICH GLA PROTEIN 2 PD059430:
BLAST_PRODOM







I95-E146







GLA DOMAIN DM00454
BLAST_DOMO







|P25155|2-80: L9-W91







|P19221|5-91: Q28-Y94







|P18292|5-91: Q28-Y94







|S49075|2-80: L7-W91







Vitamin K-dependent carboxylation domain:
MOTIFS







D54-W91


5
7509099CD1
504
S16 S35 S200
N128 N168 N184
signal_cleavage: M1-A20
SPSCAN





S225 S234 S305
N198 N232





S334 S336 S382





S402 S437 S460





S481 T152 T226





T329 Y94 Y320







Signal Peptide: M1-S16
HMMER







Signal Peptide: M1-E19
HMMER







Signal Peptide: M1-A20
HMMER







Signal Peptide: M1-K22
HMMER







Signal Peptide: M1-C23
HMMER







Signal Peptide: M1-S17
HMMER







TIR domain: A322-H472
HMMER_PFAM







Cytosolic domain: K295-G504
TMHMMER







Transmembrane domain: H272-F294







Non-cytosolic domain: M1-K271







RECEPTOR INTERLEUKIN-1 P PD02870:
BLIMPS_PRODOM







L116-F150, E169-V185, N268-K292







RECEPTOR PROTEIN PRECURSOR SIGNAL
BLAST_PRODOM







INTERLEUKIN1 TRANSMEMBRANE







GLYCOPROTEIN I IMMUNOGLOBULIN







FOLD PD002366: G317-V475







RECEPTOR INTERLEUKIN1 I PRECURSOR
BLAST_PRODOM







TRANSMEMBRANE SIGNAL TYPE IL1R1







P80







IMMUNOGLOBULIN PD011274: V216-G317







RECEPTOR TYPE I INTERLEUKIN1
BLAST_PRODOM







PRECURSOR IL1R1 P80 IMMUNOGLOBULIN







FOLD TRANSMEMBRANE PD015419:







N168-Y213







RECEPTOR PRECURSOR SIGNAL
BLAST_PRODOM







INTERLEUKIN1 IMMUNOGLOBULIN FOLD







GLYCOPROTEIN TRANSMEMBRANE TYPE







PROTEIN PD006063: L4-V97







INTERLEUKIN; ACCESSORY; INTRLEUKIN;
BLAST_DOMO







ST2L; DM02304







|P14778|323-562: A258-E498







|P13504|326-565: A258-K494







|JQ1526|326-555: A258-S488







IG-LIKE C2-TYPE DOMAIN
BLAST_DOMO







DM01362|P14778|11-227: I11-I163, D98-I163


6
7509361CD1
247
S42 S157 S208
N54 N145 N151
signal_cleavage: M1-G21
SPSCAN





S221 S237 S244





T5 T90







Signal Peptide: M1-G21
HMMER







Signal Peptide: M1-P24
HMMER







Signal Peptide: M1-G29
HMMER







TNF-receptor internal cysteine rich dom:
HMMER_INCY







C84-C125, C127-C166, C44-C81, C168-C195







TNFR/NGFR cysteine-rich region: C84-C125,
HMMER_PFAM







C44-C81, C127-C166, C168-C195







Tumor necrosis factor receptor/nerve: C84-C125,
HMMER_SMRT







C44-C81, C127-C166, C168-C195







Cytosolic domain: H33-A247
TMHMMER







Transmembrane domain: L10-P32







Non-cytosolic domain: M1-L9







TNFR/NGFR family cysteine-rich region proteins
BLIMPS_BLOCKS







BL00652: L9-L15, C58-L68, C117-C127







TUMOR NECROSIS FACTOR RECEPTOR
BLAST_PRODOM







PRECURSOR P60 TNFR1 P55







TRANSMEMBRANE GLYCOPROTEIN







PD013401: C168-S208, P214-R236







TUMOR NECROSIS FACTOR RECEPTOR
BLAST_DOMO







TYPE 1 DM04395







|P19438|120-454: D120-S208







|P50555|120-460: D120-S208, N201-A247







TNFR/NGFR FAMILY CYSTEINE-RICH
BLAST_DOMO







REGION DM00218







|P19438|39-118: K39-V119







|P50555|39-118: K39-V119







Cytochrome c family heme-binding site signature:
MOTIFS







C59-K64







EGF-like domain signature 2: C166-C179
MOTIFS







TNFR/NGFR family cysteine-rich region signature:
MOTIFS







C44-C81, C84-C125, C125-C166, C127-C166


7
7506815CD1
363
S127 S171 T270
N7 N30 N36
7 transmembrane receptor (rhodopsin family):
HMMER_PFAM







V52-Y306







Cytosolic domains: M1-H86, S158-R246,
TMHMMER







M309-G363 Transmembrane domains: A87-T109,







S135-I157, V247-Y266, A286-F308Non-cytosolic







domains: V110-W134, S267-G285







G-protein coupled receptors proteins BL00237:
BLIMPS_BLOCKS







N36-P75, F143-Y154, L242-A268, S298-R314







G-protein coupled receptors family 2 proteins
BLIMPS_BLOCKS







BL00649: R129-M150







Gastrin receptor signature PR00527: S20-N36,
BLIMPS_PRINTS







L37-S53, P75-R89, M102-P116, R117-S135,







I157-D174, A201-R217, R323-P342







Neuropeptide Y receptor PR01012: R50-A65,
BLIMPS_PRINTS







L293-N302, L304-C317







Rhodopsin-like GPCR superfamily PR00237:
BLIMPS_PRINTS







R50-I72, H86-V107, S135-S158, V247-W271,







I288-R314







G-protein coupled receptors signature: G48-T94
PROFILESCAN







Visual pigments (opsins) retinal binding site:
PROFILESCAN







G276-P341







RECEPTOR GPROTEIN COUPLED
BLAST_PRODOM







TRANSMEMBRANE GLYCOPROTEIN







LIPOPROTEIN PALMITATE







GASTRIN/CHOLECYSTOKININ TYPE B







PD005216: T109-G208







GASTRIN/CHOLECYSTOKININ TYPE B
BLAST_PRODOM







RECEPTOR CCKB CCKBR G-PROTEIN







COUPLED TRANSMEMBRANE







GLYCOPROTEIN PD009141: C307-G363







GASTRIN/CHOLECYSTOKININ TYPE B
BLAST_PRODOM







RECEPTOR CCKB CCKBR G-PROTEIN







COUPLED TRANSMEMBRANE







GLYCOPROTEIN PD007211: M1-I64







G-PROTEIN COUPLED RECEPTORS DM00013
BLAST_DOMO







|P30552|48-412: G51-A322







|P32238|35-386: G51-T320







|S17783|95-396: V52-Q182, P236-R314







|P30975|95-396: V52-Q182, P236-R314







G-protein coupled receptors signature: V56-I72
MOTIFS


8
7506814CD1
392
S82 S211 S255
N7 N30 N36
7 transmembrane receptor (rhodopsin family):
HMMER_PFAM





T299

G71-Y335







Cytosolic domains: L81-A91, R152-A171,
TMHMMER







S242-R275, M338-G392







Transmembrane domains: I58-G80,







F92-P114, A129-E151, A172-V194, S219-I241,







V276-Y295, A315-F337







Non-cytosolic domains: M1-R57,







N115-K128, V195-W218, S296-G314







G-protein coupled receptors proteins BL00237:
BLIMPS_BLOCKS







F120-P159, F227-Y238, G271-A297,







S327-R343







G-protein coupled receptors signature: S131-T178
PROFILESCAN







Visual pigments (opsins) retinal binding site:
PROFILESCAN







G305-P370







Rhodopsin-like GPCR superfamily signature
BLIMPS_PRINTS







PR00237: I56-G80, T89-F110, M134-I156,







H170-V191, S219-S242, V276-W300, I317-R343







Gastrin receptor signature PR00527:
BLIMPS_PRINTS







S20-N36, L37-E53,







F110-I125, P159-R173, M186-P200, R201-S219,







I241-D258, R352-P371







Neuropeptide Y receptor signature PR01012:
BLIMPS_PRINTS







L81-L93, T111-G123, M134-A149, L322-N331,







L333-C346







GASTRIN/CHOLECYSTOKININ TYPE B
BLAST_PRODOM







RECEPTOR CCKB CCKBR







GPROTEIN COUPLED







TRANSMEMBRANE GLYCOPROTEIN







PD007211: M1-R83







RECEPTOR GPROTEIN COUPLED
BLAST_PRODOM







TRANSMEMBRANE GLYCOPROTEIN







LIPOPROTEIN PALMITATE







GASTRIN/CHOLECYSTOKININ TYPE B







PD005216: T193-G271







GASTRIN/CHOLECYSTOKININ TYPE B
BLAST_PRODOM







RECEPTOR CCKB CCKBR







GPROTEIN COUPLED







TRANSMEMBRANE GLYCOPROTEIN







PD009141: C336-G392







RECEPTOR COUPLED GPROTEIN
BLAST_PRODOM







TRANSMEMBRANE GLYCOPROTEIN







PHOSPHORYLATION LIPOPROTEIN







PALMITATE PROTEIN FAMILY







PD000009: R83-P188







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P30552|48-412: G48-G271, A272-A351,







R8-R45







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P32238|35-386: T49-R262, L247-T349







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P25929|34-335: L52-Q344







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P25931|84-384: L60-E348







G-protein coupled receptors signature: V140-I156
MOTIFS


9
7506852CD1
125
S117 S122

signal_cleavage: M1-G59
SPSCAN







7 transmembrane receptor
HMMER_PFAM







(rhodopsin family): G26-R114







Cytosolic domains: A33-T44, V103-S125
TMHMMER







Transmembrane domains:







A10-W32, F45-I67, C80-A102







Non-cytosolic domains: M1-Q9, L68-T79







G-protein coupled receptors proteins BL00237:
BLIMPS_BLOCKS







P73-P112







G-protein coupled receptors signature: A84-S125
PROFILESCAN







Rhodopsin-like GPCR superfamily signature
BLIMPS_PRINTS







PR00237: A11-K35, T44-L65, V87-V109







Adenosine receptor signature PR00424: A10-I19,
BLIMPS_PRINTS







T79-T91







Adenosine A1 receptor signature PR00552: I5-I15,
BLIMPS_PRINTS







V34-C46, L68-C80







RECEPTOR A1 GPROTEIN COUPLED
BLAST_PRODOM







TRANSMEMBRANE GLYCOPROTEIN







ADENOSINE LIPOPROTEIN PALMITATE AS







PD007911: M1-A39







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|I48096|3-304: S4-R114







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P28190|3-303: P3-R114







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P49892|3-304: S4-R114







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|S55231|3-304: S4-R114







G-protein coupled receptors signature: S93-V109
MOTIFS


10
7503782CD1
728
S9 S200 S243
N148 N386 N582
Cytosolic domains: M1-F115, R222-S298,
TMHMMER





S248 S419 S437

Q375-N386, D469-W501, S569-Q728





S472 S536 S573

Transmembrane





S666 T60 T72

domains: L116-L138, Y199-L221,





T175 T220 T261

Y299-T321, Q352-V374,





T342 T528 Y426

L387-Q409, S446-V468, M502-I524, T546-V568







Non-cytosolic domains: R139-V198, K322-L351,







T410-L445, K525-S545







Leucine zipper pattern:
MOTIFS







L207-L228, L347-L368, L445-L466


11
7504647CD1
61
S47

signal_cleavage: M1-A23
SPSCAN







Signal Peptide: M1-G19
HMMER







Signal Peptide: M1-T21
HMMER







Signal Peptide: M1-A23
HMMER







Signal Peptide: M1-Q25
HMMER







PRECURSOR SIGNAL ADHALIN
BLAST_PRODOM







ALPHASARCOGLYCAN GLYCOPROTEIN







EPSILONSARCOGLYCAN ADHALIN35 A







DYSTROPHINASSOCIATED PD009878: M1-H51


12
7500424CD1
152


Signal Peptide: M1-G22
HMMER







Signal Peptide: M1-G25
HMMER







Signal Peptide: M1-G27
HMMER


13
7500449CD1
283
S8 S67 S139
N93 N104 N135
Domain present in hormone receptors:
HMMER_SMART





S165 S176 T111

E94-L166





T146

Hormone receptor domain: T95-K162
HMMER_PFAM







Cytosolic domain: R203-S283 Transmembrane
TMHMMER







domain: G180-F202







Non-cytosolic domain: M1-T179







G-protein coupled receptors proteins BL00237:
BLIMPS_BLOCKS







W78-A117, F181-Y192







G-protein coupled receptors family 2 signatures:
PROFILESCAN







Y74-G144







Secretin-like GPCR superfamily signature PR00249:
BLIMPS_PRINTS







T179-R203, Y211-F235







Vasoactive intestinal peptide receptor signature
BLIMPS_PRINTS







PR00491: P122-G133, N135-P150, P152-K162







RECEPTOR TRANSMEMBRANE GPROTEIN
BLAST_PRODOM







COUPLED GLYCOPROTEIN PRECURSOR







SIGNAL TYPE POLYPEPTIDE ALTERNATIVE







PD000752: C98-S244







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P32241|25-434: L68-S247







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|Q02643|16-422: S62-S244







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P41586|13-446: Q81-Q132, V136-C243







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|A53471|12-420: S66-G257







G-protein coupled receptors family 2 signature 1:
MOTIFS







C98-P122


14
7503281CD1
246
S42 S122 S202

signal_cleavage: M1-R44
SPSCAN





S222 T39 T83





T125 T226 Y172







7 transmembrane receptor (rhodopsin family):
HMMER_PFAM







G29-H246







Cytosolic domains:
TMHMMER







L38-N48, D109-R128, R194-H246







Transmembrane domains: I15-V37, L49-A71,







E86-L108, I129-K151, W171-L193 Non-cytosolic







domains: M1-A14, N72-C85, G152-A170







G-protein coupled receptors signature: A90-V136
PROFILESCAN







Rhodopsin-like GPCR superfamily signature
BLIMPS_PRINTS







PR00237: A14-L38, Q47-F68, D92-V114,







R128-I149, I173-Y196







RECEPTOR COUPLED GPROTEIN
BLAST_PRODOM







TRANSMEMBRANE GLYCOPROTEIN







PHOSPHORYLATION LIPOPROTEIN







PALMITATE PROTEIN FAMILY







PD000009: R41-Y150







ADRENERGIC RECEPTOR ADRENOCEPTOR
BLAST_PRODOM







GPROTEIN COUPLED TRANSMEMBRANE







MULTIGENE FAMILY PHOSPHORYLATION







GLYCOPROTEIN PD003999: M1-S42







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P18089|6-442: P6-S238







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|I49481|27-442: P6-A218, R205-S240







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P08913|27-442: P6-R228







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P18825|45-452: Y7-T217, K200-A245







G-protein coupled receptors signature: S98-V114
MOTIFS


15
7503292CD1
319
S5 S252 T148

signal_cleavage: M1-S53
SPSCAN







7 transmembrane receptor (rhodopsin family):
HMMER_PFAM







G49-T318







Cytosolic domains: L58-R69, E132-R151,
TMHMMER







T229-W319 Transmembrane domains: F35-V57,







H70-Y92, Y112-A131, T152-M174,







F206-V228 Non-cytosolic







domains: M1-L34, S93-Y111, G175-V205







G-protein coupled receptors signature: F113-L158
PROFILESCAN







Rhodopsin-like GPCR superfamily signature
BLIMPS_PRINTS







PR00237: L34-L58, L68-V89, H115-V137,







R151-V172, I207-V230







NEUROTENSIN RECEPTOR TYPE NTR2
BLAST_PRODOM







LEVOCABASTINE SENSITIVE GPROTEIN







COUPLED TRANSMEMBRANE LIPOPROTEIN







PD027448: M1-G66







NEUROTENSIN RECEPTOR TYPE 2 NTR2
BLAST_PRODOM







LOWAFFINITY LEVOCABASTINE SENSITIVE







NTRL GPROTEIN COUPLED







TRANSMEMBRANE LIPOPROTEIN







PALMITATE PD016080: I173-G261







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P30989|57-380: D26-Q239







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P20905|156-522: L34-L225







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P31391|41-326: D26-L219







G-PROTEIN COUPLED RECEPTORS
BLAST_DOMO







DM00013|P35371|41-345: Y39-S242







G-protein coupled receptors signature: A121-V137
MOTIFS


16
7503311CD1
284
T31 T79 T116
N62 N77 N230
Signal Peptide: M1-T25
HMMER







Domain present in hormone receptors: S57-F127
HMMER_SMART







7 transmembrane receptor
HMMER_PFAM







(Secretin family): L134-S284







Hormone receptor domain: G58-K123
HMMER_PFAM







Cytosolic domain: R163-C226 Transmembrane
TMHMMER







domains: M140-F162, V227-G249 Non-cytosolic







domains: M1-V139, G250-S284







G-protein coupled receptors family 2 proteins
BLIMPS_BLOCKS







BL00649: C61-L88, G144-S189, C216-L241







G-protein coupled receptors family 2 signatures:
PROFILESCAN







W39-G107







Secretin-like GPCR superfamily signature PR00249:
BLIMPS_PRINTS







V139-R163, Y171-P195, T218-L241, F256-P281







RECEPTOR TRANSMEMBRANE GPROTEIN
BLAST_PRODOM







COUPLED GLYCOPROTEIN PRECURSOR







SIGNAL TYPE POLYPEPTIDE ALTERNATIVE







PD000752: C61-G265







GASTRIC INHIBITORY POLYPEPTIDE
BLAST_PRODOM







RECEPTOR PRECURSOR GIPR GLUCOSE







DEPENDENT INSULINOTROPIC G PROTEIN







PD022939: Q21-L59







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P48546|21-438: Q21-A266







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P47871|18-444: L35-A266







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P43220|21-448: E34-G265







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P25107|23-499: L20-G265







G-protein coupled receptors family 2 signature 1:
MOTIFS







C61-P85


17
7510384CD1
400
T128 T347
N50
signal_cleavage: M1-G22
SPSCAN







Signal Peptide: M5-G22
HMMER







Signal Peptide: M48-G71
HMMER







Domain present in hormone receptors: T51-E121
HMMER_SMART







Hormone receptor domain: T52-E117
HMMER_PFAM







Cytosolic domain: M1-K130
TMHMMER







Transmembrane domain:







I131-A153 Non-cytosolic domain: L154-D400







G-protein coupled receptors family 2 proteins
BLIMPS_BLOCKS







BL00649: C55-F82, G136-L181







G-protein coupled receptors family 2 signatures:
PROFILESCAN







L34-G100







Secretin-like GPCR superfamily signature PR00249:
BLIMPS_PRINTS







I131-R155, Y163-F187







Vasoactive intestinal peptide receptor signature
BLIMPS_PRINTS







PR00491: P79-G90, A91-P106







RECEPTOR TRANSMEMBRANE GPROTEIN
BLAST_PRODOM







COUPLED GLYCOPROTEIN PRECURSOR







SIGNAL TYPE POLYPEPTIDE ALTERNATIVE







PD000752: C55-V200







GROWTH HORMONERELEASING HORMONE
BLAST_PRODOM







RECEPTOR PRECURSOR GHRH GRF GRFR







GPROTEIN COUPLED PD016970: M1-G54







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|Q02643|16-422: P16-M201







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P32241|25-434: M24-V200







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|P41587|13-421: M24-C195







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378|JC2532|20-434: C28-C195







G-protein coupled receptors family 2 signature 1:
MOTIFS







C55-P79


18
7509976CD1
893
S82 S182 S183
N59 N548 N637
signal_cleavage: M1-G19
SPSCAN





S188 S274 S283
N738 N746





S436 S479 S598





S671 S778 S831





T169 T248 T252





T259 T421 T614





T664 T703 T759







Signal Peptide: M1-A17
HMMER







Signal Peptide: M1-G19
HMMER







Signal Peptide: M1-R21
HMMER







Signal Peptide: M1-P22
HMMER







Signal Peptide: M1-A25
HMMER







Domain found in Plexins, Semaphorins and Int:
HMMER_SMRT







T490-V540, N637-P684, K785-S838







Plexin repeat: T490-V540, K785-S838, N637-P684
HMMER_PFAM







Sema domain: L33-Y357, A415-D471
HMMER_PFAM







Tyrosinase CuA-binding region proteins BL00497:
BLIMPS_BLOCKS







P461-K481







PLEXIN PROTEIN PRECURSOR SIGNAL
BLAST_PRODOM







KIAA0407 K04B12.1 TRANSMEMBRANE SEX







RECEPTOR GLYCOPROTEIN







PD010132: S496-H819







PLEXIN PRECURSOR SIGNAL
BLAST_PRODOM







TRANSMEMBRANE PROTEIN SEX RECEPTOR







GLYCOPROTEIN PD003973: R352-H474







SEMAPHORIN PROTEIN PRECURSOR
BLAST_PRODOM







RECEPTOR KINASE SIGNAL TYROSINE







TYROSINEPROTEIN FAMILY HEPATOCYTE







PD001844: T34-W284, I96-V454







do KINASE; TYROSINE; ATP; GROWTH;
BLAST_DOMO







DM01368|P51805|796-899: C796-R893







do KINASE; TYROSINE; HEPATOCYTE; ATP;
BLAST_DOMO







DM03653|P08581|14-526: D30-A498







do KINASE; TYROSINE; HEPATOCYTE; ATP;
BLAST_DOMO







DM03653|Q04912|17-533: V45-C497







do KINASE; TYROSINE; HEPATOCYTE; ATP;
BLAST_DOMO







DM03653|A48196|13-528: H35-L365







ATP/GTP-binding site motif A (P-loop):
MOTIFS







G168-S175


19
7510454CD1
203
S53 S198 T45
N117
Signal Peptide: M1-A15
HMMER





Y128







Signal Peptide: M1-M19
HMMER







Cytosolic domain: K27-D56 Transmembrane
TMHMMER







domains: F4-L26, I57-I75 Non-cytosolic domains:







M1-S3, C76-P203







Leucine zipper pattern: L127-L148
MOTIFS


20
8017335CD1
429
S80 S258 S287
N181 N208 N285
Cytosolic domains: L73-S83, L144-K163,
TMHMMER





S296 S343 S357

C224-V247, R323-N429 Transmembrane domains:





S366 S413 T157

S50-L72, L84-L106, F121-N143, I164-M186,







V201-I223, V248-I270, Y300-F322







Non-cytosolic domains: M1-L49,







S107-H120, L187-T200, S271-E299







PUTATIVE SEVEN PASS TRANSMEMBRANE
BLAST_PRODOM







PROTEIN TRANSMEMBRANE PD138976:







