Immune response associated proteins

TECHNICAL FIELD

[0001] The invention relates to novel nucleic acids, immune response associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of immune system, neurological, developmental, muscle, and cell proliferative disorders. The invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and immune response associated proteins.

BACKGROUND OF THE INVENTION

[0002] All vertebrates have developed sophisticated and complex immune systems that provide protection from viral, bacterial, fungal and parasitic infections. Included in these systems are the processes of humoral immunity, the complement cascade and the inflammatory response (See Paul, W. E. (1993) Fundamental Immunology, Raven Press, Ltd., New York N.Y. pp. 1-20).

[0003] The cellular components of the immune system include six different types of leukocytes, or white blood cells: monocytes, lymphocytes, polymorphonuclear granulocytes (including neutrophils, eosinophils, and basophils) and plasma cells. Additionally, fragments of megakaryocytes, a seventh type of white blood cell in the bone marrow, occur in large numbers in the blood as platelets.

[0004] Leukocytes are formed from two stem cell lineages in bone marrow. The myeloid stem cell line produces granulocytes and monocytes and the lymphoid stem cell line produces lymphocytes. Lymphoid cells travel to the thymus, spleen and lymph nodes, where they mature and differentiate into lymphocytes. Leukocytes are responsible for defending the body against invading pathogens. Neutrophils and monocytes attack invading bacteria, viruses, and other pathogens and destroy them by phagocytosis. Monocytes enter tissues and differentiate into macrophages which are extremely phagocytic. Lymphocytes and plasma cells are a part of the immune system which recognizes specific foreign molecules and organisms and inactivates them, as well as signals other cells to attack the invaders.

[0005] Granulocytes and monocytes are formed and stored in the bone marrow until needed. Megakaryocytes are produced in bone marrow, where they fragment into platelets and are released into the bloodstream. The main function of platelets is to activate the blood clotting mechanism. Lymphocytes and plasma cells are produced in various lymphogenous organs, including the lymph nodes, spleen, thymus, and tonsils.

[0006] Both neutrophils and macrophages exhibit chemotaxis towards sites of inflammation. Tissue inflammation in response to pathogen invasion results in production of chemo-attractants for leukocytes, such as endotoxins or other bacterial products, prostaglandins, and products of leukocytes or platelets. Immune recognition of microorganisms involves pattern recognition molecules that bind to specific bacterial proteins. Peptidoglycan recognition proteins, for example, bind peptidoglycan, a component of the cell wall of many bacteria, and induce immune defenses against microorganisms (Liu, C et al. (2001) J. Biol. Chem. 276:34686-34694).

[0007] Basophils participate in the release of the chemicals involved in the inflammatory process. The main function of basophils is secretion of these chemicals, to such a degree that they have been referred to as “unicellular endocrine glands.” A distinct aspect of basophilic secretion is that the contents of granules go directly into the extracellular environment, not into vacuoles as occurs with neutrophils, eosinophils, and monocytes. Basophils have receptors for the Fc fragment of immunoglobulin E (IgE) that are not present on other leukocytes. Crosslinking of membrane IgE with anti-IgE or other ligands triggers degranulation.

[0008] Eosinophils are bi- or multi-nucleated white blood cells which contain eosinophilic granules. Their plasma membrane is characterized by Ig receptors, particularly IgG and IgF. Generally, eosinophils are stored in the bone marrow until recruited for use at a site of inflammation or invasion. They have specific functions in parasitic infections and allergic reactions, and are thought to detoxify some of the substances released by mast cells and basophils which cause inflammation. Additionally, they phagocytize antigen-antibody complexes and further help prevent the spread of inflammation.

[0009] The mononuclear phagocyte system is comprised of precursor cells in the bone marrow, monocytes in circulation, and macrophages in tissues. Macrophages are monocytes that have left the blood stream to settle in tissue. Once monocytes have migrated into tissues, they do not re-enter the bloodstream. They increase several-fold in size and transform into macrophages that are characteristic of the tissue they have entered, surviving in tissues for several months. The mononuclear phagocyte system is capable of very fast and extensive phagocytosis. A macrophage may phagocytize over 100 bacteria, digest them and extrude residues, and then survive for many more months. Macrophages are also capable of ingesting large particles, including red blood cells and malarial parasites.

[0010] Mononuclear phagocytes are essential in defending the body against invasion by foreign pathogens, particularly intracellular microorganisms such as Mycobacterium tuberculosis, listeria, leishmania and toxoplasma. Macrophages can also control the growth of tumorous cells, via both phagocytosis and secretion of hydrolytic enzymes. Another important function of macrophages is that of processing antigens and presenting them in a biochemically modified form to lymphocytes.