M1-L361


21
7510197CD1
101
S40

signal_cleavage: M1-F16
SPSCAN







Signal Peptide: M1-F16
HMMER







Signal Peptide: M1-D18
HMMER







Cytosolic domain: T68-L101 Transmembrane
TMHMMER







domain: A45-L67 Non-cytosolic domain: M1-C44







Leucine zipper pattern:
MOTIFS







L39-L60, L46-L67, L53-L74







Prenyl group binding site (CAAX box): C99-L101
MOTIFS


22
7510055CD1
237
S97 S156 T55
N153 N180
signal_cleavage: M1-P20
SPSCAN





T104 T141 T165





T179







Signal Peptide: M1-P20
HMMER







Signal Peptide: M1-E28
HMMER







Signal Peptide: M1-A25
HMMER







Signal Peptide: M1-C26
HMMER







TNF-receptor internal cysteine rich domain:
HMMER_INCY







C62-C103, C146-C186,







C105-C143, C26-C59







Tumor necrosis factor receptor/nerve: C105-C143,
HMMER_SMART







C62-C103, C146-C186, C26-C59







TNFR/NGFR cysteine-rich region:
HMMER_PFAM







C26-C59, C62-C103,







C105-C143, C146-C186







TNFR/NGFR family cysteine-rich region proteins
BLIMPS_BLOCKS







BL00652: C37-L47, G95-C105







CD40L RECEPTOR PRECURSOR BCELL
BLAST_PRODOM







SURFACE ANTIGEN CD40 BP50 CDW40







GLYCOPROTEIN PD154353: P61-T104







CD40L RECEPTOR PRECURSOR BCELL
BLAST_PRODOM







SURFACE ANTIGEN CD40 BP50 CDW40







GLYCOPROTEIN TRANSMEMBRANE REPEAT







SIGNAL PD059682: M1-C59







RECEPTOR ACTIVATOR OF NFKAPPAB RANK
BLAST_PRODOM







PD173848: C8-C103







RECEPTOR FACTOR TUMOR NECROSIS
BLAST_PRODOM







HOMOLOG II PROTEIN PRECURSOR REPEAT







SIGNAL PD149629: C105-T165







TNFR/NGFR FAMILY CYSTEINE-RICH
BLAST_DOMO







REGION







DM00218|P25942|99-178: C99-T179







TNFR/NGFR FAMILY CYSTEINE-RICH
BLAST_DOMO







REGION







DM00218|P25942|22-97: P22-E98







TNFR/NGFR FAMILY CYSTEINE-RICH
BLAST_DOMO







REGION







DM00218|P27512|99-178: T99-T179







TNFR/NGFR FAMILY CYSTEINE-RICH
BLAST_DOMO







REGION







DM00218|P27512|22-97: C26-E98







EGF-like domain signature 2: C103-C116
MOTIFS







TNFR/NGFR family cysteine-rich region signature:
MOTIFS







C26-C59


23
7501754CD1
460
S134 S207 S276
N130 N205 N284
signal_cleavage: M1-G56
SPSCAN





S318 S349 T156
N355





T157 T286







Cytosolic domains: R68-G87, K237-K240,
TMHMMER







P414-H460







Transmembrane domains: L45-I67, G88-Y110,







W214-A236, W241-L263, G391-L413Non-







cytosolic







domains: M1-L44, G111-R213, E264-E390







TWEETY F42E11.2 PROTEIN PD043235:
BLAST_PRODOM







L19-S426


24
7510517CD1
218
S45 S160 S187
N7 N177
signal_cleavage: M27-A78
SPSCAN





S202 S205 Y55







7 transmembrane receptor (rhodopsin family):
HMMER_PFAM







G34-L218







Cytosolic domains: C43-T53, E116-L135
TMHMMER







Transmembrane domains: P20-L42, F54-T76,







T96-V115, A136-L158







Non-cytosolic domains: M1-S19, I77-S95,







G159-L218







G-protein coupled receptors proteins
BLIMPS_BLOCKS







BL00237: W85-A124′′







G-protein coupled receptors signature: F97-Y144
PROFILESCAN







Prostaglandin receptor signature
BLIMPS_PRINTS







PR00428: T22-V33, G68-Y80, A136-L149







Prostanoid EP4 receptor signature
BLIMPS_PRINTS







PR00586: S2-T22, C43-G60, M81-F102,







N122-T139, F171-Y191







RECEPTOR PROSTAGLANDIN E2 EP4
BLAST_PRODOM







SUBTYPE







PROSTANOID PGE G-PROTEIN COUPLED







TRANSMEMBRANE







PD014814: M1-E51







PROSTAGLANDIN; SUBTYPE; EP3;
BLAST_DOMO







PROSTACYCLIN







DM00355|P35408|11-344: S11-V200







DM00355|P43119|7-307: P20-R212







DM00355|P43253|36-335: P20-C211







DM00355|S52078|36-335: P20-C211







G-protein coupled receptors signature: S105-I121
MOTIFS


25
7511014CD1
297
S94 S144 T148
N4 N19
Signal Peptide: M3-C22, M3-T24
HMMER





Y201







7 transmembrane receptor (rhodopsin family):
HMMER_PFAM







S43-Y281







Cytosolic domains: M1-L28, V89-C108,
TMHMMER







H174-L202, L272-E297







Transmembrane domains: S29-L51, F66-A88,







S109-I131, H151-G173, L203-I225, I249-I271







Non-cytosolic domains: M52-S65,







E132-K150, T226-V248







G-protein coupled receptors proteins
BLIMPS_BLOCKS







BL00237: F101-P140, S242-Y268







G-protein coupled receptors signature: F111-F162
PROFILESCAN







Prostaglandin receptor signature
BLIMPS_PRINTS







PR00428: G80-Y92, S206-C219







Thromboxane receptor signature
BLIMPS_PRINTS







PR00429: G72-G85, F111-G126,







H174-N189, E197-G214







Prostaglandin F receptor signature
BLIMPS_PRINTS







PR00855: S2-Q23, E25-V39, K53-L68,







I171-T184, Y188-F205, L228-H244







PROSTAGLANDIN F2-ALPHA RECEPTOR
BLAST_PRODOM







PROSTANOID FP PGF PGF2 ALPHA







G-PROTEIN COUPLED







PD012201: M1-F66







PD012850: H174-Y201







PROSTAGLANDIN; SUBTYPE; EP3;
BLAST_DOMO







PROSTACYCLIN







DM00355|P43118|20-319: T20-W293







DM00355|S51281|20-319: T20-W293







DM00355|P37289|20-319: T20-L266







DM00355|P34995|26-365: I35-R238, R238-L266







G-protein coupled receptors signature: C121-V137
MOTIFS


26
7506687CD1
917
S199 S260 S509
N31 N334 N543
Signal Peptide: M1-T19
HMMER





S545 S555 S625





S646 S793 S834





S870 T173 T185





T203 T283 T411





T661 T720 T783





T856 Y142 Y341







Plexin repeat: S481-L534, R636-P682
HMMER_PFAM







domain found in Plexins, Semaphorins and
HMMER_SMART







Integrins: S481-L534, D628-P678







RECEPTOR KIAA0407 SEMAPHORIN
BLAST_PRODOM







PD184600: M1-H465







PD145279: K663-L882







PLEXIN PROTEIN PRECURSOR SIGNAL
BLAST_PRODOM







KIAA0407 K04B12.1 TRANSMEMBRANE SEX







RECEPTOR GLYCOPROTEIN







PD010132: P541-T708







RGD cell attachment sequence: R872-D874
MOTIFS


27
7510621CD1
224
S120 S216 S219
N116 N136 N158
signal_cleavage: M7-G74
SPSCAN





T138







Signal Peptide: M1-A25, M1-G32, M1-A35,
HMMER







M7-A25, M7-G27, M7-A30, M7-V31







Tetraspanin family: K11-I217
HMMER_PFAM







Cytosolic domains: M1-F12, C73-N188
TMHMMER







Transmembrane domains: L13-A35,







G50-C72, V189-C211







Non-cytosolic domains: Q36-P49, C212-M224







Transmembrane 4 family proteins
BLIMPS_BLOCKS







BL00421: K8-V26, V56-R94, M125-N136,







V151-C156, N188-I217







Transmembrane 4 family signature: T48-E100
PROFILESCAN







Transmembrane four family signature
BLIMPS_PRINTS







PR00259: F12-A35, G50-C76, V191-I217







TRANSMEMBRANE GLYCOPROTEIN SIGNAL
BLAST_PRODOM







ANCHOR PROTEIN ANTIGEN MEMBRANE







PHOTORECEPTOR VISION CD9 CELL







PD000920: K11-S145, Y80-C163, C163-I217







TRANSMEMBRANE 4 FAMILY
BLAST_DOMO







DM00947|P08962|1-232: A2-G220







DM00947|S43511|2-233: A2-G220







DM00947|P41732|2-238: C9-I217







DM00947|P27591|3-214: G6-I217







Transmembrane 4 family signature: G61-M83
MOTIFS


28
7505533CD1
114
S9 S28 T26

Signal Peptide: M1-S20
HMMER







Cornichon protein: E2-L110
HMMER_PFAM







Cytosolic domains: M1-V4, P76-L114
TMHMMER







Transmembrane domains: V5-L27, I53-L75







Non-cytosolic domain: S28-L52







C5A-anaphylatoxin receptor signature
BLIMPS_PRINTS







PR00426: L11-F23







PROTEIN TRANSMEMBRANE CORNICHON
BLAST_PRODOM







DEVELOPMENTAL CORNICHON-LIKE T09E8.3







ER-DERIVED VESICLES ERV14







ENDOPLASMIC PD008226: F6-R84







CORNICHON
BLAST_DOMO







DM04292|P53173|1-137: M1-Q87







DM04292|P38312|1-141: V5-T8


29
7511220CD1
181
S5 S143 T172

signal_cleavage: M1-A21
SPSCAN





Y32







Signal Peptide: M1-A21, M1-L22,
HMMER







M1-R24, M1-D28







Cytosolic domains: M1-C4, I55-R60, T172-K181
TMHMMER







Transmembrane domains: S5-L22, Y32-T54,







Y61-Y83, H149-F171







Non-cytosolic domains: E23-G31, L84-L148


30
7510967CD1
1753
S167 S219 S363
N71 N165 N231
Ankyrin repeat: C37-L69,
HMMER_PFAM





S381 S430 S471
N303 N315 N766
G103-L135, D236-R268,





S562 S614 S722
N971 N1309
Y170-A202, D335-K367, S269-Q301, D70-M102,





S883 S886 S1034
N1329
Y137-K169, N203-K235, D302-K334, K368-R400





S1164 S1291
N1578 N1669





S1311





S1350 S1377





S1389





S1411 S1448





S1453





S1479 S1503





S1508





S1565 S1589





S1605





S1634 S1643





S1644





S1719 T233 T432





T434 T590 T621





T791 T862 T904





T939 T950 T998





T1001 T1012





T1148 T1218





T1254 T1336





T1358 T1459





T1715 Y409





Y1442







ankyrin repeats: C37-L66, G103-V132,
HMMER_SMART







Y170-Q199, D236-I265, D335-A364,







S269-I298, Y137-C166, D70-H99, D302-I331,







N203-L232, K368-Y399







Cytosolic domains: F519-N524, R682-H687
TMHMMER







Transmembrane domains: F496-A518, L525-G547,







P659-F681, L688-L707







Non-cytosolic domains: M1-L495,







G548-L658, N708-L1753







Ankyrin repeat signature PR01415: G171-H183,
BLIMPS_PRINTS







N348-A360







Ank repeat proteins. PF00023: L42-L57,
BLIMPS_PFAM







G369-R378







Domain present in ZO-1 and Unc5-like netrin
BLIMPS_PFAM







receptor







PF00791: L42-N96, L354-P392, L983-D1025







REPEAT PROTEIN ANK NUCLEAR ANKYR.
BLIMPS_PRODOM







PD00078: D366-R378







F36H1.2 PROTEIN
BLAST_PRODOM







PD148722: D267-K304 K361-P1082 L1189-R1252







Cell attachment sequence: R1436-D1438
MOTIFS







ATP/GTP-binding site motif A (P-loop):
MOTIFS







A467-S474


31
7511298CD1
786
S50 S185 S243
N33 N38 N108
signal_cleavage: M1-T20
SPSCAN





S283 S307 S329
N322 N357 N364





S371 S384 S394
N404 N471 N774





S418 S423 S451





S453 S602 T57





T61 T105 T113





T159 T205 T670







Signal Peptide: M1-T20
HMMER







7 transmembrane receptor (Secretin family):
HMMER_PFAM







D495-V744







EGF-like domain: C120-C158, C164-G197,
HMMER_PFAM







C68-G102, C26-P58







Latrophilin/CL-1-like GPS domain: Q442-V493
HMMER_PFAM







Epidermal growth factor-like domain: E119-T159,
HMMER_SMART







E163-E208, E67-Q115, G25-D63







Calcium-binding EGF-like domain: D116-T159,
HMMER_SMART







D160-E208, D64-Q115







G-protein-coupled receptor proteolytic
HMMER_SMART







site: Q442-V493







Cytosolic domains: Q527-T532, F590-L601,
TMHMMER







W667-K685, N741-I786







Transmembrane domains: V504-I526, I533-I552,







A567-Y589, S602-I624, L644-V666, A686-L708,







L718-L740







Non-cytosolic domains: M1-R503, E553-V566,







Y625-F643, F709-V717







G-protein coupled receptors family 2 (secretin-like)
BLIMPS_BLOCKS







IPB000832: G505-A550, C563-L588, G610-Y634,







W645-S674, L689-I710, C727-V755







Calcium-binding EGF-like domain IPB001881:
BLIMPS_BLOCKS







C42-S52, C133-C144







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







A41-F51, T132-F148







EMR1 hormone receptor signature PR01128:
BLIMPS_PRINTS







K450-G468, V523-C540, S621-C635, D679-L700







CD97 protein signature PR01278: C26-C42,
BLIMPS_PRINTS







G96-Q115, D204-H222, H222-D239,







Q280-P297, K298-E317,







R331-E343, M358-K373, V388-T406,







H480-D495, E553-H571, R594-I609, K668-L683







CD97 LEUCOCYTE ANTIGEN PRECURSOR
BLAST_PRODOM







G-PROTEIN-COUPLED RECEPTOR







TRANSMEMBRANE GLYCOPROTEIN







EGF-LIKE







PD040384: W192-Q389







PD028353: M1-C114







PD005792: A752-I786







RECEPTOR TRANSMEMBRANE G-PROTEIN-
BLAST_PRODOM







COUPLED GLYCOPROTEIN PRECURSOR







SIGNAL TYPE POLYPEPTIDE ALTERNATIVE







PD000752: Q477-W751







HORMONE; EMR1; LEUCOCYTE; ANTIGEN;
BLAST_DOMO







DM05221|I37225|347-738: R391-E783







DM05221|P48960|347-738: R391-E783







DM05221|A57172|465-886: E408-T768







LEUCOCYTE; ANTIGEN; CD97;
BLAST_DOMO







DM08257|P48960|171-254: W215-G299







Aspartic acid and asparagine hydroxylation site:
MOTIFS







C82-C93, C133-C144, C177-C188







Calcium-binding EGF-like domain pattern signature:
MOTIFS







D64-C91, D116-C142, D160-C186







G-protein coupled receptors family 2 signature 2:
MOTIFS







Q729-V744


32
7510937CD1
1328
S75 S110 S161
N172 N435 N772
Cytosolic domain: M1-S6 Transmembrane domain:
TMHMMER





S188 S206 S212
N904 N1059
A7-M29 Non-cytosolic domain: K30-E1328






N1234





S213 S290 S297
N1300
Tropomyosin IPB000533: L462-E498, K612-Q655,
BLIMPS_BLOCKS





S322 S323 S397

H511-E565





S625 S632 S694

PROTEIN KINECTIN CG1 KIAA0004 A
BLAST_PRODOM





S794 S812 S906

COILEDCOIL PD017436: M1-K283





S926 S957 S975

PROTEIN KINECTIN ES/130 RIBOSOME
BLAST_PRODOM





S1002 S1017

RECEPTOR CG1 KIAA0004 A COILEDCOIL





S1061 S1081

PD013824: Q264-E400





S1142 S1156

ES/130 RIBOSOME RECEPTOR PD074881:
BLAST_PRODOM





S1215 S1290

E1040-E1235





T32 T50 T52

PROTEIN KINECTIN CG1 KIAA0004 A
BLAST_PRODOM





T200 T268

COILEDCOIL PD151414: T401-G467





T273 T275





T364 T463 T466

RIBOSOME; 160K; 180K; DM05457
BLAST_DOMO





T508 T631 T746

|S32763|1-529: M1-S531





T760 T830 T859

|A56734|660-1039: P165-E510





T878 T893 T1145

RIBOSOME; 160K; 180K; DM05456
BLAST_DOMO





T1154 T1276

|A56734|1041-1479: Q901-E1235





Y503

|S32763|1001-1356: S1002-L1327





Y1194 Y1216

|S32763|1001-1356: W1012-E1328







Leucine zipper pattern: L935-L956, L942-L963
MOTIFS


33
7511852CD1
355
S77 S129 S174
N171 N193 N274
signal_cleavage: M1-A22
SPSCAN





S221 S254 S283

Signal Peptide: M1-A20, M1-A22, M1-P24
HMMER





S305 S309 S318

TNFR/NGFR cysteine-rich region: C40-C75,
HMMER_PFAM





S326 S337 T39

C78-C118, C120-C161, C164-C200





T73 T119 T298

Tumor necrosis factor receptor/nerve growth factor
HMMER_SMART







receptor: C120-C161, C78-C118,







C40-C75, C164-C200







TNF-receptor internal cysteine rich domain:
HMMER_INCY







C120-C161, C40-C75, C78-C118, C164-C200







EGF-like domain IPB000561: C118-C126
BLIMPS_BLOCKS







TNFR/NGFR family cysteine-rich region
BLIMPS_BLOCKS







IPB001368: C53-A63, C110-C120







RECEPTOR TUMOR NECROSIS FACTOR P80
BLAST_PRODOM







PRECURSOR TNFR2 P75 TRANSMEMBRANE







GLYCOPROTEIN PD024155: V149-P166,







I190-S355, Q183-T330







RECEPTOR FACTOR TUMOR NECROSIS
BLAST_PRODOM







HOMOLOG II PROTEIN PRECURSOR REPEAT







SIGNAL PD149629: C120-H182







RECEPTOR P80 TNFALPHA
BLAST_PRODOM







TUMOR NECROSIS







FACTOR PRECURSOR TNFR2 P75







TRANSMEMBRANE PD059688: M1-Q51







TUMOR NECROSIS FACTOR RECEPTOR
BLAST_PRODOM







PRECURSOR BINDING PROTEIN TBPII P80







TNFR2 PD153238: L23-Q51







TUMOR NECROSIS FACTOR RECEPTOR
BLAST_DOMO







TYPE 2 DM06946







|P20333|195-460: V199-S355







|P25119|197-473: S195-V262, D263-S355







TNFR/NGFR FAMILY CYSTEINE-RICH
BLAST_DOMO







REGION DM00218







|P20333|113-193: E113-A194







|P20333|35-111: E35-R112







TNFR/NGFR family cysteine-rich region signature:
MOTIFS







C40-C75, C78-C118


34
7511077CD1
295
S120 T286
N34 N243
TMS membrane protein/tumour differentially
HMMER_PFAM







expressed protein (TDE): S15-L295







Cytosolic domains: C28-R39, S119-N130,
TMHMMER







H183-W201, P257-L295







Transmembrane domains: L5-S27, L40-L57,







A96-V118, G131-I150, F160-A182, Y202-F224,







F239-L256 Non-cytosolic domains: M1-C4,







S58-R95, P151-W159, M225-V238







PROTEIN PLACENTAL DIFF33
BLAST_PRODOM







DEVELOPMENTALLY REGULATED R11H6.2







PD011773: D87-P266







PD018175: C13-E73


35
7511576CD1
203
S22 S36 S91
N8 N20
Cytosolic domains: M1-K66, T158-S203
TMHMMER





S188 T60

Transmembrane domains: L67-I89, A135-L157





T65 T132

Non-cytosolic domain: L90-K134





T148 Y197


36
7511492CD1
156
S146 T4 T45 T94
N65 N92
signal_cleavage: M1-G38
SPSCAN







Cytosolic domain: M1-K21 Transmembrane
TMHMMER







domain: L22-F44







Non-cytosolic domain: T45-A156







BONE MARROW STROMAL ANTIGEN 2 BST2
BLAST_PRODOM







TRANSMEMBRANE GLYCOPROTEIN







SIGNALANCHOR PD095137: M1-G116.


37
7511141CD1
170
S130 T97
N72 N100 N106
signal_cleavage: M1-A27
SPSCAN






N128
Signal Peptide: M1-A22, M1-S24, M1-A27,
HMMER







M1-A19







Hormone receptor domain: V63-N128
HMMER_PFAM







Domain present in hormone receptors: P62-N132
HMMER_SMART







G-protein coupled receptors family 2 (secretin-like)
BLIMPS_BLOCKS







IPB000832: C66-L93







Vasoactive intestinal peptide receptor 1 signature
BLIMPS_PRINTS







PR01154: L31-E52, W76-F92







G-protein coupled receptors family 2 signatures:
PROFILESCAN







C45-G111







Secretin receptor signature PR00490: R2-L14,
BLIMPS_PRINTS







L18-V34, V37-E52, E52-V63,







P90-F104, C123-E136







G-PROTEIN COUPLED RECEPTORS FAMILY 2
BLAST_DOMO







DM00378







|JC2532|20-434: C20-L139







|P47872|20-434: C20-L139







|S47631|30-491: L12-M95 R105-S130







|P41586|13-446: E40-M95 G101-S130







G-protein coupled receptors family 2 signature 1:
MOTIFS







C66-P90


38
7511300CD1
801
S50 S234 S292
N33 N38 N108
signal_cleavage: M1-T20
SPSCAN





S332 S356 S378
N203 N371 N406
Signal Peptide: M1-T20
HMMER





S420 S433 S443
N413 N453 N520
7 transmembrane receptor (Secretin family):
HMMER_PFAM





S467 S472 S500

D544-A793





S502 S651 T57

EGF-like domain: C120-C158, C213-G246,
HMMER_PFAM





T61 T105

C164-P199, C68-G102, C26-P58





T113





T205 T254 T719

Latrophilin/CL-1-like GPS domain: Q491-V542
HMMER_PFAM







Epidermal growth factor-like domain.: E119-T159,
HMMER_SMART







E163-E208, G25-D63, E67-Q115, E212-E257







Calcium-binding EGF-like domain: D116-T159,
HMMER_SMART







D160-E208, D209-E257, D64-Q115







G-protein-coupled receptor proteolytic site:
HMMER_SMART







Q491-V542







Cytosolic domains: Q576-T581, Q646-S651,
TMHMMER







K717-R736, C787-I801







Transmembrane domains: V553-I575,







I582-I601, F623-F645, T652-Y674, W694-W716,







A737-I759, S764-H786 Non-cytosolic







domains: M1-R552, E602-C622,







S675-L693, F760-R763







Calcium-binding EGF-like domain IPB001881:
BLIMPS_BLOCKS







C142-P152, C133-C144







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







A41-F51 A41-F51







G-protein coupled receptors family IPB000832:
BLIMPS_BLOCKS







G554-A599, C612-L637, G659-S683,







W694-S723, L738-I759, C776-I801







Type II EGF-like signature PR00010: D116-C127,
BLIMPS_PRINTS







G138-F148







EMR1 hormone receptor signature PR01128:
BLIMPS_PRINTS







K499-G517, V572-C589, S670-C684, D728-L749







CD97 protein signature PR01278: C26-C42,
BLIMPS_PRINTS







G96-Q115, D253-H271, H271-D288,







Q329-P346, K347-E366, R380-E392,







M407-K422, V437-T455, H529-D544,







E602-H620, R643-I658, K717-L732







CD97 LEUCOCYTE ANTIGEN PRECURSOR
BLAST_PRODOM







GPROTEIN COUPLED RECEPTOR







TRANSMEMBRANE GLYCOPROTEIN EGF-







LIKE; PD028353: M1-C114







PD040384: W192-V210 W241-Q438







RECEPTOR TRANSMEMBRANE GPROTEIN
BLAST_PRODOM







COUPLED GLYCOPROTEIN PRECURSOR







SIGNAL TYPE POLYPEPTIDE ALTERNATIVE







PD000752: Q526-K792







HORMONE; EMR1; LEUCOCYTE; ANTIGEN;
BLAST_DOMO







DM05221







|I37225|347-738: R440-K792







|A57172|465-886: E457-N790







|P48960|347-738: R440-K792







LEUCOCYTE; ANTIGEN; CD97; DM08257
BLAST_DOMO







|P48960|171-254: W264-G348







Calcium-binding EGF-like domain pattern signature:
MOTIFS







D64-C91, D116-C142, D160-C186, D209-C235

















TABLE 4





Polynucleotide



SEQ ID NO:/


Incyte ID/Sequence


Length
Sequence Fragments







39/3048626CB1/
1-631, 10-543, 15-646, 80-703, 157-686, 190-754, 205-683, 205-714, 205-741,


2714
218-917, 224-720, 236-885, 256-871, 310-981, 335-894,



335-1009, 351-998, 372-915, 374-1057, 375-948, 378-883, 378-1017, 



382-1063, 384-1049, 385-1037, 396-1028, 404-1011, 431-1040, 438-1035,



460-1045, 462-898, 492-1178, 502-1222, 519-1154, 527-714, 533-1174,



553-1057, 558-1195, 566-1041, 566-1190, 575-895, 586-869,



586-1187, 586-1253, 605-1039, 605-1305, 616-1324, 623-1248, 625-1251,



638-1215, 641-1230, 649-1061, 650-1170, 662-1329, 665-1253,



668-1220, 682-1265, 685-845, 687-1258, 695-1360, 703-1354,



704-1230, 709-1240, 724-1314, 726-1402, 735-1335, 747-1435, 751-1329,



777-1430, 781-1263, 787-1233, 789-1532, 794-1549, 804-1351, 812-1353,



824-1138, 824-1375, 824-1386, 824-1399, 824-1402, 858-1314,



861-1378, 911-1556, 916-1547, 930-1551, 935-1427, 935-1643,



936-1617, 946-1628, 968-1639, 974-1481, 977-1549, 1001-1671,



1017-1671, 1031-1640, 1037-1588, 1038-1217, 1038-1361, 1043-1558,



1044-1651, 1087-1669, 1095-1463, 1096-1671, 1102-1671, 1109-1408,



1109-1523, 1151-1671, 1576-2268, 2045-2308, 2178-2714


40/2684425CB1/
1-298, 19-268, 29-320, 37-883, 55-575, 55-704, 94-356, 99-613,


2858
110-755, 237-1049, 445-1099, 509-1105, 533-785, 533-954, 541-785, 541-813



541-1174, 544-1223, 546-809, 664-955, 666-1270, 668-1274, 719-1337,



723-1303, 783-1358, 797-1357, 802-1274, 813-1417, 820-1134,



832-1439, 854-1362, 881-1235, 916-1412, 920-1100, 984-1630,



1013-1675, 1015-1272, 1047-1664, 1087-1706, 1106-1704, 1119-1621,



1129-1722, 1215-1749, 1235-1533, 1235-1815, 1304-1886, 1329-1921,



1347-1587, 1360-1621, 1362-1638, 1362-1740, 1369-1872,



1443-1934, 1443-1984, 1445-2045, 1569-2199, 1596-1761, 1599-1980,



1627-1898, 1632-1861, 1639-2223, 1649-2167, 1675-1938, 1675-2151,



1681-1950, 1746-1901, 1753-2427, 1770-2010, 1787-2397, 1904-2180,



1904-2581, 1951-2331, 2181-2777, 2224-2782, 2275-2521,



2310-2790, 2317-2817, 2317-2832, 2413-2819, 2610-2858, 2650-2858, 2714-2855


41/7505960CB1/
1-315, 1-340, 1-457, 1-479, 1-487, 1-640, 1-646, 1-2239, 2-597,


2445
3-330, 3-626, 5-518, 5-551, 5-620, 8-260, 8-493, 8-521, 8-637, 10-536, 11-571,



12-281, 14-111, 17-274, 18-285, 19-194, 19-259, 19-620, 19-630,



19-636, 20-273, 22-305, 22-584, 24-288, 24-479, 25-288, 25-397,



77-325, 91-364, 91-578, 104-561, 106-868, 131-433, 141-676, 161-784, 171-487, 171-489,



188-330, 194-708, 197-784, 210-411, 210-853,



219-485, 266-879, 269-625, 271-901, 282-814, 294-561, 294-899, 295-805,



298-531, 300-581, 312-861, 335-920, 335-927, 343-962, 387-1114,



418-1146, 450-1185, 450-1289, 454-1009, 457-703, 461-1147, 464-1051,



478-827, 479-611, 482-1072, 532-1163, 534-1057, 538-1216,



545-1070, 583-1165, 585-1107, 602-1241, 630-1190, 645-1202,



656-1237, 657-1243, 658-1054, 663-859, 673-1270, 691-1289, 691-1302,



695-1223, 720-1301, 770-1062, 776-1242, 787-1028, 808-991, 843-976,



865-1275, 902-1017, 903-1276, 922-1190, 932-1073, 955-1253,



961-1222, 964-1246, 966-1214, 971-1208, 985-1245, 1013-1286,



1028-1313, 1040-1313, 1058-1313, 1067-1297, 1247-1515,



1247-1592, 1247-1643, 1247-1646, 1247-1803, 1247-1836, 1254-1876,



1310-1603, 1310-1780, 1310-1785, 1310-1880, 1310-1949, 1312-1586,



1312-1808, 1313-1534, 1313-1877, 1324-1642, 1345-1848, 1356-1717,



1357-1749, 1359-1618, 1369-1637, 1369-1808, 1369-1917,



1371-1884, 1373-2020, 1375-1649, 1379-1588, 1379-1918, 1382-1586,



1385-1946, 1390-1619, 1398-1935, 1400-1777, 1405-1671, 1405-1943,



1408-1775, 1410-1902, 1413-1902, 1428-2133, 1431-2018, 1435-1622,



1442-1758, 1448-2178, 1456-1719, 1456-1764, 1467-1738,



1467-1758, 1474-1967, 1474-2071, 1477-1891, 1479-1603, 1483-1753,



1487-2007, 1492-1762, 1497-2191, 1498-1923, 1507-1769, 1507-1888,



1510-1776, 1511-1758, 1516-1792, 1521-2143, 1526-1853, 1536-1808,



1540-2085, 1542-2053, 1544-2231, 1548-2011, 1548-2083,



1551-1951, 1551-2218, 1552-1991, 1552-2124, 1554-1822, 1554-2378,



1570-1859, 1580-1780, 1580-2360, 1604-1928, 1607-2098, 1608-2229,



1610-1824, 1620-1885, 1626-1895, 1632-1996, 1634-1890, 1634-1898,



1636-2284, 1637-1904, 1637-1915, 1638-2231, 1655-2372,



1658-2207, 1673-2202, 1673-2241, 1677-2269, 1686-2273, 1690-1931,



1691-1934, 1693-2015, 1697-2355, 1701-2147, 1703-2063,



1705-1922, 1706-2141, 1713-2321, 1714-2359, 1715-2146, 1731-1963,



1743-2032, 1744-2102, 1755-1960, 1759-2271, 1760-2163, 1761-2012,



1765-2039, 1765-2040, 1767-2176, 1776-2251, 1782-2421, 1787-2046,



1787-2278, 1788-2187, 1791-2244, 1794-1985, 1796-2375,



1799-2060, 1799-2101, 1815-2388, 1818-2419, 1820-2359, 1821-2417,



1823-2443, 1827-2061, 1828-2383, 1830-2416, 1831-2358, 1835-2422,



1844-2101, 1849-2062, 1849-2434, 1853-2157, 1862-2357, 1862-2442,



1862-2444, 1873-2283, 1877-2158, 1878-2419, 1886-2033,



1888-2438, 1898-2187, 1903-2444, 1911-2221, 1916-2418, 1927-2101,



1929-2324, 1931-2324, 1933-2101, 1936-2445, 1937-2417, 1941-2101,



1941-2182, 1944-2239, 1959-2161, 1961-2222, 1966-2233, 1981-2399,



1983-2101, 1989-2222, 1989-2425, 1991-2101, 1996-2212,



2002-2101, 2008-2101, 2017-2101, 2021-2077, 2021-2087, 2023-2135,



2028-2101, 2029-2101, 2030-2101, 2031-2101, 2033-2101, 2040-2101,



2043-2322, 2046-2101, 2046-2125, 2118-2147


42/7507021CB1/
1-137, 1-223, 1-250, 1-357, 1-390, 1-424, 1-433, 1-532, 1-545,


1248
1-573, 1-1248, 3-584, 4-584, 5-224, 5-585, 11-244, 11-299, 11-530, 11-538,



11-554, 11-579, 24-585, 28-585, 56-537, 93-585, 115-328, 115-565,



115-715, 115-767, 116-258, 117-258, 137-359, 137-585, 144-585, 152-566,



203-501, 210-456, 251-513, 292-448, 577-1108, 585-730, 585-816,



585-854, 590-1226, 593-1248, 627-1156, 628-841, 629-1248, 640-1248,



647-868, 648-767, 657-1082, 661-767, 677-1132, 678-912, 678-1197,



699-908, 702-836, 715-1119, 726-1246, 741-767, 743-1248,



746-1228, 748-1247, 755-1248, 762-1248, 804-944, 818-1248, 829-1248,



830-1248, 838-1248, 839-1248, 845-1069, 853-1248, 862-928,



892-1228, 894-1248, 906-1248, 918-1248, 926-1248, 928-1248, 962-1248,



973-1248, 979-1248, 1023-1238, 1025-1248, 1048-1248, 1104-1248


43/7509099CB1/
1-243, 1-1842, 10-761, 10-767, 37-242, 92-365, 374-712, 385-1112,


1989
409-1028, 409-1130, 425-945, 453-1317, 466-1051, 585-1182, 588-1290,



691-1682, 698-1682, 734-1681, 751-1368, 757-1018, 770-1438, 788-1682,



821-1681, 825-1352, 837-1682, 838-1682, 840-1682, 841-1682,



842-1681, 843-1681, 847-1438, 856-1682, 863-1682, 877-1681, 878-1110,



890-1681, 890-1682, 899-1681, 905-1681, 906-1681, 906-1682,



918-1438, 985-1681, 991-1677, 993-1384, 1041-1438, 1055-1752,



1134-1729, 1172-1736, 1317-1910, 1383-1686, 1412-1675, 1429-1633,



1475-1989, 1681-1924, 1686-1855, 1733-1989, 1833-1866


44/7509361CB1/
1-242, 1-268, 1-361, 1-1863, 3-207, 4-242, 4-261, 5-105, 5-241,


1863
6-258, 7-193, 7-263, 7-284, 7-303, 7-445, 8-294, 8-433, 9-256, 12-237, 15-270,