[0011] The immune system responds to invading microorganisms in two major ways: antibody production and cell mediated responses. Antibodies are immunoglobulin proteins produced by B-lymphocytes which bind to specific antigens and cause inactivation or promote destruction of the antigen by other cells. Cell-mediated immune responses involve T-lymphocytes (T cells) that react with foreign antigens on the surface of infected host cells. Depending on the type of T cell, the T cell either kills the infected cell itself, or secretes signals which activate macrophages and other cells to destroy the infected cell (Paul, supra).

[0012] T-lymphocytes originate in the bone marrow or liver in fetuses. Precursor cells migrate via the blood to the thymus, where they are processed to mature into T-lymphocytes. This processing is crucial because it involves positive and negative selection of T cells for those that will react with foreign antigen and not with self molecules. After processing, T cells continuously circulate in the blood and secondary lymphoid tissues, such as lymph nodes, spleen, certain epithelium-associated tissues in the gastrointestinal tract, respiratory tract and skin. When T-lymphocytes are presented with the complementary antigen, they are stimulated to proliferate and release large numbers of activated T cells into the lymph system and the blood system. These activated T cells can survive and circulate for several days. At the same time, T memory cells are created, which remain in the lymphoid tissue for months or years. Upon subsequent exposure to that specific antigen, these memory cells will respond more rapidly and with a stronger response than induced by the original antigen. This creates an “immunological memory” that can provide immunity for years.

[0013] Adult liver gives rise to extrathymic T cells, natural killer (NK)cells, and granulocytes. Extrathymic T cells generated in mouse liver are intermediate T-cell receptor (TCR(int)) cells, including NK1.1+TCR(int) (NKT) and NK1.1−TCR(int) cells. Extrathymic T cells increase in number with aging or stress such as infection, malignancy, pregnancy, autoimmune disease, chronic graft-versus-host diseases. Under these conditions, T-cell differentiation in the thymus, which produces conventional T cells, is suppressed. Extrathymic T cells comprise self-reactive clones and mediate cytotoxicity against abnormal self-cells (e.g. malignant tumor cells, microbially infected hepatocytes, and regenerating hepatocytes). Hyperactivation of extrathymic T cells may result in onset of autoimmune diseases (Abo, T. et al. (2000) Immunol. Rev. 174:135-149).

[0014] There are two major types of T cells: cytotoxic T cells destroy infected host cells, and helper. T cells activate other white blood cells via chemical signals. One class of helper cell, TH1, activates macrophages to destroy ingested microorganisms, while another, TH2, stimulates the production of antibodies by B cells.

[0015] Cytotoxic T cells directly attack the infected target cell. Receptors on the surface of T cells bind to antigen presented by MHC molecules on the surface of the infected cell. Once activated by binding to antigen, T cells secrete γ-interferon, a signal molecule that induces the expression of genes necessary for presenting viral (or other) antigens to cytotoxic T cells. Cytotoxic T cells kill the infected cell by stimulating programmed cell death.

[0016] Helper T cells constitute up to 75% of the total T cell population. They regulate the immune functions by producing a variety of lymphokines that act on other cells in the immune system and on bone marrow. Among these lymphokines are interleukins 2 through 6, granulocyte-monocyte colony stimulating factor, and γ-interferon.

[0017] Helper T cells are required for most B cells to respond to antigen. When an activated helper cell contacts a B cell, its centrosome and Golgi apparatus become oriented toward the B cell, aiding the directing of signal molecules, such as a transmembrane-bound protein called CD40 ligand, onto the B cell surface to interact with the CD40 transmembrane protein. Secreted signals also help B cells to proliferate and mature and, in some cases, to switch the class of antibody being produced.

[0018] B-lymphocytes (B cells) produce antibodies which react with specific antigenic proteins presented by pathogens. Once activated, B cells become filled with extensive rough endoplasmic reticulum and are known as plasma cells. As with T cells, interaction of B cells with antigen stimulates proliferation of only those B cells which produce antibody specific to that antigen. There are five classes of antibodies, known as immunoglobulins, which together comprise about 20% of total plasma protein. Each class mediates a characteristic biological response after antigen binding. Upon activation by specific antigen B cells switch from making the membrane-bound antibody to the secreted form of that antibody.