25-232, 28-736, 28-815, 28-843, 28-863, 29-755, 39-275, 39-282, 39-321,



39-323, 40-280, 40-730, 46-309, 46-321, 46-434, 47-475,



48-694, 50-536, 50-607, 50-632, 53-662, 54-286, 54-357, 54-363, 54-367,



54-639, 55-302, 57-272, 57-329, 57-684, 59-171, 60-305, 61-324,



61-340, 61-364, 62-300, 64-363, 69-317, 69-347, 70-540, 72-445,



74-359, 74-745, 76-712, 79-210, 79-372, 79-382, 80-276, 82-198,



82-303, 83-207, 83-301, 84-651, 90-294, 90-372, 92-281, 96-626,



97-391, 99-400, 102-406, 103-645, 110-702, 111-294, 113-253, 123-703,



129-420, 136-445, 146-347, 153-630, 183-816, 200-261, 206-396,



207-581, 239-734, 239-780, 239-876, 239-877, 239-952, 257-445, 271-679,



280-869, 294-877, 311-532, 354-590, 446-465, 446-800, 446-825,



446-877, 467-722, 467-746, 527-833, 554-679, 566-803, 573-784,



573-786, 585-816, 612-841, 612-856, 658-1441, 692-860, 875-1040,



875-1085, 875-1118, 875-1191, 875-1306, 875-1523,



875-1569, 875-1577, 880-1034, 881-1149, 884-1441, 897-1441,



899-1154, 900-1173, 918-1152, 920-1159, 935-1167, 942-1148, 952-1159,



952-1219, 957-1117, 961-1557, 962-1205, 968-1270, 973-1596, 974-1243,



994-1258, 997-1089, 1000-1259, 1011-1315, 1028-1403,



1040-1275, 1058-1299, 1078-1337, 1085-1351, 1112-1309, 1117-1335,



1123-1388, 1154-1815, 1155-1846, 1160-1848, 1175-1441, 1180-1778,



1187-1853, 1203-1457, 1209-1482, 1213-1514, 1219-1510, 1223-1496,



1238-1501, 1240-1502, 1240-1507, 1253-1478, 1264-1824,



1271-1848, 1276-1772, 1281-1855, 1291-1565, 1297-1537, 1302-1594,



1302-1776, 1304-1777, 1304-1851, 1314-1539, 1314-1658, 1315-1539,



1315-1557, 1316-1466, 1319-1612, 1323-1562, 1323-1797, 1324-1579,



1327-1863, 1328-1790, 1337-1577, 1337-1585, 1337-1594,



1339-1718, 1340-1579, 1342-1602, 1347-1514, 1347-1584, 1351-1789,



1354-1790, 1357-1658, 1365-1847, 1366-1837, 1367-1718, 1369-1862,



1377-1526, 1377-1851, 1379-1609, 1379-1806, 1386-1633, 1386-1847,



1386-1855, 1387-1859, 1387-1863, 1389-1655, 1389-1700,



1391-1674, 1394-1657, 1398-1777, 1400-1654, 1402-1854, 1406-1850,



1413-1853, 1418-1682, 1418-1850, 1419-1663, 1421-1669,



1422-1654, 1422-1674, 1424-1851, 1425-1857, 1427-1849, 1432-1825,



1433-1848, 1435-1685, 1436-1850, 1438-1848, 1440-1848, 1443-1848,



1447-1602, 1452-1689, 1457-1848, 1457-1851, 1460-1715, 1469-1715,



1472-1650, 1474-1839, 1482-1851, 1483-1862, 1487-1863,



1491-1848, 1492-1863, 1495-1850, 1502-1789, 1513-1848, 1513-1853,



1514-1851, 1520-1863, 1522-1776, 1522-1809, 1522-1848, 1523-1848,



1526-1846, 1526-1848, 1531-1848, 1537-1848, 1537-1850, 1544-1803,



1550-1863, 1554-1785, 1559-1851, 1562-1846, 1562-1848,



1562-1849, 1564-1800, 1565-1848, 1567-1848, 1572-1852, 1575-1778,



1577-1848, 1584-1843, 1589-1799, 1593-1851, 1602-1848, 1604-1848,



1605-1846, 1605-1848, 1605-1855, 1609-1797, 1620-1863, 1625-1855,



1627-1731, 1631-1846, 1633-1861, 1662-1848, 1662-1850,



1662-1858, 1673-1848, 1678-1848, 1678-1850, 1683-1848, 1687-1848,



1688-1848, 1689-1818, 1689-1851, 1689-1863, 1692-1826, 1692-1848,



1693-1863, 1696-1848, 1696-1861, 1708-1838, 1709-1814, 1711-1853,



1712-1851, 1720-1855, 1723-1840, 1752-1858, 1753-1862,



1753-1863, 1757-1860, 1757-1863, 1765-1863, 1768-1850, 1777-1849,



1784-1851, 1785-1863, 1786-1851, 1794-1848


45/7506815CB1/
1-1734, 123-827, 309-1275, 443-1275, 464-1274, 466-1274, 540-1275,


1734
547-1274, 613-1274, 866-1096, 866-1180, 866-1348, 866-1363,



866-1395, 866-1431, 866-1484, 866-1543, 866-1650, 866-1658, 904-1221,



940-1731, 971-1541, 973-1367, 974-1371, 975-1222, 975-1331,



975-1440, 975-1595, 988-1284, 988-1563, 998-1607, 1020-1427, 1020-1517,



1051-1566, 1071-1427, 1099-1734, 1103-1362, 1109-1604,



1115-1432, 1152-1408, 1159-1694, 1180-1365, 1206-1635, 1228-1734,



1263-1675, 1264-1560, 1293-1691, 1322-1510, 1331-1734,



1352-1620, 1428-1575, 1466-1709, 1507-1702


46/7506814CB1/
1-785, 1-825, 1-883, 1-901, 1-902, 1-1786, 87-758, 87-804, 87-813,


1786
87-824, 87-904, 87-980, 88-900, 242-820, 363-1327, 740-1328, 918-1148,



918-1232, 918-1400, 918-1415, 918-1447, 918-1483, 918-1536, 918-1595,



918-1702, 918-1710, 956-1273, 992-1783, 1023-1593,



1025-1419, 1026-1423, 1027-1274, 1027-1383, 1027-1492, 1027-1647,



1040-1336, 1040-1615, 1050-1659, 1072-1479, 1072-1569, 1103-1618,



1123-1479, 1151-1786, 1155-1414, 1161-1656, 1167-1484, 1204-1460,



1211-1746, 1232-1417, 1258-1687, 1280-1786, 1315-1727,



1316-1612, 1345-1743, 1374-1562, 1383-1786, 1404-1672, 1480-1627,



1518-1761, 1559-1754


47/7506852CB1/
1-2193, 56-668, 56-787, 56-793, 532-1066


2193


48/7503782CB1/
1-3673, 532-1000, 716-895, 1524-2351, 1525-1791, 1863-2448, 1863-2471,


3696
1888-2305, 1903-2671, 1904-2671, 1939-2201, 1979-2671,



2023-2671, 2187-2671, 2211-2453, 2211-2471, 2321-2671, 2321-2735,



2321-2792, 2366-2627, 2371-2786, 2379-2623, 2420-2503, 2421-2696,



2501-2830, 2575-2752, 2735-3211, 2784-3282, 2848-3200, 2899-3384,



2966-3536, 3033-3535, 3037-3671, 3038-3650, 3040-3289,



3064-3673, 3067-3521, 3095-3194, 3104-3682, 3129-3535, 3132-3696,



3140-3668, 3147-3662, 3149-3673, 3185-3420, 3185-3642, 3187-3659,



3191-3535, 3205-3391, 3205-3673, 3281-3535, 3595-3647


49/7504647CB1/
1-132, 1-169, 1-297, 1-439, 1-442, 4-814, 4-1260, 10-166, 12-168,


1283
169-419, 169-552, 169-630, 169-702, 180-777, 235-919, 235-1034, 271-791,



277-497, 277-967, 278-501, 286-520, 313-846, 324-882, 332-844, 339-1004,



342-1151, 346-838, 350-806, 354-625, 357-1121, 362-855,



384-949, 412-1250, 418-1250, 424-1087, 431-893, 434-1175, 457-1132,



465-1159, 466-1101, 469-954, 471-872, 472-599, 472-1000,



480-1011, 487-844, 492-933, 502-763, 502-1075, 521-1170, 537-1214,



555-1151, 562-1222, 563-1050, 570-1218, 595-1110, 596-849, 596-1259,



597-1255, 602-883, 603-1175, 612-1222, 614-1067, 622-1065, 626-1218,



628-1207, 652-1143, 653-1182, 685-1222, 690-1222, 694-1153,



698-1222, 701-1222, 704-1010, 705-1148, 707-1222, 713-971, 718-1208,



718-1222, 723-1214, 725-978, 736-1218, 737-1222, 751-1197,



791-1226, 795-1283, 808-1057, 809-833, 810-1226, 811-1275,



836-1222, 841-1222, 875-1275, 891-1133, 910-1250, 910-1254, 951-1257,



999-1221, 1003-1258, 1099-1224


50/7500424CB1/
1-245, 1-248, 1-325, 1-377, 1-441, 1-463, 1-1142, 7-61, 18-437,


1142
59-134, 59-283, 59-753, 59-761, 59-879, 59-944, 59-955, 60-732, 100-392,



126-1040, 164-1040, 199-1040, 241-1040, 290-563, 388-1040, 401-1040,



425-1040, 467-678, 467-699, 467-714, 467-882, 467-956,



467-968, 467-1040, 467-1058, 467-1083, 467-1119, 467-1123, 468-718,



468-1116, 469-1086, 469-1116, 469-1122, 471-721, 471-1065,



474-1040, 480-658, 484-707, 484-922, 490-750, 490-761, 491-1092,



493-796, 494-760, 495-710, 495-1065, 496-943, 496-1034, 499-694,



500-851, 503-851, 513-1125, 514-720, 514-1072, 515-1118, 516-1002,



528-792, 528-805, 528-1113, 530-745, 530-967, 531-976, 531-1080,



531-1136, 535-1113, 535-1118, 536-820, 539-818, 540-768, 541-784,



543-849, 544-877, 549-867, 552-1116, 556-1142, 559-851,



559-1114, 561-851, 566-1114, 567-853, 575-1142, 579-832, 579-891,



579-1142, 580-851, 585-1123, 586-1114, 587-885, 596-1119, 598-834,



601-1078, 606-851, 608-1117, 609-1040, 611-1129, 614-949, 615-833,



615-865, 615-1142, 622-1121, 623-861, 624-1123,



625-1123, 626-898, 635-979, 636-1115, 636-1142, 644-851, 646-1112,



648-940, 650-1138, 652-1142, 654-1142, 656-1132, 657-1133, 658-851,



662-1131, 664-1142, 668-1142, 679-1142, 680-1142, 686-1132, 690-989,



690-1131, 691-1114, 697-1138, 697-1142, 700-932, 704-1121,



704-1134, 705-1139, 709-1142, 710-1131, 711-1133, 712-1084, 714-1131,



715-1130, 715-1132, 716-1129, 716-1135, 717-1129, 718-1127,



719-1042, 720-1129, 721-1123, 721-1129, 721-1142, 722-1133, 722-1136,



723-1121, 724-1121, 724-1133, 725-984, 726-1132, 727-1132,



729-1129, 729-1132, 730-1128, 733-946, 734-851, 736-1130, 739-983,



739-1042, 739-1121, 740-1000, 741-1120, 742-1129, 744-1114,



745-1135, 750-851, 753-1002, 755-1142, 756-1113, 756-1114, 756-1132,



756-1142, 762-1129, 764-1142, 766-1128, 769-1123, 770-1142,



773-1134, 777-1025, 780-1137, 782-1141, 783-1022, 784-1128, 787-1142,



800-1130, 801-1130, 802-1075, 804-1042, 804-1133, 806-1130,



808-1114, 808-1132, 808-1133, 815-1132, 823-1111, 823-1123,



836-1086, 837-1042, 842-1042, 847-1132, 849-1042, 851-1132, 859-1042,



862-1142, 863-1124, 865-1042, 870-1048, 870-1130, 870-1132, 873-1132,



874-1042, 884-1130, 886-1119,



886-1142, 887-1132, 893-1142, 895-1142, 902-1129, 904-1129, 904-1142,



905-1123, 905-1130, 910-1142, 912-1132, 913-1132, 915-1075,



915-1129, 918-1132, 920-1137, 925-1132, 932-1132, 933-1137, 934-1132,



936-1142, 938-1142, 939-1129, 972-1132, 984-1142, 988-1130,



989-1129, 994-1136, 995-1125, 1004-1142, 1005-1132, 1006-1132,



1022-1142, 1030-1132, 1040-1142, 1068-1142


51/7500449CB1/
1-261, 7-270, 44-271, 47-1477, 52-163, 52-183, 52-199, 52-271,


1477
60-183, 109-217, 273-761, 447-514, 513-1237, 515-757, 515-923, 515-928,



515-970, 515-1000, 515-1033, 515-1074, 515-1173, 515-1210, 515-1211,



515-1212, 515-1332, 515-1344, 515-1350, 517-1236, 519-1107,



809-1364, 899-1448, 1030-1319, 1177-1417, 1211-1400, 1269-1477,



1282-1477


52/7503281CB1/
1-665, 1-687, 1-696, 1-719, 1-730, 1-731, 1-738,


1097
1-739, 1-775, 1-817, 1-838, 1-1097, 139-523, 293-1059, 298-1059,



311-1059, 334-1059, 419-1059, 432-1059


53/7503292CB1/
1-1493, 173-754, 173-769, 173-786, 173-812, 173-822, 173-1016,


1501
175-840, 333-1159, 356-674, 405-1140, 484-1101, 548-715, 661-849,



686-1221, 800-1344, 806-1384, 848-1498, 849-1154, 849-1287, 849-1337,



849-1341, 849-1348, 849-1416, 849-1475, 936-1497, 940-1428,



955-1465, 965-1498, 967-1234, 968-1245, 975-1498, 978-1498, 982-1497,



987-1498, 994-1497, 996-1387, 998-1490, 999-1495,



1019-1501, 1025-1501, 1035-1491, 1037-1418, 1042-1497, 1045-1501,



1052-1495, 1057-1374, 1059-1413, 1062-1245, 1069-1496, 1075-1498,



1091-1501, 1115-1424, 1141-1388, 1144-1462, 1146-1497, 1147-1501,



1157-1424, 1172-1416, 1173-1390, 1179-1487, 1190-1496,



1215-1459, 1231-1380, 1242-1494, 1300-1501, 1310-1424


54/7503311CB1/
1-1613, 240-338, 240-555, 240-570, 240-632, 240-690, 240-715,


1613
240-739, 240-899, 255-1038, 465-729, 584-1466, 628-723, 902-1132,



913-1441, 968-1499, 1050-1152, 1092-1584


55/7510384CB1/
1-1523, 68-557, 181-329, 224-318, 237-449, 243-418, 527-1059,


1523
532-1059, 1062-1185


56/7509976CB1/
1-6826, 693-1218, 709-1289, 866-1146, 866-1217, 1001-1539, 1258-1887,


6826
1857-2171, 1960-2223, 2291-2502, 2419-3005, 2419-3012,



2864-3322, 5428-5856, 5452-5888, 5452-5949, 5454-5984, 5471-5969,



5477-5746, 5494-5753, 5515-6064, 5518-5766, 5528-6062, 5540-6086,



5551-5692, 5553-5800, 5559-6119, 5566-6097, 5570-5821, 5578-5842,



5581-5975, 5589-5848, 5594-6130, 5612-5819, 5612-5881,



5614-6218, 5621-5863, 5645-6069, 5655-6226, 5657-5952, 5720-6221,



5742-6119, 5749-5980, 5780-5954, 5788-6179, 5795-6072, 5826-6004,



5834-6071, 5868-6219, 5871-6150, 5884-5985, 5913-6207, 5935-6232,



5944-6491, 5945-6222, 5952-6542, 5970-6209, 6009-6216,



6013-6272, 6033-6242, 6034-6229, 6054-6573, 6064-6333, 6094-6322,



6101-6408, 6101-6750, 6105-6361, 6147-6424, 6165-6431, 6171-6439,



6183-6406, 6194-6816, 6198-6550, 6200-6759, 6201-6796, 6207-6818,



6210-6467, 6210-6498, 6210-6503, 6212-6822, 6213-6516,



6219-6458, 6248-6501, 6254-6684, 6267-6500, 6267-6795, 6272-6611,



6278-6826, 6317-6570, 6317-6801, 6319-6815, 6326-6532, 6333-6546,



6333-6594, 6341-6778, 6352-6826, 6356-6816, 6362-6820, 6372-6821,



6378-6825, 6387-6816, 6394-6817, 6394-6820, 6397-6645,



6398-6661, 6399-6816, 6401-6542, 6406-6816, 6407-6816, 6409-6815,



6411-6824, 6423-6826, 6431-6703, 6432-6821, 6434-6655, 6435-6821,



6446-6815, 6451-6815, 6456-6746, 6458-6824, 6459-6803, 6460-6739,



6461-6741, 6491-6816, 6505-6821, 6521-6823, 6539-6816,



6558-6826, 6595-6819, 6601-6815, 6614-6818, 6627-6816, 6631-6826,



6638-6821, 6643-6816, 6668-6816, 6669-6808, 6669-6809, 6721-6819,



6724-6826


57/7510454CB1/
1-270, 1-423, 1-432, 1-447, 1-520, 1-532, 1-535, 1-552, 1-565,


2481
1-568, 1-571, 1-602, 1-605, 1-611, 1-623, 1-631, 1-634, 1-663, 1-2481, 309-1087,



334-1085, 393-1006, 402-1085, 406-1085, 423-1085, 431-1085, 454-1212,



455-1085, 455-1179, 456-1085, 459-1085, 475-1075, 475-1085,



479-1085, 482-733, 482-1085, 497-1073, 530-1465, 531-1347, 531-1409,



558-1343, 558-1437, 558-1505, 562-1417, 720-1661, 748-1661,



752-1661, 825-1661, 825-1663, 827-1661, 827-1663, 829-1661, 886-1663,



895-1136, 895-1195, 895-1302, 895-1391, 895-1475, 895-1477,



895-1485, 895-1513, 895-1523, 898-1595, 905-1544, 964-1165, 965-1523,



976-1661, 988-1563, 1077-1609, 1102-1682, 1150-1706,



1160-1800, 1210-1865, 1277-1856, 1334-1917, 1347-1941, 1351-2015,



1384-1934, 1386-1952, 1390-1978, 1393-1960, 1423-2010, 1463-2048,



1464-2112, 1477-2096, 1482-1992, 1491-2031, 1504-2170, 1517-1912,



1522-2023, 1537-2162, 1547-2481, 1549-1723, 1549-1977,



1549-2251, 1550-2010, 1562-2104, 1614-2282, 1616-2114, 1624-2191,



1626-2218, 1626-2219, 1633-1900, 1637-2170, 1658-1962, 1667-2299,



1670-2043, 1811-2064, 2214-2324, 2222-2313


58/8017335CB1/
1-2498, 34-594, 62-374, 544-862, 544-932, 544-1013, 544-1021,


2512
544-1063, 544-1073, 544-1082, 544-1092, 544-1114, 544-1120, 544-1123,



544-1131, 560-1147, 856-1581, 856-1589, 907-1529, 909-1500, 1065-1926,



1084-1925, 1092-1926, 1099-1926, 1106-1924, 1106-1926,



1114-1925, 1114-1926, 1135-1924, 1180-1892, 1182-1924, 1208-1549,



1230-1926, 1239-1926, 1304-1925, 1495-1841, 1737-2008,



1747-2330, 1763-2324, 1814-2134, 1823-2345, 1904-2427, 1941-2218,



1966-2461, 1968-2512, 1996-2397, 2039-2512, 2061-2504, 2203-2512,



2206-2484


59/7510197CB1/
1-270, 1-343, 1-552, 1-754


754


60/7510055CB1/
1-1660, 113-420, 430-480, 472-685, 544-751, 545-826, 585-1147,


1660
585-1259, 676-817, 676-848, 681-931, 690-883, 720-984, 732-951, 763-1204,



775-1071, 792-1207, 793-1045, 794-1163, 806-1015, 806-1026, 858-1368,



886-1147, 886-1160, 923-1143, 923-1355, 923-1358, 923-1405,



976-1398, 976-1405, 983-1210, 983-1239, 990-1246, 991-1219, 1031-1204,



1039-1207, 1048-1422, 1070-1315, 1092-1295, 1115-1356,



1167-1405, 1228-1315, 1298-1377, 1369-1453, 1400-1489, 1404-1453,



1406-1453, 1410-1453, 1424-1453, 1449-1475, 1449-1479,



1449-1481


61/7501754CB1/
1-241, 1-271, 1-340, 1-486, 1-625, 2-645, 4-291, 6-667, 13-527,


2118
14-625, 19-678, 19-685, 21-261, 21-1822, 28-272, 33-612, 35-287, 35-656,



45-741, 74-306, 80-309, 146-597, 193-451, 193-705, 233-512, 248-457,



249-833, 260-829, 280-951, 302-583, 318-528, 363-932, 369-813,



385-898, 390-977, 413-978, 415-595, 423-632, 423-632, 423-940, 428-668,



428-693, 429-912, 463-937, 476-1151, 493-953, 498-621, 524-1192,



543-1113, 548-1116, 548-1151, 552-1125, 578-1229, 591-1187, 598-1248,



603-886, 606-1146, 649-1239, 651-900, 664-914, 664-1023,



666-1177, 668-1027, 671-1163, 690-1171, 693-958, 704-1223, 707-1140,



707-1239, 714-1362, 715-1132, 726-1142, 729-1316, 737-1184,



738-1266, 750-1035, 754-1176, 763-1039, 764-1389, 771-1298, 780-1023,



793-1046, 799-1313, 804-1281, 814-1029, 814-1116, 814-1347,



815-895, 815-927, 819-1240, 819-1425, 821-1099, 836-1143, 840-1444,



847-1365, 861-1084, 865-993, 885-1348, 891-1287, 891-1316,



897-1170, 897-1325, 906-1389, 914-1161, 914-1167, 914-1177,



926-1372, 928-1207, 937-1195, 940-1179, 942-1190, 942-1195, 942-1226,



942-1243, 942-1344, 945-1286, 950-1209, 950-1217, 950-1520, 953-1428,



953-1629, 969-1230, 972-1073, 972-1228,



972-1407, 976-1418, 982-1290, 983-1609, 984-1537, 993-1250, 997-1238,



998-1515, 1024-1300, 1024-1506, 1026-1331, 1055-1579, 1070-1323,



1071-1356, 1073-1277, 1080-1569, 1082-1325, 1084-1755, 1091-1545,



1110-1664, 1131-1349, 1133-1617, 1151-1410, 1151-1416,



1167-1811, 1174-1747, 1179-1779, 1184-1698, 1188-1825, 1213-1761,



1217-1482, 1220-1543, 1265-1707, 1280-1546, 1324-1807, 1340-1464,



1347-1632, 1347-1746, 1347-1780, 1347-1792, 1347-1808, 1347-1824,



1350-1551, 1351-1830, 1354-1469, 1355-1611, 1355-1782,



1355-1814, 1356-1824, 1357-1602, 1358-1814, 1362-1814, 1365-1601,



1368-1814, 1369-1814, 1369-1844, 1377-1796, 1380-1833, 1382-1812,



1384-1881, 1385-1812, 1389-1823, 1391-1616, 1391-1812, 1392-1873,



1393-1592, 1393-1679, 1394-1831, 1399-1809, 1400-1636,



1401-1631, 1404-1650, 1404-1882, 1412-1812, 1414-1825, 1419-1540,



1419-1715, 1430-1620, 1430-1811, 1434-1666, 1434-1670, 1444-1874,



1451-1814, 1454-2118, 1456-1725, 1457-1798, 1459-1725, 1459-1791,



1463-1813, 1467-1814, 1468-1743, 1484-1814, 1485-1811,



1499-1811, 1502-1826, 1507-1816, 1509-1735, 1509-1756, 1511-1811,



1512-1903, 1523-1815, 1526-1810, 1548-1810, 1555-1877,



1560-1854, 1569-1778, 1569-1789, 1569-1797, 1569-1827, 1572-1811,



1572-1814, 1575-1814, 1576-1803, 1578-1810, 1580-1814, 1581-1843,



1588-1793, 1592-1803, 1593-1831, 1594-1826, 1606-1811, 1634-1812,



1639-1814, 1652-1814, 1657-1814, 1660-1817, 1696-1810,



1697-1800, 1697-1827, 1718-1830, 1718-1904


62/7510517CB1/
1-224, 1-286, 1-416, 1-437, 1-454, 1-465, 1-524, 1-587, 1-603,


2800
1-616, 1-697, 4-287, 4-2800, 16-831, 22-466, 22-606, 22-640, 22-793, 22-818,



22-821, 22-831, 25-831, 32-803, 35-778, 59-546, 60-619, 61-205,



62-516, 62-569, 73-499, 82-638, 86-577, 134-453, 149-705, 208-683,



219-653, 220-647, 228-520, 259-486, 263-484, 360-668, 427-578,



454-885, 847-1295, 879-1327, 891-1152, 900-1114, 900-1150, 900-1224,



900-1473, 900-1532, 900-1775, 977-1269, 1006-1241, 1006-1511, 1008-1322,



1008-1345, 1095-1677, 1151-2034, 1223-1470, 1223-1653,



1227-1887, 1235-1430, 1287-1530, 1297-1582, 1308-1607, 1436-1677,



1439-1702, 1441-2064, 1467-1793, 1469-1989, 1475-1988,



1485-1744, 1492-1758, 1497-2397, 1498-1989, 1509-1748, 1542-1772,



1554-1787, 1579-1850, 1589-1827, 1596-1808, 1596-2112, 1600-1988,



1602-2177, 1665-1934, 1679-2129, 1679-2196, 1682-1977, 1713-1958,



1714-1932, 1721-1974, 1728-1950, 1730-2150, 1733-2256,



1734-2397, 1735-2237, 1741-2394, 1762-2010, 1785-2608, 1799-2286,



1799-2517, 1800-2209, 1800-2220, 1800-2321, 1800-2364,



1800-2394, 1805-2293, 1824-2394, 1826-2607, 1833-2394, 1843-2102,



1843-2344, 1868-2395, 1871-2608, 1877-2604, 1881-2149, 1899-2133,



1899-2145, 1918-2394, 1934-2205, 1947-2604, 1952-2229, 1963-2092,



1969-2273, 1971-2534, 1976-2538, 1991-2493, 1992-2608,



1995-2537, 2003-2537, 2011-2575, 2033-2239, 2038-2291, 2038-2329,



2040-2296, 2040-2553, 2050-2456, 2050-2566, 2052-2245, 2067-2570,



2075-2637, 2081-2791, 2083-2536, 2091-2214, 2116-2309, 2166-2382,



2174-2758, 2177-2793, 2187-2462, 2210-2784, 2215-2760,



2233-2720, 2253-2523, 2272-2752, 2291-2754, 2300-2751, 2301-2789,



2311-2791, 2322-2575, 2322-2600, 2322-2731, 2322-2793, 2324-2606,



2325-2800, 2336-2452, 2350-2798, 2353-2800, 2362-2791, 2374-2794,



2445-2794, 2455-2800, 2480-2789, 2550-2800, 2702-2800


63/7511014CB1/
1-477, 9-1750, 10-295, 611-1452, 890-1719


1750


64/7506687CB1/
1-487, 88-7106, 246-807, 446-710, 578-852, 662-1293, 688-1178,


7106
694-1282, 797-1490, 925-1439, 931-1292, 950-1288, 1131-1378, 1131-1426,



1395-1422, 1921-2491, 2447-2687, 2475-2787, 2508-3180, 2613-3272,



2701-3232, 2712-3395, 2742-3405, 2764-3021, 2789-3203,



2814-3396, 2828-3402, 2861-3457, 2873-3235, 2873-3237, 2899-3296,



2924-3464, 3124-3265, 3141-3395, 3251-3801, 3358-4003, 3361-3957,



3491-4067, 3508-4096, 3530-4167, 3555-4093, 3588-4004, 3612-4174,



3678-3977, 3706-3977, 3735-4136, 3771-4055, 3775-4476,



3782-4000, 3804-4067, 3810-4563, 3846-4297, 3847-4297, 3848-4545,



3853-4195, 3853-4238, 3859-4331, 3861-4490, 3900-4056, 3913-4617,



3917-4433, 3919-4307, 3946-4297, 3967-4793, 3992-4328, 4004-4518,



4086-4355, 4124-4714, 4165-4765, 4169-4404, 4169-4413,



4171-4417, 4175-4829, 4251-4856, 4269-4831, 4319-4666, 4322-4729,



4352-4956, 4355-4950, 4372-4869, 4383-4957, 4392-5302, 4479-5089,



4499-4750, 4525-4709, 4528-4811, 4533-5128, 4543-4686, 4557-5004,



4562-4763, 4576-4812, 4597-5169, 4599-4885, 4645-4864,



4646-5190, 4653-5233, 4720-4933, 4735-5016, 4765-4921, 4779-5160,



4780-5137, 4785-5089, 4785-5132, 4785-5144, 4787-5227,



4792-5158, 4798-5153, 4800-5070, 4810-5634, 4816-5062, 4816-5066,



4829-5044, 4850-5348, 4850-5553, 4851-5150, 4855-5561, 4861-5104,



4861-5466, 4869-5460, 4869-5507, 4873-5166, 4878-5561, 4897-5540,



4920-5248, 4937-5253, 4959-5180, 4960-5521, 4965-5217,



4969-5130, 4971-5214, 4979-5166, 4996-5203, 4996-5274, 5014-5666,



5033-5296, 5033-5546, 5038-5271, 5043-5309, 5050-5184, 5070-5723,



5076-5594, 5089-5510, 5094-5291, 5095-5653, 5096-5707, 5101-5498,



5110-5292, 5110-5311, 5116-5773, 5127-5328, 5127-5344,



5141-5412, 5143-5838, 5148-5743, 5148-5753, 5151-5462, 5152-5434,



5156-5333, 5156-5385, 5157-5718, 5159-5747, 5161-5510, 5193-5838,



5194-5736, 5221-6083, 5232-5710, 5260-5697, 5263-5571, 5316-5562,



5336-5874, 5341-5969, 5342-5608, 5342-5989, 5345-5906,



5350-5594, 5377-5989, 5380-5628, 5380-5647, 5382-5495, 5390-5632,



5390-5637, 5390-5830, 5423-5652, 5425-5602, 5425-5698, 5429-5695,



5444-6079, 5450-5546, 5450-5740, 5460-5691, 5463-5820, 5473-5830,



5515-5763, 5521-5770, 5526-5918, 5526-6214, 5535-5773,



5551-5847, 5562-6071, 5566-6041, 5572-5838, 5575-5831, 5585-5816,



5600-6032, 5601-5856, 5605-5833, 5615-5801, 5634-5882,



5634-5964, 5642-6345, 5651-6071, 5655-6151, 5658-5950, 5673-5964,



5685-5968, 5712-6184, 5715-6362, 5716-5964, 5725-6042, 5738-5808,



5744-6089, 5751-6014, 5761-6116, 5762-5923, 5765-5886, 5765-5922,



5767-6109, 5776-6061, 5776-6464, 5776-6648, 5784-6151,



5785-6041, 5795-6082, 5812-5990, 5812-6035, 5820-6094, 5842-6129,



5859-6100, 5860-6134, 5869-6147, 5877-6167, 5877-6183, 5878-6144,



5888-6109, 5907-6145, 5908-6637, 5916-6548, 5917-6183, 5926-6184,



5926-6508, 5928-6119, 5929-6209, 5943-6221, 5945-6371,



5945-6533, 5961-6220, 5971-6210, 5971-6301, 5971-6450, 5978-6271,



5984-6192, 5987-6628, 5991-6149, 6011-6245, 6012-6256, 6012-6337,



6027-6656, 6029-6281, 6038-6613, 6048-6479, 6053-6378, 6064-6295,



6068-6340, 6072-6348, 6081-6536, 6081-6656, 6106-6372,



6107-6336, 6107-6393, 6108-6382, 6113-6298, 6116-6464, 6119-6406,



6128-6246, 6128-6345, 6134-6545, 6135-6758, 6137-6800, 6171-6388,



6171-6779, 6176-6497, 6186-6453, 6190-6827, 6192-6624, 6193-6387,



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66/7505533CB1/
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67/7511220CB1
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685


68/7510967CB1/
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69/7511298CB1/
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2023-2158, 2028-2683, 2030-2307, 2033-2310, 2037-2585, 2043-2334,



2048-2248, 2048-2344, 2048-2636, 2054-2305, 2058-2312,



2058-2319, 2061-2310, 2061-2394, 2068-2752, 2072-2645, 2072-2657,



2079-2349, 2089-2236, 2091-2341, 2095-2362, 2095-2700, 2102-2375,



2102-2700, 2103-2343, 2110-2374, 2111-2702, 2113-2358, 2114-2404,



2115-2294, 2116-2388, 2127-2301, 2127-2386, 2127-2406,



2127-2545, 2132-2559, 2133-2685, 2137-2435, 2143-2413, 2156-2570,



2159-2700, 2159-3000, 2160-2630, 2165-2700, 2171-2700,



2180-2700, 2181-2343, 2188-2453, 2189-2452, 2189-2462, 2189-2792,



2203-2651, 2203-2691, 2209-2809, 2210-2700, 2214-2489, 2215-2506,



2215-2803, 2217-2837, 2227-2943, 2230-2700, 2231-2497, 2231-2500,



2232-2709, 2236-2361, 2241-2505, 2243-2521, 2250-2517,



2251-2700, 2261-2594, 2268-2513, 2268-2516, 2273-2506, 2273-2559,



2273-2650, 2277-2826, 2282-2623, 2282-3027, 2283-2526, 2283-2615,



2283-3027, 2284-2700, 2289-3027, 2290-2508, 2293-2666, 2293-2910,



2303-2701, 2303-3027, 2304-2880, 2306-3025, 2306-3027,



2311-2989, 2311-3027, 2318-3028, 2321-3025, 2330-2908, 2353-2557,



2357-2862, 2362-2911, 2363-2609, 2363-3027, 2366-2799, 2369-2569,



2369-2928, 2373-2570, 2373-2913, 2373-2926, 2374-2548, 2374-2881,



2375-2771, 2376-2568, 2376-2613, 2376-2619, 2376-2632,



2378-2868, 2378-2928, 2380-2700, 2381-2639, 2383-2667, 2386-2729,



2387-2966, 2389-2864, 2391-2662, 2391-2912, 2391-2935, 2393-2678,



2394-2927, 2396-2863, 2396-2881, 2396-2999, 2398-3028, 2400-2926,



2405-2702, 2405-3029, 2406-3002, 2410-2949, 2411-2921,



2415-2631, 2423-2606, 2428-2685, 2430-2649, 2432-2585, 2432-2715,



2435-2915, 2435-3029, 2439-3030, 2439-3044, 2441-2695,



2441-2821, 2447-2994, 2448-2697, 2448-2830, 2448-2927, 2451-3044,



2455-2721, 2455-2880, 2458-2808, 2462-3044, 2472-2774, 2472-2948,



2476-3006, 2478-2996, 2484-2992, 2485-2772, 2485-3031, 2496-2775,



2504-2996, 2520-2779, 2521-2923, 2521-2993, 2522-3040,



2533-2745, 2537-2681, 2543-3006, 2544-2880, 2551-2994, 2552-2776,



2552-2781, 2552-2830, 2553-3036, 2556-2994, 2557-2823, 2558-2637,



2561-2822, 2564-2955, 2565-2796, 2573-2839, 2584-2834, 2584-2873,



2584-2991, 2587-3044, 2590-2991, 2590-3035, 2591-2871,



2591-2916, 2592-2854, 2592-2861, 2593-2846, 2593-2859, 2593-3031,



2594-3037, 2596-3030, 2597-2865, 2601-2846, 2609-3033, 2610-2884,



2613-3040, 2616-3034, 2621-3033, 2633-2993, 2633-3031, 2633-3033,



2633-3034, 2634-3043, 2638-2852, 2638-2875, 2639-3040,



2648-2872, 2649-2854, 2653-2873, 2653-2909, 2653-2933, 2653-3031,



2659-3031, 2661-3023, 2661-3029, 2663-2900, 2663-3032, 2665-3039,



2666-3031, 2667-3035, 2669-3031, 2670-2891, 2673-2909, 2673-2962,



2673-3025, 2673-3036, 2677-3043, 2677-3044, 2678-3032,



2678-3035, 2681-2995, 2682-3033, 2689-2923, 2693-2982, 2701-2923,



2701-2993, 2709-3031, 2716-2776, 2725-2994, 2726-3044,



2727-3044, 2728-3023, 2729-3035, 2737-2994, 2739-2996, 2740-3043,



2740-3044, 2741-2985, 2742-3043, 2747-2999, 2758-3041, 2760-3029,



2760-3031, 2765-3044, 2772-2981, 2776-2993, 2786-3030, 2788-3030,



2790-3029, 2794-3040, 2802-3028, 2803-3034, 2812-3044,



2814-3034, 2817-3029, 2817-3030, 2820-3031, 2821-2970, 2821-3030,



2825-3036, 2826-3033, 2827-3040, 2847-3038, 2879-3044, 2880-3037,



2893-3044, 2918-3044, 2955-3044


70/7510937CB1/
1-191, 1-4449, 33-482, 35-298, 36-487, 37-917, 38-326, 40-338, 41-297,


4455
41-322, 41-491, 41-494, 41-497, 42-828, 46-315, 49-119, 49-321,



49-322, 49-326, 49-327, 49-332, 49-486, 49-487, 52-323, 53-325,



54-246, 54-307, 59-178, 60-327, 64-327, 65-327, 68-325, 72-487, 74-323



75-166, 75-300, 75-307, 75-487, 76-325, 77-329, 77-371, 77-604,



78-328, 81-317, 84-145, 88-322, 88-803, 91-444, 92-333, 100-327,



105-487, 108-490, 108-537, 109-487, 110-487, 119-370, 120-487,



124-320, 124-488, 125-487, 125-652, 125-811, 130-475, 140-488, 141-475



145-487, 154-507, 156-487, 169-320, 173-355, 173-382, 173-419,



173-491, 198-486, 199-322, 199-479, 212-487, 219-480, 225-491,



242-497, 252-710, 319-606, 330-604, 346-794, 351-578, 351-601,



351-619, 364-714, 374-670, 378-1113, 392-1192, 401-1135, 409-644,



416-703, 441-1161, 465-1050, 486-779, 499-895, 504-808, 507-774,



513-790, 522-901, 583-1225, 598-880, 618-925, 620-1199, 648-893,



653-1252, 674-1146, 696-951, 707-1214, 708-1199, 733-1286, 772-1241,



776-1241, 852-1191, 871-1245, 884-1199, 894-1346,



898-1216, 903-1241, 935-1211, 1001-1200, 1006-1232, 1006-1272,



1032-1600, 1063-1844, 1065-1500, 1073-1835, 1084-1218, 1089-1447,



1104-1382, 1137-1976, 1151-1245, 1153-1766, 1167-1425, 1184-1817,



1200-1730, 1220-1530, 1228-1406, 1231-1991, 1256-1830,



1259-1730, 1284-1554, 1293-2080, 1300-1570, 1354-1591, 1361-1595,



1394-1550, 1398-1634, 1403-2014, 1403-2033, 1422-1942, 1429-2190



1472-1953, 1494-1827, 1496-1765, 1505-1779, 1568-1985, 1585-2219,



1611-1883, 1615-1874, 1615-1895, 1675-1892, 1692-1942,



1704-2227, 1709-1954, 1711-2568, 1711-2654, 1711-2679, 1712-2074,



1718-2363, 1718-2562, 1728-2281, 1732-2319, 1745-1864, 1756-2331,



1780-1997, 1805-2337, 1811-2582, 1815-2122, 1830-2387, 1840-2076,



1840-2100, 1844-2690, 1851-2567, 1867-2681, 1875-2452,



1877-2166, 1883-2125, 1887-2201, 1892-2160, 1921-2528, 1942-2212,



1981-2215, 1981-2597, 1985-2273, 2001-2223, 2019-2286, 2032-2255,



2035-2626, 2045-2269, 2054-2503, 2070-2168, 2093-2346, 2101-2338,



2102-2682, 2106-2374, 2147-2329, 2158-2410, 2164-2424,



2188-2435, 2198-2419, 2198-2477, 2210-2520, 2211-2457, 2216-2839,



2233-2478, 2243-2494, 2268-2498, 2268-2547, 2288-2439,



2300-2454, 2311-2580, 2317-2556, 2342-2721, 2348-2616, 2354-2647,



2359-2588, 2359-2594, 2363-2753, 2391-2635, 2479-3076, 2480-3047,



2480-3100, 2481-2871, 2510-2805, 2531-3133, 2542-2798, 2545-2801,



2554-2833, 2563-2833, 2563-2876, 2565-2826, 2571-2762,



2571-2876, 2585-2824, 2585-2839, 2589-2799, 2593-2817, 2595-3266,



2599-2899, 2604-2863, 2648-2914, 2648-2915, 2674-3076, 2694-2948,



2722-2970, 2734-3002, 2742-3320, 2754-3015, 2755-2991, 2765-3023,



2794-3059, 2794-3064, 2801-3010, 2803-3057, 2803-3091,



2809-3112, 2815-3109, 2819-3231, 2830-3069, 2843-3440, 2867-3110,



2875-3032, 2884-3144, 2893-3132, 2893-3167, 2893-3169, 2893-3236,



2904-3171, 2904-3206, 2905-3178, 2916-3222, 2934-3358, 2957-3222,



2961-3365, 2964-3207, 2964-3220, 2964-3559, 2964-3755,



2973-3240, 2978-3491, 2982-3244, 2982-3537, 2991-3226, 2998-3637,



3011-3316, 3011-3358, 3013-3291, 3014-3141, 3015-3373, 3046-3547,



3063-3314, 3074-3208, 3074-3333, 3074-3591, 3088-3297, 3093-3668,



3116-3373, 3131-3538, 3133-3541, 3161-3477, 3166-3419,



3172-3755, 3179-3402, 3181-3460, 3192-3432, 3237-3369, 3237-3490,



3237-3510, 3242-3503, 3242-3733, 3243-3512, 3246-3511,



3246-3550, 3248-3710, 3251-3527, 3255-3492, 3256-3433, 3258-3350,



3258-3532, 3260-3514, 3268-3687, 3270-3559, 3278-3498, 3294-3540,



3316-3607, 3318-3566, 3320-3484, 3320-3555, 3320-3584, 3322-3430,



3322-3571, 3338-3639, 3340-3613, 3341-3641, 3344-3602,



3354-3650, 3372-3635, 3378-3755, 3378-3966, 3382-3675, 3383-3606,



3392-3755, 3396-3642, 3411-3664, 3415-3602, 3417-3764, 3421-3732,



3426-3671, 3427-3683, 3428-3722, 3434-3684, 3456-3755, 3494-3746,



3498-3749, 3504-3776, 3508-3673, 3513-3746, 3559-3755,



3591-3863, 3598-4390, 3599-3755, 3602-4391, 3606-3865, 3607-3860,



3611-4387, 3614-3734, 3617-3911, 3617-3972, 3631-3894, 3632-3814,



3641-4141, 3641-4186, 3641-4216, 3643-3843, 3643-3933, 3654-4391,



3660-4091, 3674-3931, 3702-3973, 3714-3978, 3716-4250,



3718-4269, 3721-4195, 3777-3977, 3789-4391, 3805-4096, 3812-4390,



3819-3934, 3826-4306, 3833-4287, 3833-4388, 3834-3886, 3834-3935,



3834-3956, 3834-4042, 3834-4055, 3837-4241, 3840-4390, 3841-4288,



3849-4152, 3851-4162, 3851-4178, 3859-4165, 3859-4277,



3859-4328, 3861-4100, 3863-4305, 3863-4395, 3865-4304, 3868-4140,



3869-4261, 3875-4364, 3875-4388, 3878-4080, 3879-4305,



3882-4375, 3886-4157, 3889-4291, 3896-4291, 3903-4288, 3904-4278,



3905-4175, 3912-4015, 3914-4423, 3927-4204, 3931-4185, 3931-4188,



3933-4227, 3940-4171, 3940-4212, 3944-4194, 3944-4288, 3945-4288,



3955-4424, 3959-4239, 3961-4233, 3962-4249, 3975-4391,



3977-4245, 3977-4306, 3992-4227, 3993-4266, 3995-4436, 3997-4271,



4004-4134, 4012-4435, 4025-4455, 4027-4313, 4027-4320, 4032-4435,



4037-4308, 4038-4276, 4042-4443, 4043-4299, 4045-4434, 4049-4291,



4061-4339, 4073-4288, 4094-4333, 4095-4340, 4099-4310,



4109-4272, 4114-4410, 4121-4377, 4136-4288, 4138-4385, 4139-4424,



4141-4402, 4141-4418, 4153-4435, 4174-4357, 4183-4448, 4185-4435,



4186-4435, 4202-4432, 4207-4417, 4214-4435, 4220-4388, 4228-4435,



4261-4372


71/7511852CB1/
1-252, 1-502, 1-505, 1-548, 1-551, 1-555, 1-570, 1-578, 1-581, 1-594,


1949
1-621, 1-622, 1-626, 1-633, 1-636, 1-640, 1-641, 1-656, 1-657, 1-658,



1-678, 1-680, 1-689, 1-705, 1-711, 1-714, 1-731, 1-735, 1-738, 1-746,



1-751, 1-752, 1-753, 1-755, 1-785, 1-786, 1-790, 1-814, 2-409,



2-509, 2-643, 2-679, 2-738, 2-745, 2-762, 2-1949, 4-640, 21-250,



82-250, 126-767, 154-250, 161-249, 161-251, 215-492, 217-490, 217-500,



320-554, 320-694, 320-757, 331-583, 368-1085, 388-650, 800-1037,



800-1410, 809-1083, 818-1300, 869-1418, 888-1037, 910-1158,



937-1207, 993-1084, 1010-1683, 1037-1321, 1099-1374, 1099-1560,



1107-1313, 1110-1386, 1119-1420, 1179-1469, 1191-1449, 1258-1495,



1299-1565, 1319-1530, 1319-1680, 1339-1472, 1361-1613, 1361-1619,



1361-1624, 1366-1619, 1409-1644, 1432-1860, 1444-1667,



1459-1685, 1491-1693, 1515-1766, 1521-1756, 1524-1805, 1526-1782,



1577-1845, 1591-1693


72/7511077CB1/
1-280, 1-341, 127-588, 330-685, 377-1066, 383-953, 392-941,


1322
398-858, 411-1128, 475-1137, 490-943, 499-947, 516-1187, 533-1024, 538-980,



538-1075, 565-1066, 575-1296, 613-1025, 625-1042, 654-1088, 663-1248,



847-1322, 854-1319, 854-1322, 899-1322, 1011-1282, 1062-1292


73/7511576CB1/
1-244, 1-339, 5-292, 6-1364, 15-230, 15-265, 15-281, 16-298,


1381
42-286, 46-249, 46-310, 47-191, 47-297, 50-339, 52-331, 52-339, 54-303,



54-309, 54-315, 59-307, 61-339, 62-339, 62-340, 63-339, 65-295,



65-339, 71-339, 72-339, 75-217, 84-333, 84-381, 86-307, 86-333, 90-212,



90-219, 90-334, 90-339, 91-339, 93-328, 95-174, 95-244, 95-306,



95-312, 95-323, 95-327, 95-339, 96-319, 96-320, 96-330, 97-339,



99-327, 99-339, 100-332, 103-339, 104-339, 115-339, 116-320, 117-324,



148-339, 194-339, 339-499, 340-538, 340-540, 340-557, 340-569,



340-583, 340-607, 340-799, 340-814, 340-921, 340-930, 340-948,



343-948, 346-605, 346-621, 348-588, 348-596, 348-614, 348-941,



348-948, 349-546, 353-583, 353-879, 357-948, 359-551, 360-575,



361-943, 367-948, 369-909, 371-667, 374-609, 383-795, 387-662, 387-784,



397-933, 398-905, 403-653, 406-903, 418-687, 418-948, 419-668,



420-679, 421-841, 431-670, 437-670, 445-684, 447-691, 451-947,



453-942, 460-745, 461-948, 468-658, 469-741, 476-948, 483-948,



484-948, 485-948, 486-947, 489-947, 489-951, 492-947, 492-948,



493-947, 494-947, 496-947, 497-948, 498-927, 498-948, 499-923,



504-947, 506-943, 507-948, 508-942, 509-950, 512-948, 514-943, 515-947,



515-948, 516-798, 517-941, 518-952, 526-942, 531-733, 531-948,



532-947, 534-947, 536-948, 539-948, 539-951, 540-951, 541-947,



541-948, 542-947, 543-917, 543-947, 544-948, 545-945, 546-941,



547-947, 547-948, 548-910, 550-941, 553-941, 558-948, 563-875, 568-857,



574-828, 579-830, 579-893, 579-923, 581-800, 590-947, 592-941,



595-771, 595-948, 607-948, 611-872, 611-899, 611-947, 615-946,



617-948, 618-947, 622-939, 622-948, 623-947, 627-769, 627-907,



629-948, 630-946, 637-941, 670-948, 682-911, 695-948, 700-947, 708-947,



713-943, 759-948, 798-947, 807-947, 811-948, 821-953, 822-948, 839-947,



843-948, 867-951, 878-948, 885-948, 922-1381, 1058-1084, 1112-1138


74/7511492CB1/
1-162, 1-265, 1-269, 1-270, 1-275, 1-289, 1-314, 1-316, 1-1027,


1027
10-238, 10-263, 10-291, 11-254, 11-304, 14-184, 14-214, 14-337, 16-249,



16-281, 19-247, 19-287, 19-300, 20-249, 20-261, 20-271, 20-282, 25-229,



25-250, 25-269, 25-341, 27-254, 27-263, 27-270, 27-278, 27-285,



27-287, 27-310, 27-321, 27-349, 28-328, 30-229, 30-269, 31-323,



32-298, 33-199, 33-325, 34-313, 35-295, 35-301, 36-263, 36-299,



37-246, 37-264, 37-275, 37-319, 39-267, 40-273, 40-294, 40-296,



40-322, 40-325, 40-328, 42-242, 45-212, 45-325, 45-346, 46-226, 46-240,



46-285, 46-294, 46-297, 46-298, 46-302, 46-309, 46-312, 46-316,



46-319, 46-333, 46-334, 47-197, 47-282, 47-292, 47-295, 47-300,



47-320, 47-322, 47-329, 47-333, 47-334, 47-335, 47-338, 47-342,



47-349, 48-248, 48-322, 48-323, 48-324, 48-325, 48-328, 48-343, 48-346,



48-349, 49-279, 49-293, 49-322, 51-248, 51-301, 51-349, 52-190,



52-202, 52-294, 52-306, 52-310, 52-317, 52-319, 52-320, 52-325,



52-326, 52-332, 52-339, 52-349, 53-178, 53-205, 53-271, 53-274,



53-282, 53-283, 53-286, 53-293, 53-294, 53-300, 53-302, 53-304,



53-306, 53-310, 53-311, 53-317, 53-318, 53-323, 53-328, 53-335,



53-349, 54-139, 54-257, 54-264, 54-265, 54-271, 54-280, 54-307, 54-310,



54-316, 54-323, 54-349, 55-250, 55-270, 55-288, 55-295, 55-305,



55-318, 55-346, 55-348, 55-349, 56-349, 57-197, 57-211, 57-250,



57-258, 57-292, 57-295, 57-298, 57-304, 57-306, 57-308, 57-310,



57-318, 57-320, 57-324, 57-325, 57-326, 57-328, 57-330, 57-337, 57-341,



57-349, 58-250, 58-253, 58-275, 58-282, 58-286, 58-295, 58-296,



58-306, 58-309, 58-313, 58-322, 58-329, 58-336, 58-346, 58-349,



59-298, 59-304, 59-315, 59-316, 59-326, 59-349, 60-183, 60-226,



60-253, 60-264, 60-277, 60-282, 60-292, 60-293, 60-294, 60-296, 60-297,



60-298, 60-301, 60-302, 60-307, 60-308, 60-309, 60-311, 60-316,



60-317, 60-327, 60-334, 60-335, 60-338, 60-339, 60-342, 60-349



61-223, 61-251, 61-295, 61-300, 61-304, 61-319, 61-337, 61-342,



61-347, 61-349, 62-266, 62-304, 62-306, 62-314, 62-318, 62-321, 62-331



62-346, 62-349, 63-314, 64-215, 64-264, 64-343, 64-349, 66-285,



66-315, 66-329, 66-338, 66-343, 67-321, 67-345, 69-315, 69-329



69-338, 70-296, 70-329, 70-343, 70-349, 72-201, 72-294, 72-336,



74-296, 86-311, 86-341, 89-166, 89-317, 89-326, 89-333, 89-337, 89-348



90-309, 94-271, 102-294, 102-349, 102-392, 105-295, 108-337,



108-349, 108-356, 109-289, 112-349, 118-327, 132-349, 141-304



142-251, 166-338, 183-310, 343-460, 343-481, 343-483, 343-490, 343-503,



343-525, 343-545, 343-546, 343-549, 343-554, 343-555, 343-565



343-569, 343-575, 343-577, 343-580, 343-585, 343-586, 343-598,



343-599, 343-601, 343-608, 343-610, 343-611, 343-642, 343-647



343-712, 343-718, 344-588, 344-633, 348-585, 348-603, 348-615, 351-610,



353-584, 353-635, 358-618, 369-607, 371-654, 371-688, 372-559



377-633, 377-636, 377-643, 380-659, 381-636, 381-648, 382-655,



383-614, 385-625, 394-496, 406-604, 406-654, 409-642, 409-711



412-717, 414-670, 416-717, 421-691, 443-557, 446-562, 458-696, 460-680,



460-683, 460-693, 460-694, 460-717, 461-653, 462-716, 463-717



469-626, 469-637, 486-675, 489-717, 506-672, 510-717, 512-644,



530-679, 535-637, 578-717, 593-677


75/7511141CB1/
1-228, 1-518, 1-538, 5-710, 250-824, 250-826, 292-1101, 343-914,


3040
359-903, 450-1096, 528-1167, 561-1252, 673-1397, 773-1481, 776-1427,



828-1553, 829-1557, 837-1461, 846-1436, 851-1469, 853-1707, 919-1605,



930-1670, 958-1752, 959-1514, 970-1544, 986-1588



1116-1494, 1270-1832, 1389-2037, 1402-2184, 1554-2212, 1602-2231, 1676-2200,



1780-2403, 1799-2294, 2053-2619, 2053-2621, 2270-2736, 2271-3040, 2376-2807


76/7511300CB1/
1-157, 1-202, 1-237, 1-262, 1-275, 1-386, 1-513, 1-528, 1-550,


3158
1-555, 1-619, 1-3088, 2-241, 2-248, 2-303, 6-273, 6-455, 6-627, 6-666, 9-466



13-313, 13-417, 16-239, 16-250, 18-257, 18-269, 25-243, 27-407, 28-556,



33-310, 63-392, 63-445, 107-382, 107-445, 107-456, 139-629,



145-609, 174-677, 188-823, 215-346, 217-672, 336-594, 356-1261,



378-886, 393-885, 411-710, 466-697, 466-931, 545-1467, 547-615



547-646, 554-1077, 560-646, 560-752, 560-808, 582-953, 587-646,



690-1245, 690-1416, 694-793, 697-793, 703-1350, 707-793, 732-793



734-1282, 750-1136, 772-1147, 784-1252, 792-1511, 792-1630, 802-1136,



802-1321, 819-1415, 846-1116, 846-1303, 847-1439, 858-1527



907-1132, 924-1430, 935-1640, 941-1416, 943-1598, 945-1212, 952-1411,



976-1622, 977-1444, 987-1778, 1006-1697, 1016-1169



1016-1181, 1016-1500, 1018-1269, 1020-1422, 1039-1288, 1084-1384,



1084-1395, 1084-1685, 1084-1715, 1084-1867, 1084-1917, 1084-1959



1084-1964, 1086-1830, 1088-1333, 1088-1336, 1092-1339, 1094-1341,



1094-1502, 1097-1342, 1097-1601, 1098-1367, 1106-1839



1119-1964, 1140-1794, 1148-1428, 1153-1744, 1156-1388, 1156-2028,



1172-1847, 1175-1409, 1178-1430, 1178-1466, 1178-1575, 1190-1791,



1194-1425, 1200-1800, 1202-1701, 1211-1480, 1219-2084, 1220-1682,



1222-1896, 1223-1707, 1227-1653, 1231-1491, 1253-1695,



1255-1853, 1270-1878, 1274-2124, 1275-2011, 1278-1791, 1284-1841,



1286-1590, 1286-1766, 1286-1799, 1286-1874, 1286-1937, 1288-1549,



1290-1496, 1293-1522, 1315-1848, 1325-2148, 1327-1961, 1328-1897,



1348-1599, 1352-1614, 1354-2148, 1369-1635, 1369-1648,



1369-1896, 1371-2031, 1374-1514, 1374-1604, 1397-2059, 1400-1678,



1400-2001, 1428-1632, 1429-1633, 1435-1939, 1437-2148, 1442-2125,



1444-1754, 1452-2177, 1466-1971, 1481-1860, 1485-1655, 1485-2175,



1496-2111, 1506-2000, 1514-1750, 1518-1799, 1534-1897,



1563-2241, 1570-1766, 1573-1854, 1575-1829, 1575-1833, 1582-2067,



1591-1854, 1598-1686, 1623-1873, 1627-2289, 1645-2266, 1674-2016,



1683-1962, 1686-2419, 1699-1908, 1699-1931, 1699-2258, 1699-2259,



1701-2419, 1702-1968, 1702-1970, 1707-2312, 1710-2149,



1714-2385, 1721-2240, 1725-2219, 1728-2283, 1729-2445, 1735-2080,



1738-2403, 1743-2246, 1744-2246, 1745-1993, 1747-2023,



1747-2343, 1750-2134, 1752-2029, 1757-2244, 1767-2285, 1767-2341,



1779-2297, 1785-2388, 1790-2059, 1790-2065, 1790-2401, 1801-2024,



1803-2056, 1804-2013, 1811-2105, 1811-2401, 1821-2100, 1834-2084,



1834-2095, 1862-2094, 1865-2121, 1866-2099, 1868-2051,



1873-2115, 1874-2183, 1877-2102, 1877-2112, 1877-2412, 1898-2258,



1910-2172, 1917-2118, 1935-2329, 1940-2361, 1942-2225, 1959-2411,



1963-2236, 1964-2214, 1964-2217, 1964-2236, 1964-2447, 1971-2211,



1984-2230, 1986-2273, 1986-2394, 1997-2180, 1999-2083,



1999-2262, 1999-2302, 1999-2441, 2002-2241, 2005-2251, 2017-2279,



2018-2266, 2019-2389, 2021-2319, 2046-2280, 2048-2334, 2128-2378,



2131-2397, 2158-2393, 2158-2445, 2166-2412, 2170-2305, 2177-2447,



2180-2447, 2195-2395, 2201-2447, 2205-2447, 2208-2447,



2236-2383, 2262-2441, 2274-2447, 2328-2447, 2425-2711, 2446-2593,



2446-2613, 2446-2615, 2446-2658, 2446-2664, 2446-2677, 2446-2684,



2446-2707, 2446-2712, 2446-2723, 2446-2774, 2446-2909, 2446-2926,



2446-2971, 2446-2972, 2446-2973, 2446-2980, 2446-3011,



2446-3034, 2446-3044, 2446-3072, 2446-3073, 2446-3088, 2450-2747,



2450-3074, 2451-3047, 2455-2994, 2456-2966, 2460-2676,



2468-2651, 2473-2730, 2475-2696, 2477-2630, 2477-2760, 2480-2960,



2480-3074, 2484-3075, 2484-3089, 2486-2740, 2486-2866, 2492-3039,



2493-2742, 2493-2875, 2493-2972, 2496-3089, 2500-2766, 2500-2925,



2503-2853, 2507-3091, 2517-2819, 2517-2993, 2521-3051,



2523-3041, 2529-3037, 2530-2817, 2530-3076, 2541-2820, 2547-2884,



2549-3041, 2565-2824, 2566-2968, 2566-3038, 2567-3085, 2578-2790,



2582-2726, 2588-3051, 2589-2925, 2596-3039, 2597-2821, 2597-2826,



2597-2875, 2598-3081, 2601-3039, 2602-2868, 2603-2682,



2606-2867, 2609-3000, 2610-2841, 2629-2879, 2629-2918, 2629-3036,



2632-3110, 2635-3036, 2635-3080, 2636-2916, 2636-2961, 2637-2899,



2637-2906, 2638-2891, 2638-2904, 2638-3076, 2639-3082, 2641-3075,



2642-2910, 2646-2891, 2654-3078, 2655-2929, 2658-3085,



2661-3079, 2666-3078, 2676-3076, 2676-3079, 2678-3038, 2678-3078,



2679-3088, 2683-2897, 2683-2920, 2684-3085, 2693-2917, 2694-2899,



2698-2918, 2698-2954, 2698-2978, 2698-3076, 2704-3076, 2706-3068,



2706-3074, 2708-2945, 2708-3077, 2710-3084, 2711-3076,



2712-3080, 2714-3076, 2715-2936, 2718-2954, 2718-3007, 2718-3070,



2718-3081, 2722-3093, 2723-3077, 2723-3080, 2726-3040,



2727-3078, 2734-2968, 2738-3027, 2746-2968, 2746-3038, 2754-3076,



2761-2946, 2770-3039, 2771-3093, 2772-3092, 2773-3068, 2774-3080,



2782-3039, 2784-3041, 2785-3088, 2785-3089, 2786-3030, 2787-3088,



2792-3044, 2803-3086, 2805-3074, 2805-3076, 2810-3090,



2817-3026, 2821-3038, 2831-3075, 2833-3075, 2835-3074, 2839-3085,



2847-3073, 2848-3079, 2857-3097, 2859-3079, 2862-3074, 2862-3075,



2865-3076, 2866-3015, 2866-3075, 2870-3081, 2871-3078, 2872-3085,



2892-3083, 2924-3158, 2925-3095, 2938-3090, 2963-3143, 3000-3137


















TABLE 5





Polynucleotide SEQ




ID NO:
Incyte Project ID:
Representative Library







39
3048626CB1
FIBRUNT02


40
2684425CB1
PONSAZT01


41
7505960CB1
PROSTUT20


42
7507021CB1
THYRNOT02


43
7509099CB1
MIXDTUE01


44
7509361CB1
LIVRTUE01


45
7506815CB1
BRAINOT11


46
7506814CB1
BRAINOT11


47
7506852CB1
BRAINOT20


48
7503782CB1
TMLR2DT01


49
7504647CB1
COLNNOT23


50
7500424CB1
THYRNOT03


51
7500449CB1
BRSTNOT16


53
7503292CB1
BRAINOT18


54
7503311CB1
CONNNOT01


55
7510384CB1
PITUDIR01


56
7509976CB1
FIBRTXS07


57
7510454CB1
BRAINOT18


58
8017335CB1
LATRTUT02


59
7510197CB1
PANCNOT17


60
7510055CB1
SINTBST01


61
7501754CB1
BRAITUT03


62
7510517CB1
BRSTNOT01


63
7511014CB1
BRAIFET01


64
7506687CB1
CORPNOT02


65
7510621CB1
FIBRUNT02


66
7505533CB1
MIXDTME02


67
7511220CB1
BRAITUT12


68
7510967CB1
MLP000032


69
7511298CB1
EOSINOT01


70
7510937CB1
UTRSTMR01


71
7511852CB1
SCOMDIT01


72
7511077CB1
COLNTUT03


73
7511576CB1
UCMCL5T01


74
7511492CB1
PROSTUS23


75
7511141CB1
PANCNOT15


76
7511300CB1
BRAVUNT02


















TABLE 6





Library
Vector
Library Description







BRAIFET01
pINCY
Library was constructed using RNA isolated from brain tissue removed




from a Caucasian male fetus, who was stillborn with a




hypoplastic left heart at 23 weeks' gestation.


BRAINOT11
pINCY
Library was constructed using RNA isolated from brain tissue




removed from the right temporal lobe of a 5-year-old Caucasian




male during a hemispherectomy. Pathology indicated extensive




polymicrogyria and mild to moderate gliosis (predominantly




subpial and subcortical), consistent with chronic seizure




disorder. Family history included a cervical neoplasm.


BRAINOT18
pINCY
Library was constructed using RNA isolated from left temporal




lobe brain tissue removed from a 34-year-old Caucasian male




during cerebral meninges lesion excision. Pathology for the




associated tumor tissue indicated metastatic malignant melanoma.




Neoplastic cells strongly expressed HMB-45. Patient history included




malignant melanoma of skin of the trunk. Family history




included liver cancer, acute myocardial infarction, atherosclerotic




coronary artery disease, and cerebrovascular disease.


BRAINOT20
pINCY
Library was constructed using RNA isolated from diseased brain




tissue removed from the left temporal lobe of a 27-year-old




Caucasian male during a brain lobectomy. Pathology for the




left temporal lobe, including the mesial temporal structures,




indicated focal, marked pyramidal cell loss and gliosis in




hippocampal sector CA1, consistent with mesial temporal sclerosis.




The left frontal lobe showed a focal deep white matter lesion,




characterized by marked gliosis, calcifications, and hemosiderin-




laden macrophages, consistent with a remote perinatal injury.




This frontal lobe tissue also showed mild to moderate




generalized gliosis, predominantly subpial and subcortical,




consistent with chronic seizure disorder. GFAP was positive for




astrocytes. Family history included brain cancer.


BRAITUT03
PSPORT1
Library was constructed using RNA isolated from




brain tumor tissue removed from the left frontal lobe of a 17-year-old




Caucasian female during excision of a cerebral meningeal lesion.




Pathology indicated a grade 4 fibrillary giant and small-cell




astrocytoma. Family history included benign hypertension




and cerebrovascular disease.


BRAITUT12
pINCY
Library was constructed using RNA isolated from




brain tumor tissue removed from the left frontal lobe of a 40-year-old




Caucasian female during excision of a cerebral meningeal lesion.




Pathology indicated grade 4 gemistocytic astrocytoma.


BRAVUNT02
PSPORT1
Library was constructed using pooled RNA isolated




from separate populations of unstimulated astrocytes.


BRSTNOT01
PBLUESCRIPT
Library was constructed using RNA isolated from




the breast tissue of a 56-year-old Caucasian female who died in a motor




vehicle accident.


BRSTNOT16
pINCY
Library was constructed using RNA isolated from diseased




breast tissue removed from a 59-year-old Caucasian female during




a unilateral extended simple mastectomy. Pathology for the associated




tumor tissue indicated an invasive lobular carcinoma




with extension into ducts. Patient history included liver cirrhosis,




esophageal ulcer, hyperlipidemia, and neuropathy.


COLNNOT23
pINCY
Library was constructed using RNA isolated from diseased




colon tissue removed from a 16-year-old Caucasian male during a




total colectomy with abdominal/perineal resection. Pathology




indicated gastritis and pancolonitis consistent with the acute




phase of ulcerative colitis. Inflammation was more severe in the




transverse colon, with inflammation confined to the mucosa.




There was only mild involvement of the ascending and sigmoid




colon, and no significant involvement of the cecum, rectum, or




terminal ileum. Family history included irritable bowel syndrome.


COLNTUT03
pINCY
Library was constructed using RNA isolated from colon tumor




tissue obtained from the sigmoid colon of a 62-year-old




Caucasian male during a sigmoidectomy and permanent




colostomy. Pathology indicated invasive grade 2 adenocarcinoma. One




lymph node contained metastasis with extranodal extension. Patient




history included hyperlipidemia, cataract disorder, and




dermatitis. Family history included benign hypertension,




atherosclerotic coronary artery disease, hyperlipidemia, breast cancer,




and prostate cancer.


CONNNOT01
pINCY
Library was constructed using RNA isolated from




mesentery fat tissue obtained from a 71-year-old Caucasian male during a




partial colectomy and permanent colostomy. Family history included




atherosclerotic coronary artery disease, myocardial




infarction, and extrinsic asthma.


CORPNOT02
pINCY
Library was constructed using RNA isolated from diseased




corpus callosum tissue removed from the brain of a 74-year-old




Caucasian male who died from Alzheimer's disease.


EOSTNOT01
pINCY
Library was constructed using RNA isolated from




microscopically normal eosinophils from 31 non-allergic donors. Donors




abstained from prescription and over-the-counter drug use for at




least one week prior to donating 200 ml of peripheral venous




blood.


FTBRTXS07
pINCY
This subtracted library was constructed using 1.3 million




clones from a dermal fibroblast library and was subjected to two




rounds of subtraction hybridization with 2.8 million clones from




an untreated dermal fibroblast tissue library. The starting




library for subtraction was constructed using RNA isolated from




treated dermal fibroblast tissue removed from the breast of a




31-year-old Caucasian female. The cells were treated with 9CIS




retinoic acid. The hybridization probe for subtraction was




derived from a similarly constructed library from RNA isolated from




untreated dermal fibroblast tissue from the same donor.




Subtractive hybridization conditions were based on the methodologies




of Swaroop et al, NAR (1991) 19: 1954 and Bonaldo, et




al., Genome Research (1996) 6: 791.


FIBRUNT02
pINCY
Library was constructed using RNA isolated from an




untreated MG-63 cell line derived from an osteosarcoma removed from a




14-year-old Caucasian male.


LATRTUT02
pINCY
Library was constructed using RNA isolated from a




myxoma removed from the left atrium of a 43-year-old Caucasian male




during annuloplasty. Pathology indicated atrial myxoma. Patient




history included pulmonary insufficiency, acute myocardial




infarction, atherosclerotic coronary artery disease, hyperlipidemia,




and tobacco use. Family history included benign




hypertension, acute myocardial infarction, atherosclerotic




coronary artery disease, and type II diabetes.


LIVRTUE01
PCDNA2.1
This 5′ biased random primed library was constructed




using RNA isolated from liver tumor tissue removed from a 72-year-old




Caucasian male during partial hepatectomy. Pathology indicated




metastatic grade 2 (of 4) neuroendocrine carcinoma forming a




mass. The patient presented with metastatic liver cancer. Patient




history included benign hypertension, type I diabetes, prostatic




hyperplasia, prostate cancer, alcohol abuse in remission, and




tobacco abuse in remission. Previous surgeries included




destruction of a pancreatic lesion, closed prostatic biopsy,




transurethral prostatectomy, removal of bilateral testes and total




splenectomy. Patient medications included Eulexin, Hytrin,




Proscar, Ecotrin, and insulin. Family history included




atherosclerotic coronary artery disease and acute myocardial




infarction in the mother; atherosclerotic coronary artery disease




and type II diabetes in the father.


MIXDTME02
PBK-CMV
This 5′ biased random primed library was constructed




using pooled cDNA from five donors. cDNA was generated using




mRNA isolated from heart tissue removed from a Caucasian




male fetus who died after 20 weeks gestation from Patau's




syndrome (donor A); adrenal gland removed from a 43-year-old




Caucasian male (donor B) during nephroureterectomy,




regional lymph node excision and unilateral adrenalectomy;




kidney cortex removed from a 65-year-old male (donor C) during




nephroureterectomy; lung tissue removed from a 14-month-old




Caucasian female who died from drowning (donor D); and




kidney tissue removed from an 8-year-old Caucasian female




who died from a motor vehicle accident (donor E). For donor B,




pathology for the associated tumor indicated grade 2 (of 4) renal




cell carcinoma in the left kidney with invasion into the renal




pelvis. Patient presented with hematuria and anemia. Patient




history included benign hypertension and obesity. Previous




surgeries included adenotonsillectomy and indirect inguinal




hernia repair. The patient was not taking any medications. Family




history included benign hypertension and atherosclerotic coronary




artery disease in the father. For donor C pathology for the




associated tumor shows grade 3 (of 4) renal cell carcinoma, clear




cell type, within the mid-portion of the kidney. For donor D,




serologies were negative. For donor E, medications included




respiradol.


MIXDTUE01
PBK-CMV
This 5′ biased random primed library was constructed




using pooled cDNA from seven donors. cDNA was generated using




mRNA isolated from placental tissue removed from a Caucasian




fetus (A), who died after 16 weeks' gestation from fetal




demise and hydrocephalus; from placental tissue removed from




a Caucasian male fetus (B), who died after 18 weeks' gestation




from fetal demise; from an untreated LNCaP cell line, derived




from prostate carcinoma with metastasis to the left




supraclavicular lymph nodes, removed from a 50-year-old




Caucasian male (C); from endometrial tissue removed from a 32-




year-old female (D); from diseased right ovary tissue removed




from a 45-year-old Caucasian female (E); from diseased right




ovary tissue removed from a 47-year-old Caucasian female




(donor F) and from right fallopian tube tumor tissue removed from




an 85-year-old Caucasian female (donor G). For donor A,




patient history included umbilical cord wrapped around the head (3




times) and the shoulders (1 time). Serology was positive for




anti-CMV. Family history included multiple pregnancies and live




births, and an




abortion in the mother. For donor B, serologies were negative.




For donor D, pathology indicated the endometrium was in




secretory phase. For donor E, pathology indicated stromal




hyperthecosis of the right and left ovaries. For donor F, pathology




indicated endometriosis. For donor G, pathology indicated poorly




differentiated mixed endometrioid (80%) and serous (20%)




adenocarcinoma of the right fallopian tube. Patient history




included medullary carcinoma of the thyroid.


MLP000032
PCR2-TOPOTA
Library was constructed using pooled cDNA from




different donors. cDNA was generated using mRNA isolated from the




following: aorta, cerebellum, lymph nodes, muscle, tonsil (lymphoid




hyperplasia), bladder tumor (invasive grade 3 transitional




cell carcinoma.), breast (proliferative fibrocystic changes without




atypia characterized by epithelial ductal hyperplasia, testicle




tumor (embryonal carcinoma), spleen, ovary, parathyroid,




ileum, breast skin, sigmoid colon, penis tumor (fungating invasive




grade 4 squamous cell carcinoma), fetal lung,, breast, fetal small




intestine, fetal liver, fetal pancreas, fetal lung, fetal skin, fetal




penis, fetal bone, fetal ribs, frontal brain tumor (grade 4 gemistocytic




astrocytoma), ovary (stromal hyperthecosis), bladder,




bladder tumor (invasive grade 3 transitional cell carcinoma),




stomach, lymph node tumor (metastatic basaloid squamous cell




carcinoma), tonsil (reactive lymphoid hyperplasia), periosteum




from the tibia, fetal brain, fetal spleen, uterus tumor,




endometrial (grade 3 adenosquamous carcinoma), seminal




vesicle, liver, aorta, adrenal gland, lymph node




(metastatic grade 3 squamous cell carcinoma), glossal muscle,




esophagus, esophagus tumor (invasive grade 3




adenocarcinoma), ileum, pancreas, soft tissue tumor from the




skull (grade 3 ependymoma), transverse colon, (benign familial




polyposis), rectum tumor (grade 3 colonic adenocarcinoma), rib




tumor, (metastatic grade 3 osteosarcoma), lung, heart,




placenta, thymus, stomach, spleen (splenomegaly with congestion),




uterus, cervix (mild chronic cervicitis with focal squamous




metaplasia), spleen tumor (malignant lymphoma, diffuse large




cell type, B-cell phenotype with abundant reactive T-cells and




marked granulomatous response), umbilical cord blood mononuclear




cells, upper lobe lung tumor, (grade 3 squamous cell




carcinoma), endometrium (secretory phase), liver, liver tumor




(metastatic grade 2 neuroendocrine carcinoma), colon, umbilical




cord blood, Th1 cells, nonactivated, umbilical cord blood,




Th2 cells, nonactivated, coronary artery endothelial cells




(untreated), coronary artery smooth muscle cells, (untreated),




coronary artery smooth muscle cells.(treated with TNF & IL-1




10 ng/ml each for 20 hours), bladder (mild chronic cystitis),




epiglottis, breast skin, small intestine, fetal prostate stroma




fibroblasts, prostate epithelial cells (PrEC cells), fetal adrenal




glands, fetal liver, kidney transformed embryonal cell line (293-




EBNA) (untreated), kidney transformed embryonal cell line




(293-EBNA) (treated with 5Aza-2deoxycytidine for 72 hours),




mammary epithelial cells, (HMEC cells), peripheral blood monocytes




(treated with IL-10 at time 0, 10 ng/ml, LPS was added at




1 hour at 5 ng/ml. Incubation 24 hours), peripheral blood monocytes




(treated with anti-IL-10 at time 0, 10 ng/ml, LPS was added




at 1 hour at 5 ng/ml. Incubation 24 hours), spinal cord, base of




medulla (Huntington's chorea), thigh and arm muscle (ALS),




breast skin fibroblast (untreated), breast skin fibroblast (treated




with 9CIS Retinoic Acid 1 μM for 20 hours), breast skin




fibroblast (treated with TNF-alpha & IL-1 beta, 10 ng/ml each for




20 hours), fetal liver mast cells, hematopoietic (Mast cells




prepared from human fetal liver hematopoietic progenitor cells




(CD34+ stem cells) cultured in the presence of




hIL-6 and hSCF for 18 days), epithelial layer of colon, bronchial




epithelial cells (treated for 20hours with 20% smoke




conditioned media), lymph node, pooled peripheral blood




mononuclear cells (untreated), pooled brain segments: striatum,




globus pallidus and posterior putamen (Alzheimer's Disease),




pituitary gland, umbilical cord blood, CD34+ derived dendritic




cells (treated with SCF, GM-CSF & TNF alpha, 13 days), umbilical




cord blood, CD34+ derived dendritic cells (treated with




SCF, GM-CSF & TNF alpha, 13 days followed by PMA/Ionomycin




for 5 hours), small intestine, rectum, bone marrow




neuroblastoma cell line (SH-SY5Y cells, treated with 6-




Hydroxydopamine 100 uM for 8 hours), bone marrow, neuroblastoma




cell line (SH-SY5Y cells, untreated), brain segments from one




donor: amygdala, entorhinal cortex, globus pallidus, substantia




innominata, striatum, dorsal caudate nucleus, dorsal putamen,




ventral nucleus accumbens, archaecortex (hippocampus anterior




and posterior), thalamus, nucleus raphe magnus, periaqueductal




gray, midbrain, substantia nigra, and dentate nucleus,




pineal gland (Alzheimer's Disease), preadipocytes (untreated),




preadipocytes (treated with a peroxisome proliferator-activated




receptor gamma agonist, 1microM, 4 hours), pooled prostate




(adenofibromatous hyperplasia), pooled kidney, pooled




adipocytes (untreated), pooled adipocytes (treated with human




insulin), pooled mesentaric and abdomenal fat, pooled adrenal




glands, pooled thyroid (normal and adenomatous hyperplasia),




pooled spleen (normal and with changes consistent with




idiopathic thrombocytopenic purpura), pooled right and left breast,




pooled lung, pooled nasal polyps, pooled fat, pooled




synovium (normal and rhumatoid arthritis), pooled brain




(meningioma, gemistocytic astrocytoma. and Alzheimer's disease),




pooled fetal colon, pooled colon: ascending, descending (chronic




ulcerative colitis), and rectal tumor (adenocarcinoma),




pooled esophagus, normal and tumor (invasive grade 3




adenocarcinoma), pooled breast skin fibroblast (one treated w/9CIS




Retinoic Acid and the other with TNF-alpha & IL-1 beta),




pooled gallbladder (acute necrotizing




cholecystitis with cholelithiasis (clinically hydrops), acute




hemorrhagic cholecystitis with cholelithiasis, chronic cholecystitis




and cholelithiasis), pooled fetal heart, (Patau's and fetal




demise), pooled neurogenic tumor cell line, SK-N-MC,




(neuroepitelioma, metastasis to supra-orbital area, untreated)




and neuron, NT-2 cell line, (treated with mouse leptin at 1 μg/ml




and 9cis retinoic acid at 3.3 μM for 6 days), pooled ovary




(normal and polycystic ovarian disease), pooled prostate,




(adenofibromatous hyperplasia), pooled seminal vesicle,




pooled small intestine, pooled fetal small intestine, pooled stomach




and fetal stomach, prostate epithelial cells, pooled testis (normal




and embryonal carcinoma), pooled uterus, pooled uterus




tumor (grade 3 adenosquamous carcinoma and leiomyoma),




pooled uterus, endometrium, and myometrium, (normal and




adenomatous hyperplasia with squamous metaplasia and focal




atypia), pooled brain: (temporal lobe meningioma, cerebellum




and hippocampus (Alzheimer's Disease), pooled skin,




fetal lung, adrenal tumor (adrenal cortical carcinoma), prostate tumor




(adenocarcinoma), fetal heart,




fetal small intestine, ovary tumor (mucinous cystadenoma),




ovary, ovary tumor (transitional cell carcinoma), disease prostate




(adenofibromatous hyperplasia), fetal colon, uterus tumor




(leiomyoma), temporal brain, submandibular gland, colon tumor




(adenocarcinoma), ascending and transverse colon, ovary tumor




(endometrioid carcinoma), lung tumor (squamous cell




carcinoma), fetal brain, fetal lung, ureter tumor (transitional cell




carcinoma), untreated HNT cells, para-aortic soft tissue, testis,




seminal vesicle, diseased ovary (endometriosis), temporal




lobe, myometrium, diseased gallbladder (cholecystitis,




cholelithiasis), placenta, breast tumor (ductal adenocarcinoma),




breast, lung tumor (liposarcoma), endometrium, abdominal fat,




cervical spine dorsal root ganglion, thoracic spine dorsal root




ganglion, diseased thyroid (adenomatous hyperplasia), liver,




kidney, fetal liver, NT-2 cells (treated with mouse leptin and




9cis RA), K562 cells (treated with 9cis RA), cerebellum, corpus




callosum, hypothalamus, fetal brain astrocytes (treated with




TNFa and IL-1b), inferior parietal cortex, posterior hippocampus,




pons,




thalamus, C3A cells (untreated), C3A cells (treated with 3-




methylcholanthrene), testis, colon epithelial layer, pooled prostate,




pooled liver, substantia nigra, thigh muscle, rib bone, fallopian




tube tumor (endometrioid and serous adenocarcinoma),




diseased lung (idiopathic pulmonary disease), cingulate anterior




allocortex and neocortex, cingulate posterior allocortex,




auditory neocortex, frontal neocortex, orbital inferior neocortex,




parietal superior neocortex, visual primary neocortex, dentate




nucleus, posterior cingulate, cerebellum, vermis, inferior temporal




cortex, medulla, posterior parietal cortex, colon polyp,




pooled breast, anterior and posterior hippocampus, mesenteric




and abdominal fat, pooled esophagus, pooled fetal kidney,




pooled fetal liver, ileum, small intestine, pooled gallbladder, frontal




and superior temporal cortex, pooled ovary, pooled




endometrium, pooled prostate, pooled kidney, fetal femur, sacrum




tumor (giant cell tumor), pooled kidney and kidney tumor




(renal cell carcinoma clear-cell type), pooled liver and liver tumor




(neuroendocrine carcinoma), pooled fetal liver,




pooled lung, fetal pancreas, pancreas, parotid gland, parotid




tumor (sebaceous lymphadenoma), retroperitoneal and suprglottic




soft tissue, spleen, fetal spleen, spleen tumor (malignant lymphoma),




diseased spleen (idiopathic thrombocytopenic purpura),




parathyroid, thyroid, thymus, tonsil ureter tumor (transitional cell




carcinoma), pooled adrenal gland and adrenal tumor




(pheochromocytoma), pooled lymph node tumor (Hodgkin's




disease and metastatic adenocarcinoma), pooled neck and calf




muscles, and pooled bladder.


PANCNOT15
pINCY
Library was constructed using RNA isolated from diseased




pancreatic tissue removed from a 15-year-old Caucasian male




during a exploratory laparotomy with distal pancreatectomy and




total splenectomy. Pathology indicated islet cell hyperplasia.




Family history included prostate cancer and cardiovacular disease.


PANCNOT17
pINCY
Library was constructed using RNA isolated from pancreatic




tissue removed from a 65-year-old female. Pathology for the




associated tumor tissue indicated well-differentiated, metastatic,




neuroendocrine carcinoma (nuclear grade 1).


PITUDIR01
PCDNA2.1
This random primed library was constructed using RNA isolated




from pituitary gland tissue removed from a 70-year-old




female who died from metastatic adenocarcinoma.


PONSAZT01
pINCY
Library was constructed using RNA isolated from diseased pons tissue




removed from the brain of a 74-year-old Caucasian




male who died from Alzheimer's disease.


PROSTUS23
pINCY
This subtracted prostate tumor library was constructed using 10 million




clones from a pooled prostate tumor library that was




subjected to 2 rounds of subtractive hybridization with 10 million




clones from a pooled prostate tissue library. The starting




library for subtraction was constructed by pooling equal numbers




of clones from 4 prostate tumor libraries using mRNA




isolated from prostate tumor removed from Caucasian males




at ages 58 (A), 61 (B), 66 (C), and 68 (D) during prostatectomy




with lymph node excision. Pathology indicated adenocarcinoma




in all donors. History included elevated PSA, induration and




tobacco abuse in donor A; elevated PSA, induration, prostate




hyperplasia, renal failure, osteoarthritis, renal artery stenosis,




benign HTN, thrombocytopenia, hyperlipidemia, tobacco/alcohol




abuse and hepatitis C (carrier) in donor B; elevated PSA,




induration, and tobacco abuse in donor C; and elevated PSA,




induration, hypercholesterolemia, and kidney calculus in donor




D. The hybridization probe for subtraction was constructed by




pooling equal numbers of cDNA clones from 3 prostate




tissue libraries derived from prostate tissue, prostate epithelial




cells, and fibroblasts from prostate stroma from 3 different




donors. Subtractive hybridization conditions were based on the




methodologies of Swaroop et al., NAR 19 (1991): 1954 and




Bonaldo, et al. Genome Research 6 (1996): 791.


PROSTUT20
pINCY
The library was constructed using RNA isolated from prostate




umor tissue removed from a 58-year-old Caucasian male during




radical prostatectomy, regional lymph node excision, and




prostate needle biopsy. Pathology indicated adenocarcinoma




(Gleason grade 3 + 2) of the prostate, which formed a




predominant mass involving primarily the right side and focally involved




the left side, peripherally and anteriorly. The patient presented




with elevated prostate specific antigen (PSA)and induration.




Family history included breast cancer.


SCOMDIT01
pINCY
Library was constructed using RNA isolated from diseased spinal




cord tissue removed from the base of the medulla of a 57-




year-old Caucasian male who died from a cerebrovascular




accident. Patient history included Huntington's disease and




emphysema.


SINTBST01
pINCY
Library was constructed using RNA isolated from ileum




tissue obtained from an 18-year-old Caucasian female during bowel




anastomosis. Pathology indicated Crohn's disease of the ileum,




involving 15 cm of the small bowel. Family history included




cerebrovascular disease and atherosclerotic coronary artery disease.


THYRNOT02
PSPORT1
Library was constructed using RNA isolated from the diseased




thyroid tissue of a 16-year-old Caucasian female with Graves'




disease (hyperthyroidism).


THYRNOT03
pINCY
Library was constructed using RNA isolated from thyroid




tissue removed from the left thyroid of a 28-year-old Caucasian




female during a complete thyroidectomy. Pathology indicated a




small nodule of adenomatous hyperplasia present in the left




thyroid. Pathology for the associated tumor tissue indicated




dominant follicular adenoma, forming a well-encapsulated mass in




the left thyroid.


TMLR2DT01
PBLUESCRIPT
Library was constructed using RNA isolated from




non-adherent peripheral blood mononuclear cells. The blood was obtained




from unrelated male and female donors. Cells from each donor




were purified on Ficoll Hypaque, then co-cultured for 24 hours




in medium containing normal human serum at a cell




density of 2million cells/ml.


UCMCL5T01
PBLUESCRIPT
Library was constructed using RNA isolated from




mononuclear cells obtained from the umbilical cord blood of 12 individuals.




The cells were cultured for 12 days with IL-5 before RNA was




obtained from the pooled lysates.


UTRSTMR01
pINCY
Library was constructed using RNA isolated from uterine




myometrial tissue removed from a 41-year-old Caucasian female




during a vaginal hysterectomy. The endometrium was secretory




and contained fragments of endometrial polyps. Pathology for




associated tumor tissue indicated uterine leiomyoma. Patient




history included ventral hernia and a benign ovarian neoplasm.



















TABLE 7








Parameter


Program
Description
Reference
Threshold







ABI FACTURA
A program that removes vector sequences and masks
Applied Biosystems, Foster City, CA.




ambiguous bases in nucleic acid sequences.


ABI/PARACEL FDF
A Fast Data Finder useful in comparing and
Applied Biosystems, Foster City, CA;
Mismatch <50%



annotating amino acid or nucleic acid sequences.
Paracel Inc., Pasadena, CA.


ABI AutoAssembler
A program that assembles nucleic acid sequences.
Applied Biosystems, Foster City, CA.


BLAST
A Basic Local Alignment Search Tool useful in
Altschul, S. F. et al. (1990) J. Mol. Biol.
ESTs: Probability



sequence similarity search for amino acid and nucleic
215: 403-410; Altschul, S. F. et al. (1997)
value = 1.0E−8



acid sequences. BLAST includes five functions:
Nucleic Acids Res. 25: 3389-3402.
or less;



blastp, blastn, blastx, tblastn, and tblastx.

Full Length





sequences:





Probability





value = 1.0E−10





or less


FASTA
A Pearson and Lipman algorithm that searches for
Pearson, W. R. and D. J. Lipman (1988) Proc.
ESTs: fasta E



similarity between a query sequence and a group of
Natl. Acad Sci. USA 85: 2444-2448; Pearson,
value = 1.06E−6;



sequences of the same type. FASTA comprises as
W. R. (1990) Methods Enzymol. 183: 63-98;
Assembled ESTs:



least five functions: fasta, tfasta, fastx, tfastx, and
and Smith, T. F. and M. S. Waterman (1981)
fasta Identity =



ssearch.
Adv. Appl. Math. 2: 482-489.
95% or greater





and Match





length = 200





bases or greater;





fastx E value =





1.0E−8 or less;





Full Length





sequences: fastx





score = 100





or greater


BLIMPS
A BLocks IMProved Searcher that matches a
Henikoff, S. and J. G. Henikoff (1991)
Probability



sequence against those in BLOCKS, PRINTS,
Nucleic Acids Res. 19: 6565-6572; Henikoff, J. G.
value = 1.0E−3



DOMO, PRODOM, and PFAM databases to search
and S. Henikoff (1996) Methods
or less



for gene families, sequence homology, and structural
Enzymol. 266: 88-105; and Attwood, T. K. et



fingerprint regions.
al. (1997) J. Chem. Inf. Comput. Sci. 37: 417-424.


HMMER
An algorithm for searching a query sequence against
Krogh, A. et al. (1994) J. Mol. Biol.
PFAM, INCY,



hidden Markov model (HMM)-based databases of
235: 1501-1531; Sonnhammer, E. L. L. et al.
SMART or



protein family consensus sequences, such as PFAM,
(1988) Nucleic Acids Res. 26: 320-322;
TIGRFAM hits:



INCY, SMART and TIGRFAM.
Durbin, R. et al. (1998) Our World View, in
Probability




a Nutshell, Cambridge Univ. Press, pp. 1-350.
value =





1.0E−3 or less;





Signal peptide





hits: Score =





0 or greater


ProfileScan
An algorithm that searches for structural and
Gribskov, M. et al. (1988) CABIOS 4: 61-66;
Normalized



sequence motifs in protein sequences that match
Gribskov, M. et al. (1989) Methods
quality score ≧



sequence patterns defined in Prosite.
Enzymol. 183: 146-159; Bairoch, A. et al.
GCG specified




(1997) Nucleic Acids Res. 25: 217-221.
“HIGH” value





for that





particular Prosite





motif. Generally,





score = 1.4-2.1.


Phred
A base-calling algorithm that examines automated
Ewing, B. et al. (1998) Genome Res. 8: 175-185;



sequencer traces with high sensitivity and probability.
Ewing, B. and P. Green (1998) Genome




Res. 8: 186-194.


Phrap
A Phils Revised Assembly Program including
Smith, T. F. and M. S. Waterman (1981) Adv.
Score = 120 or



SWAT and CrossMatch, programs based on efficient
Appl. Math. 2: 482-489; Smith, T. F. and
greater; Match



implementation of the Smith-Waterman algorithm,
M. S. Waterman (1981) J. Mol. Biol. 147: 195-197;
length = 56 or



useful in searching sequence homology and
and Green, P., University of
greater



assembling DNA sequences.
Washington, Seattle, WA.


Consed
A graphical tool for viewing and editing Phrap
Gordon, D. et al. (1998) Genome Res. 8: 195-202.



assemblies.


SPScan
A weight matrix analysis program that scans protein
Nielson, H. et al. (1997) Protein Engineering
Score = 3.5 or



sequences for the presence of secretory signal
10: 1-6; Claverie, J. M. and S. Audic (1997)
greater



peptides.
CABIOS 12: 431-439.


TMAP
A program that uses weight matrices to delineate
Persson, B. and P. Argos (1994) J. Mol. Biol.



transmembrane segments on protein sequences and
237: 182-192; Persson, B. and P. Argos



determine orientation.
(1996) Protein Sci. 5: 363-371.


TMHMMER
A program that uses a hidden Markov model (HMM)
Sonnhammer, E. L. et al. (1998) Proc. Sixth



to delineate transmembrane segments on protein
Intl. Conf. On Intelligent Systems for Mol.



sequences and determine orientation.
Biol., Glasgow et al., eds., The Am. Assoc.




for Artificial Intelligence (AAAI) Press,




Menlo Park, CA, and MIT Press, Cambridge,




MA, pp. 175-182.


Motifs
A program that searches amino acid sequences for
Bairoch, A. et al. (1997) Nucleic Acids Res.



patterns that matched those defined in Prosite.
25: 217-221; Wisconsin Package Program




Manual, version 9, page M51-59, Genetics




Computer Group, Madison, WI.





























TABLE 8

















Asian



SEQ









Caucasian
African
Allele 1
Hispanic


ID



EST
CB1
EST


Amino
Allele 1
Allele 1
fre-
Allele 1


NO:
PID
EST ID
SNP ID
SNP
SNP
Allele
Allele 1
Allele 2
acid
frequency
frequency
quency
frequency




























39
3048626
3504115H1
SNP00142220
215
249
T
T
C
S68
n/a
n/a
n/a
n/a


39
3048626
3517533H1
SNP00038815
179
382
T
C
T
S113
n/a
n/a
n/a
n/a


39
3048626
7409370H1
SNP00142220
376
252
T
T
C
P69
n/a
n/a
n/a
n/a


40
2684425
2120782H1
SNP00096166
164
709
T
T
C
I165
n/a
n/a
n/a
n/a


40
2684425
2291826H1
SNP00027861
175
2682
T
C
T
noncoding
n/a
n/a
n/a
n/a


40
2684425
4379356H1
SNP00096166
169
708
T
T
C
I165
n/a
n/a
n/a
n/a


40
2684425
4380813H1
SNP00096166
165
706
T
T
C
N164
n/a
n/a
n/a
n/a


40
2684425
4574989H1
SNP00073769
107
2351
T
C
T
C713
n/d
n/d
n/d
n/d


40
2684425
5814109H1
SNP00105399
56
2524
G
G
A
E770
n/a
n/a
n/a
n/a


40
2684425
5817776H1
SNP00105399
57
2525
G
G
A
E771
n/a
n/a
n/a
n/a


40
2684425
5821814H1
SNP00105399
55
2523
G
G
A
G770
n/a
n/a
n/a
n/a


40
2684425
6196749H1
SNP00073769
105
2354
C
C
T
H714
n/d
n/d
n/d
n/d


40
2684425
6725305H1
SNP00105399
330
2527
G
G
A
Q771
n/a
n/a
n/a
n/a


41
7505960
2699329H1
SNP00113522
36
1589
A
A
C
K528
n/d
n/d
n/d
n/d


41
7505960
2743044H1
SNP00060643
34
820
T
C
T
Y271
n/a
n/a
n/a
n/a


41
7505960
4443424H1
SNP00113522
8
1587
A
A
C
D527
n/d
n/d
n/d
n/d


41
7505960
4860526H1
SNP00113522
79
1588
A
A
C
A527
n/d
n/d
n/d
n/d


41
7505960
6298990H1
SNP00122314
88
387
G
G
A
G127
n/d
n/d
n/d
n/d


41
7505960
6455382H1
SNP00122314
391
390
G
G
A
G128
n/d
n/d
n/d
n/d


42
7507021
6041339H1
SNP00058963
323
903
C
T
C
noncoding
n/d
n/d
n/a
n/d


43
7509099
7196339H1
SNP00116698
483
1254
A
A
C
R391
0.97
0.96
n/d
0.99


44
7509361
1307093H1
SNP00076254
169
753
C
C
G
T167
n/a
n/a
n/a
n/a


44
7509361
1314858H1
SNP00148375
150
1207
G
G
A
noncoding
n/a
n/a
n/a
n/a


44
7509361
1442760H1
SNP00011095
243
288
G
G
A
P12
n/a
n/a
n/a
n/a


44
7509361
1554337H1
SNP00011095
144
289
A
G
A
M13
n/a
n/a
n/a
n/a


44
7509361
1560591H1
SNP00148375
94
1205
G
G
A
noncoding
n/a
n/a
n/a
n/a


44
7509361
1620178H1
SNP00148375
90
1206
G
G
A
noncoding
n/a
n/a
n/a
n/a


44
7509361
2697396H1
SNP00011095
279
287
G
G
A
R12
n/a
n/a
n/a
n/a


44
7509361
2841966H1
SNP00100840
34
500
G
G
C
G83
n/d
n/d
n/a
n/d


44
7509361
2842095H1
SNP00100840
35
501
G
G
C
E83
n/d
n/d
n/a
n/d


44
7509361
2848215H1
SNP00011095
184
286
G
G
A
A12
n/a
n/a
n/a
n/a


44
7509361
3112588H1
SNP00011095
239
285
G
G
A
L11
n/a
n/a
n/a
n/a


44
7509361
3498684H1
SNP00148375
29
1200
G
G
A
noncoding
n/a
n/a
n/a
n/a


44
7509361
3551794H1
SNP00076254
116
752
C
C
G
T167
n/a
n/a
n/a
n/a


44
7509361
5120295H1
SNP00076254
115
741
G
C
G
K163
n/a
n/a
n/a
n/a


44
7509361
7432084H1
SNP00148375
235
1198
G
G
A
noncoding
n/a
n/a
n/a
n/a


47
7506852
1401073H1
SNP00036002
67
1691
T
C
T
noncoding
n/a
n/a
n/a
n/a


47
7506852
3724338H1
SNP00036002
254
1687
C
C
T
noncoding
n/a
n/a
n/a
n/a


47
7506852
3854972H1
SNP00036002
17
1689
C
C
T
noncoding
n/a
n/a
n/a
n/a


47
7506852
4205312H1
SNP00036002
192
1677
C
C
T
noncoding
n/a
n/a
n/a
n/a


48
7503782
6366326H1
SNP00096612
255
2675
C
C
A
H627
0.45
0.28
0.50
0.38


48
7503782
6587229H1
SNP00058173
320
3251
A
A
G
noncoding
0.62
n/a
n/a
n/a


48
7503782
7036138H1
SNP00096612
125
2677
A
C
A
Q628
0.45
0.28
0.50
0.38


49
7504647
5051517H1
SNP00065898
155
1110
A
A
G
noncoding
n/a
n/a
n/a
n/a


49
7504647
5051533H1
SNP00065898
155
1111
A
A
G
noncoding
n/a
n/a
n/a
n/a


50
7500424
1335968H1
SNP00001367
118
696
G
G
A
noncoding
n/a
n/a
n/a
n/a


50
7500424
1335968H1
SNP00149783
49
627
A
A
G
noncoding
n/a
n/a
n/a
n/a


50
7500424
1445563H1
SNP00121362
107
574
G
G
C
noncoding
n/a
n/a
n/a
n/a


50
7500424
3407118H1
SNP00001367
154
694
G
G
A
noncoding
n/a
n/a
n/a
n/a


50
7500424
3407118H1
SNP00149783
85
625
A
A
G
noncoding
n/a
n/a
n/a
n/a


50
7500424
3484021H1
SNP00148356
184
193
C
C
T
P33
n/a
n/a
n/a
n/a


50
7500424
3614711H1
SNP00148356
112
112
C
C
T
P6
n/a
n/a
n/a
n/a


50
7500424
3646188H1
SNP00001367
137
693
G
G
A
noncoding
n/a
n/a
n/a
n/a


50
7500424
3714764H1
SNP00149783
131
624
A
A
G
noncoding
n/a
n/a
n/a
n/a


50
7500424
4201810H1
SNP00121362
24
573
G
G
C
noncoding
n/a
n/a
n/a
n/a


50
7500424
4457406H1
SNP00148356
18
117
C
C
T
Q8
n/a
n/a
n/a
n/a


50
7500424
4822857H1
SNP00121362
34
572
G
G
C
noncoding
n/a
n/a
n/a
n/a


50
7500424
4992535H1
SNP00148356
129
109
C
C
T
S5
n/a
n/a
n/a
n/a


50
7500424
5810876H1
SNP00121362
204
571
G
G
C
noncoding
n/a
n/a
n/a
n/a


51
7500449
2376438H1
SNP00037402
81
185
T
T
C
R39
n/a
n/a
n/a
n/a


51
7500449
6880929J1
SNP00142781
103
930
C
C
A
noncoding
n/a
n/a
n/a
n/a


53
7503292
7076246H1
SNP00111578
128
1217
G
A
G
noncoding
0.03
n/a
n/a
n/a


54
7503311
7455178H2
SNP00126507
378
1463
C
C
A
noncoding
n/a
n/a
n/a
n/a


54
7503311
8007727H1
SNP00137525
212
1326
G
G
A
noncoding
n/a
n/a
n/a
n/a


55
7510384
6908872J1
SNP00062894
404
528
C
T
C
P165
n/d
n/a
n/a
n/a


55
7510384
6912987J1
SNP00062894
374
520
T
T
C
N162
n/d
n/a
n/a
n/a


58
8017335
1351856H1
SNP00120957
54
1790
C
C
T
noncoding
n/a
n/a
n/a
n/a


58
8017335
4776360H1
SNP00031314
199
2140
C
A
C
noncoding
0.87
0.87
0.88
0.84


58
8017335
6839382H1
SNP00031314
379
2141
A
A
C
noncoding
0.87
0.87
0.88
0.84


60
7510055
1432995H1
SNP00015901
1
1031
G
G
A
noncoding
n/a
n/a
n/a
n/a


60
7510055
1432995H1
SNP00015902
56
1086
C
C
G
noncoding
0.91
n/a
n/a
n/a


60
7510055
1452055H1
SNP00000585
7
77
C
C
T
noncoding
0.83
0.97
0.58
0.81


60
7510055
1536017H1
SNP00000585
20
76
C
C
T
noncoding
0.83
0.97
0.58
0.81


60
7510055
2610356H1
SNP00000585
59
75
C
C
T
noncoding
0.83
0.97
0.58
0.81


60
7510055
2970103H2
SNP00015901
154
1032
G
G
A
noncoding
n/a
n/a
n/a
n/a


60
7510055
2970103H2
SNP00015902
209
1087
C
C
G
noncoding
0.91
n/a
n/a
n/a


60
7510055
3520995H1
SNP00000585
18
74
C
C
T
noncoding
0.83
0.97
0.58
0.81


60
7510055
3679678H1
SNP00000585
44
73
T
C
T
noncoding
0.83
0.97
0.58
0.81


60
7510055
4689661H1
SNP00000585
38
71
C
C
T
noncoding
0.83
0.97
0.58
0.81


61
7501754
1287052H1
SNP00020471
223
1622
C
C
T
noncoding
n/a
n/a
n/a
n/a


61
7501754
1366741H1
SNP00020470
16
957
C
C
T
L292
n/a
n/a
n/a
n/a


61
7501754
1366741H1
SNP00144758
230
1171
T
T
C
L364
n/a
n/a
n/a
n/a


61
7501754
1412435H1
SNP00020469
240
267
C
C
T
I62
n/a
n/a
n/a
n/a


61
7501754
1416519H1
SNP00020471
188
1621
C
C
T
noncoding
n/a
n/a
n/a
n/a


61
7501754
1477407H1
SNP00115549
182
609
A
A
G
R176
n/a
n/a
n/a
n/a


61
7501754
1592982H1
SNP00144757
103
916
G
G
A
D279
n/a
n/a
n/a
n/a


61
7501754
1593474H1
SNP00060616
79
396
T
T
G
I105
n/a
n/a
n/a
n/a


61
7501754
2110613H1
SNP00002430
142
1269
A
A
G
L396
0.55
0.30
0.67
0.69


61
7501754
2286353H1
SNP00002429
69
732
C
C
T
Y217
n/a
n/a
n/a
n/a


61
7501754
3268502H1
SNP00020469
199
233
C
C
T
A51
n/a
n/a
n/a
n/a


61
7501754
3603324H1
SNP00020470
16
956
C
C
T
P292
n/a
n/a
n/a
n/a


61
7501754
3603324H1
SNP00144758
230
1170
T
T
C
H363
n/a
n/a
n/a
n/a


61
7501754
3665554H1
SNP00002430
31
1266
G
A
G
L395
0.55
0.30
0.67
0.69


61
7501754
3782483H1
SNP00002429
129
731
T
C
T
F217
n/a
n/a
n/a
n/a


61
7501754
3782483H1
SNP00115549
6
608
A
A
G
Q176
n/a
n/a
n/a
n/a


61
7501754
3825869H1
SNP00020469
249
251
C
C
T
P57
n/a
n/a
n/a
n/a


61
7501754
4061686H1
SNP00144757
220
913
G
G
A
A278
n/a
n/a
n/a
n/a


61
7501754
4205282H1
SNP00119977
113
1541
A
A
C
noncoding
n/a
n/a
n/a
n/a


61
7501754
4259242H1
SNP00020470
15
955
C
C
T
L292
n/a
n/a
n/a
n/a


61
7501754
4259242H1
SNP00144758
229
1169
T
T
C
L363
n/a
n/a
n/a
n/a


61
7501754
4753963H1
SNP00020470
4
952
C
C
T
R291
n/a
n/a
n/a
n/a


61
7501754
4753963H1
SNP00144758
217
1165
T
T
C
Y362
n/a
n/a
n/a
n/a


61
7501754
5269726H1
SNP00144758
167
1163
T
T
C
F361
n/a
n/a
n/a
n/a


61
7501754
5662745H1
SNP00144757
129
915
G
G
A
P278
n/a
n/a
n/a
n/a


61
7501754
5832111H1
SNP00020471
101
1607
C
C
T
noncoding
n/a
n/a
n/a
n/a


61
7501754
5847218H1
SNP00144757
93
914
G
G
A
R278
n/a
n/a
n/a
n/a


61
7501754
5847579H1
SNP00020470
164
940
C
C
T
Q287
n/a
n/a
n/a
n/a


61
7501754
5847579H1
SNP00144757
123
898
G
G
A
D273
n/a
n/a
n/a
n/a


61
7501754
6882567J1
SNP00119977
271
1543
C
A
C
noncoding
n/a
n/a
n/a
n/a


61
7501754
6997953H1
SNP00020469
121
265
C
C
T
L62
n/a
n/a
n/a
n/a


61
7501754
6997953H1
SNP00060616
250
394
T
T
G
F105
n/a
n/a
n/a
n/a


62
7510517
1252418F6
SNP00053146
95
2574
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
1252418T6
SNP00053146
135
2575
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
1253746H1
SNP00016073
86
2252
A
A
G
noncoding
n/a
n/a
n/a
n/a


62
7510517
1560944H1
SNP00016072
183
1896
G
A
G
noncoding
0.12
n/a
n/a
n/a


62
7510517
2137250T6
SNP00053146
110
2600
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
2771793T6
SNP00053146
155
2581
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
3009863T7
SNP00053146
147
2594
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
3472133T6
SNP00053146
131
2579
T
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
415685T6
SNP00053146
138
2616
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
461386T6
SNP00053146
105
2605
C
C
T
noncoding
n/a
n/a
n/a
n/a


62
7510517
6521830H1
SNP00053145
453
432
C
C
T
T53
0.91
n/a
n/a
n/a


62
7510517
950639T6
SNP00053146
160
2593
C
C
T
noncoding
n/a
n/a
n/a
n/a


63
7511014
1439526F7
SNP00144232
42
1598
C
C
T
noncoding
n/a
n/a
n/a
n/a


63
7511014
1439526F7
SNP00144233
87
1643
A
G
A
noncoding
n/a
n/a
n/a
n/a


63
7511014
1446257F6
SNP00144233
389
1646
G
G
A
noncoding
n/a
n/a
n/a
n/a


63
7511014
7612633H1
SNP00144232
444
1600
C
C
T
noncoding
n/a
n/a
n/a
n/a


64
7506687
1415884H1
SNP00041797
78
5120
T
T
C
noncoding
n/d
n/a
n/a
n/a


64
7506687
1477444H1
SNP00076239
52
4874
T
T
C
noncoding
n/d
n/d
n/d
n/d


64
7506687
1485053H1
SNP00076240
117
5540
C
C
T
noncoding
n/d
n/d
n/d
n/d


64
7506687
1965096R6
SNP00041797
87
5119
T
T
C
noncoding
n/d
n/a
n/a
n/a


64
7506687
3679061H1
SNP00011057
18
6519
C
C
T
noncoding
n/d
n/a
n/a
n/a


65
7510621
076193H1
SNP00039731
93
502
T
T
C
V38
n/d
n/a
n/a
n/a


65
7510621
076193H1
SNP00069420
159
568
T
T
G
V60
n/d
n/d
n/d
n/d


65
7510621
076193H1
SNP00135470
154
563
C
C
T
I58
n/a
n/a
n/a
n/a


65
7510621
1002877H1
SNP00069735
152
807
T
T
G
W140
n/a
n/a
n/a
n/a


65
7510621
1281484H1
SNP00074872
32
932
T
T
G
I181
n/a
n/a
n/a
n/a


65
7510621
1636307H1
SNP00132688
94
734
C
C
T
N115
n/a
n/a
n/a
n/a


65
7510621
1731529F6
SNP00124792
79
79
C
C
T
noncoding
n/a
n/a
n/a
n/a


65
7510621
3216409T6
SNP00069735
269
823
T
T
G
F145
n/a
n/a
n/a
n/a


65
7510621
3216409T6
SNP00074872
144
948
T
T
G
stop187
n/a
n/a
n/a
n/a


65
7510621
3905994H1
SNP00039731
153
504
T
T
C
L39
n/d
n/a
n/a
n/a


65
7510621
3905994H1
SNP00069420
219
570
T
T
G
C61
n/d
n/d
n/d
n/d


65
7510621
3905994H1
SNP00135470
214
565
C
C
T
A59
n/a
n/a
n/a
n/a


65
7510621
5020934T1
SNP00074872
113
966
T
T
G
S193
n/a
n/a
n/a
n/a


65
7510621
6804058J1
SNP00069735
356
790
G
T
G
G134
n/a
n/a
n/a
n/a


65
7510621
6804058J1
SNP00074872
481
915
G
T
G
G176
n/a
n/a
n/a
n/a


65
7510621
7618082H1
SNP00132688
410
735
C
C
T
H116
n/a
n/a
n/a
n/a


66
7505533
5210141H1
SNP00152657
57
307
T
T
C
D92
n/a
n/a
n/a
n/a


66
7505533
5210141H1
SNP00152658
94
344
A
A
G
T105
n/a
n/a
n/a
n/a


66
7505533
5210141H1
SNP00152659
218
468
T
T
C
noncoding
n/a
n/a
n/a
n/a


66
7505533
5210183H1
SNP00152657
56
304
T
T
C
A91
n/a
n/a
n/a
n/a


66
7505533
5210183H1
SNP00152658
93
340
A
A
G
stop103
n/a
n/a
n/a
n/a


67
7511220
6863270H1
SNP00062114
182
437
A
A
C
D131
0.23
0.10
0.33
0.16


67
7511220
6863270H1
SNP00065517
55
310
A
C
A
I89
n/a
n/a
n/a
n/a


68
7510967
1269923F6
SNP00068898
197
5398
A
A
G
E1750
n/a
n/a
n/a
n/a


68
7510967
1269923F6
SNP00116132
65
5265
T
T
C
I1706
n/d
n/d
n/d
n/d


68
7510967
1269923F6
SNP00116133
310
5511
C
C
T
noncoding
n/a
n/a
n/a
n/a


68
7510967
2215706F6
SNP00004638
161
4918
G
G
T
Q1590
n/a
n/a
n/a
n/a


68
7510967
2215706F6
SNP00025482
195
4952
G
G
C
D1602
n/d
n/d
n/d
n/d


68
7510967
4252319F6
SNP00134721
171
452
A
A
G
M102
n/a
n/a
n/a
n/a


68
7510967
5089321H1
SNP00134719
13
7
C
C
G
noncoding
n/a
n/a
n/a
n/a


68
7510967
6977458H1
SNP00134720
282
275
A
A
G
M43
n/a
n/a
n/a
n/a


69
7511298
1359892H1
SNP00114894
62
1791
T
T
C
F574
n/a
n/a
n/a
n/a


69
7511298
1389872F6
SNP00136492
371
2647
C
C
A
noncoding
n/a
n/a
n/a
n/a


69
7511298
1391007F6
SNP00114895
532
2085
A
A
G
K672
n/d
n/d
n/d
n/d


69
7511298
1391007F6
SNP00148080
277
1828
G
G
A
L586
n/a
n/a
n/a
n/a


69
7511298
1429445F6
SNP00016801
294
1546
C
C
T
D492
n/a
n/a
n/a
n/a


69
7511298
1429445F6
SNP00151717
252
1504
C
C
T
C478
n/a
n/a
n/a
n/a


69
7511298
1501745F6
SNP00058189
420
2271
C
C
T
S734
n/a
n/a
n/a
n/a


69
7511298
1519651F6
SNP00123884
164
676
A
A
G
Q202
n/d
n/a
n/a
n/a


69
7511298
1877054H1
SNP00058189
23
2272
C
C
T
Y734
n/a
n/a
n/a
n/a


69
7511298
1878525H1
SNP00016803
213
2588
T
C
T
noncoding
n/a
n/a
n/a
n/a


69
7511298
2044874F6
SNP00062888
285
596
G
C
G
V176
n/d
n/a
n/a
n/a


69
7511298
2082962T6
SNP00136492
332
2654
C
C
A
noncoding
n/a
n/a
n/a
n/a


69
7511298
2437515F6
SNP00016802
456
2185
C
C
T
V705
n/a
n/a
n/a
n/a


69
7511298
2803211H1
SNP00126602
25
2370
C
T
C
T767
n/a
n/a
n/a
n/a


69
7511298
5615830F6
SNP00016801
407
1540
T
C
T
H490
n/a
n/a
n/a
n/a


69
7511298
5615830F6
SNP00151717
365
1498
C
C
T
C476
n/a
n/a
n/a
n/a


69
7511298
7226075H1
SNP00123884
182
675
A
A
G
Q202
n/d
n/a
n/a
n/a


69
7511298
7607396J1
SNP00062884
266
690
C
C
A
S207
n/d
n/a
n/a
n/a


70
7510937
1213711H1
SNP00001099
182
4086
G
G
A
A1312
n/a
n/a
n/a
n/a


70
7510937
1233991H1
SNP00046056
492
149
G
G
C
E667
n/a
n/a
n/a
n/a


70
7510937
1294381H1
SNP00016843
76
4277
G
G
A
noncoding
n/d
n/a
n/a
n/a


70
7510937
1702543F6
SNP00108395
156
3932
T
T
C
L1261
n/a
n/a
n/a
n/a


70
7510937
183926H1
SNP00046055
148
1856
T
T
C
M569
n/a
n/a
n/a
n/a


70
7510937
1867984H1
SNP00108393
161
2808
A
A
G
K886
n/d
n/d
n/d
n/d


70
7510937
2056164T6
SNP00016843
279
4281
G
G
A
noncoding
n/d
n/a
n/a
n/a


70
7510937
2070471H1
SNP00046057
28
2631
G
G
A
K827
n/a
n/a
n/a
n/a


70
7510937
2705113T6
SNP00016843
274
4279
G
G
A
noncoding
n/d
n/a
n/a
n/a


70
7510937
2786224T6
SNP00001099
276
4087
G
G
A
V1313
n/a
n/a
n/a
n/a


70
7510937
2903742T6
SNP00016843
180
4374
G
G
A
noncoding
n/d
n/a
n/a
n/a


70
7510937
3773791H1
SNP00046054
183
1287
G
A
G
R379
0.86
0.93
n/d
0.95


70
7510937
3790871H1
SNP00016841
4
3240
G
G
C
K1030
n/a
n/a
n/a
n/a


70
7510937
6597222H1
SNP00108394
384
3239
A
A
C
N1030
n/a
n/a
n/a
n/a


70
7510937
7459195H1
SNP00046054
99
1282
A
A
G
N378
0.86
0.93
n/d
0.95


71
7511852
1674771H1
SNP00124011
23
1466
C
C
T
noncoding
n/a
n/a
n/a
n/a


71
7511852
2330339H1
SNP00004236
163
1261
A
A
G
noncoding
0.64
0.82
0.57
n/a


71
7511852
2330339H1
SNP00004237
190
1288
C
T
C
noncoding
0.31
0.19
0.09
0.26


71
7511852
3555370H1
SNP00024881
213
1393
C
C
T
noncoding
n/a
n/a
n/a
n/a


72
7511077
1223450H1
SNP00124213
14
30
G
G
A
noncoding
n/a
n/a
n/a
n/a


72
7511077
1315226H1
SNP00124215
130
1229
G
G
A
noncoding
0.98
n/a
n/a
n/a


72
7511077
1434590H1
SNP00124214
208
399
C
C
T
R122
n/d
n/a
n/a
n/a


72
7511077
1581204H1
SNP00017009
17
524
T
C
T
G163
n/d
n/a
n/a
n/a


72
7511077
1705518H1
SNP00017008
155
485
T
T
C
I150
n/a
n/a
n/a
n/a


72
7511077
1843956R6
SNP00017008
328
486
C
T
C
P151
n/a
n/a
n/a
n/a


72
7511077
1843956R6
SNP00124214
242
400
C
C
T
P122
n/d
n/a
n/a
n/a


72
7511077
4586519H1
SNP00124216
223
1251
C
C
T
noncoding
n/a
n/a
n/a
n/a


72
7511077
6481807H1
SNP00001218
40
1248
G
G
C
noncoding
n/a
n/a
n/a
n/a


72
7511077
7639431J2
SNP00017008
354
457
C
T
C
A141
n/a
n/a
n/a
n/a


72
7511077
7639431J2
SNP00017009
393
496
C
C
T
S154
n/d
n/a
n/a
n/a


72
7511077
7639431J2
SNP00124214
269
371
C
C
T
T112
n/d
n/a
n/a
n/a


73
7511576
034843H1
SNP00098584
220
335
T
T
C
I48
n/a
n/a
n/a
n/a


73
7511576
1493422H1
SNP00034873
58
384
G
A
G
L64
n/a
n/a
n/a
n/a


73
7511576
1520967H1
SNP00033391
154
442
C
C
T
L84
n/a
n/a
n/a
n/a


73
7511576
1724713F6
SNP00033392
303
1128
A
A
C
noncoding
n/a
n/a
n/a
n/a


73
7511576
1724713T6
SNP00033392
160
1134
A
A
C
noncoding
n/a
n/a
n/a
n/a


73
7511576
8588603T1
SNP00154298
467
395
G
A
G
W68
n/a
n/a
n/a
n/a


73
7511576
8588603T1
SNP00154299
363
500
G
A
G
R103
n/a
n/a
n/a
n/a


74
7511492
008076H1
SNP00001520
204
306
T
T
C
F81
n/a
n/a
n/a
n/a


74
7511492
1216191H1
SNP00050705
215
565
G
G
A
noncoding
n/a
n/a
n/a
n/a


74
7511492
1693356H1
SNP00064907
56
648
C
C
A
noncoding
n/a
n/a
n/a
n/a


74
7511492
1907176H1
SNP00001521
222
703
C
C
G
noncoding
n/a
n/a
n/a
n/a


75
7511141
1435538T6
SNP00003434
193
2614
T
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
1435538T6
SNP00065777
28
2777
A
A
G
noncoding
n/a
n/a
n/a
n/a


75
7511141
1513489T6
SNP00003434
145
2602
C
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
1631170F6
SNP00003434
381
2601
T
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
1631170F6
SNP00065776
212
2432
C
C
T
noncoding
n/a
n/a
n/a
n/a


75
7511141
1631170T6
SNP00003434
165
2629
T
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
1631170T6
SNP00065776
335
2459
C
C
T
noncoding
n/a
n/a
n/a
n/a


75
7511141
1632763T6
SNP00003434
193
2605
T
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
1632763T6
SNP00065776
363
2435
C
C
T
noncoding
n/a
n/a
n/a
n/a


75
7511141
1632763T6
SNP00065777
28
2769
A
A
G
noncoding
n/a
n/a
n/a
n/a


75
7511141
2091984H2
SNP00065777
206
2767
A
A
G
noncoding
n/a
n/a
n/a
n/a


75
7511141
2216503T6
SNP00003434
139
2654
C
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
2640569T6
SNP00003434
177
2631
C
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
2640569T6
SNP00065776
347
2461
C
C
T
noncoding
n/a
n/a
n/a
n/a


75
7511141
2640569T6
SNP00065777
12
2794
A
A
G
noncoding
n/a
n/a
n/a
n/a


75
7511141
2676369F6
SNP00065777
381
2766
A
A
G
noncoding
n/a
n/a
n/a
n/a


75
7511141
2676369T6
SNP00003434
121
2685
C
T
C
noncoding
n/a
n/a
n/a
n/a


75
7511141
2676369T6
SNP00065776
291
2515
C
C
T
noncoding
n/a
n/a
n/a
n/a


75
7511141
3835977T6
SNP00065776
377
2431
C
C
T
noncoding
n/a
n/a
n/a
n/a


76
7511300
1359892H1
SNP00114894
62
1938
T
T
C
F623
n/a
n/a
n/a
n/a


76
7511300
1389872F6
SNP00136492
371
2692
C
C
A
noncoding
n/a
n/a
n/a
n/a


76
7511300
1391007F6
SNP00114895
532
2232
A
A
G
K721
n/d
n/d
n/d
n/d


76
7511300
1391007F6
SNP00148080
277
1975
G
G
A
L635
n/a
n/a
n/a
n/a


76
7511300
1429445F6
SNP00016801
294
1693
C
C
T
D541
n/a
n/a
n/a
n/a


76
7511300
1429445F6
SNP00151717
252
1651
C
C
T
C527
n/a
n/a
n/a
n/a


76
7511300
1501745F6
SNP00058189
420
2418
C
C
T
S783
n/a
n/a
n/a
n/a


76
7511300
1519651F6
SNP00123884
164
823
A
A
G
Q251
n/d
n/a
n/a
n/a


76
7511300
1878525H1
SNP00016803
213
2633
T
C
T
noncoding
n/a
n/a
n/a
n/a


76
7511300
2044874F6
SN000062888
285
743
G
C
G
V225
n/d
n/a
n/a
n/a


76
7511300
2082962T6
SNP00136492
332
2699
C
C
A
noncoding
n/a
n/a
n/a
n/a


76
7511300
2242331H1
SNP00058189
214
2419
C
C
T
Y783
n/a
n/a
n/a
n/a


76
7511300
2437515F6
SNP00016802
456
2332
C
C
T
V754
n/a
n/a
n/a
n/a


76
7511300
5615830F6
SNP00016801
407
1687
T
C
T
H539
n/a
n/a
n/a
n/a


76
7511300
5615830F6
SNP00151717
365
1645
C
C
T
C525
n/a
n/a
n/a
n/a


76
7511300
7226075H1
SNP00123884
182
822
A
A
G
Q251
n/d
n/a
n/a
n/a

## Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 22.

2. The isolated polypeptide of claim 1, wherein said polypeptide is biologically active.

3. The isolated polypeptide of claim 1, produced recombinantly.

4. An isolated polypeptide consisting of SEQ ID NO: 22.

5. An isolated polypeptide comprising amino acids 188-237 of SEQ ID NO: 22.

6. A polypeptide consisting of (a) an amino acid sequence comprising SEQ ID NO: 22; fused to (b) a heterologous fusion polypeptide.