[0019] Antibodies, or immunoglobulins, are the founding members of the immunoglobulin (Ig) superfamily and the central components of the humoral immune response. Antibodies are either expressed on the surface of B cells or secreted by B cells into the circulation. Antibodies bind and neutralize blood-borne foreign antigens. The prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition. The five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the α, δ, ε, γ, and μ H-chain types. There are two types of L-chains, κ and λ, either of which may associate as a pair with any H-chain pair. IgG, the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure.

[0020] H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region. The constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains. The amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class. The variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class. Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing, New York, N.Y., pp. 1206-1213 and 1216-1217.)

[0021] The immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens. Such antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site-specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population.

[0022] Both H-chains and L-chains contain repeated Ig domains. For example, a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site. Likewise, a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region. In addition, H chains such as μ have been shown to associate with other polypeptides during differentiation of the B-cell.

[0023] Antibodies can be described in terms of their two main functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. Binding of antibody to an antigen, such as a bacterium, triggers the destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface receptors that specifically bind to the antibody Fc region and allow the phagocytic cells to engulf, ingest, and degrade the antibody-bound antigen. The Fc receptors expressed by phagocytic cells are single-pass transmembrane glycoproteins of about 300 to 400 amino acids (Sears, D. W. et al. (1990) J. Immunol. 144:371-378). The extracellular portion of the Fc receptor typically contains two or three Ig domains.

[0024] Diseases which cause over- or under-abundance of any one type of leukocyte usually result in the entire immune defense system becoming involved. The most notorious autoimmune disease is AIDS (Acquired Immunodeficiency Syndrome). This disease depletes the number of helper T cells and leaves the patient susceptible to infection by microorganisms and parasites.

[0025] Another widespread medical condition attributable to the immune system is that of allergic reactions to certain antigens. Delayed reaction allergy is experienced by many genetically normal people. In the case of atopic allergies, there is a genetic origin, such that large quantities of IgE antibodies are produced. IgEs have a strong tendency to attach to mast cells and basophils, up to half million each (IgE/mast) which then rupture and release histamine, leukotrienes, eosinophil chemotactic substance, protease, neutrophil chemotactic substance, heparin, and platelet activation factors. Tissues can respond in a number of ways to these substances resulting in what are commonly known as allergic reactions: hay fever, asthma, anaphylaxis, and urticaria (hives).

[0026] Leukemias are an excess production of white blood cells, to the point where a major portion of the body's metabolic resources are directed solely at proliferation of white blood cells, leaving other tissues to starve. With lymphogenous leukemias, cancerous lymphogenous cells spread from a lymph node to other body parts. Excess T- and B-lymphocytes are produced. In myelogenous leukemias, cancerous young myelogenous cells spread from the bone marrow to other organs, especially the spleen, liver, lymph nodes and other highly vascularized regions. Usually, the extra leukemic cells released are immature, incapable of function, and undifferentiated. Occasionally, partially differentiated cells are produced, leading to classification of the disease as neutrophilic leukemia, eosinophilic leukemia, basophilic leukemia, or monocytic leukemia. Leukemias may be caused by exposure to environmental factors such as radiation or toxic chemicals or by genetic aberration.

[0027] Leukopenia or agranulocytosis occurs when the bone marrow stops producing white blood cells. This leaves the body unprotected against foreign microorganisms, including those which normally inhabit skin, mucous membranes, and gastrointestinal tract. If all white blood cell production stops completely, infection will occur within two days and death may follow only 1 to 4 days later. Acute leukopenia can be caused by exposure to radiation or chemicals containing benzene. Occasionally, drugs such as chloramphenicol and thiouracil can suppress blood cell production by the bone marrow and initiate the onset of agranulocytosis. In cases of monoblastic leukemia, primitive monocytes in blood and bone marrow do not mature. Clinical symptoms reflect this abnormality: high lysozyme levels in blood serum, renal tubular dysfunction, and high fevers.

[0028] Impaired phagocytosis occurs in several diseases, including monocytic leukemia, systemic lupus, and granulomatous disease. In such a situation, macrophages can phagocytize normally, but the enveloped organism is not killed. There is a defect in the plasma membrane enzyme which converts oxygen to lethally reactive forms. This results in abscess formation in liver, lungs, spleen, lymph nodes, and beneath the skin.

[0029] Eosinophilia is an excess of eosinophils commonly observed in patients with allergies (hay fever, asthma), allergic reactions to drugs, rheumatoid arthritis, and cancers (Hodgkins disease, lung, and liver cancer). The mechanism for elevated levels of eosinophils in these diseases is unknown (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, Inc., New York, N.Y.).

[0030] Host defense is further augmented by the complement system. The complement system serves as an effector system and is involved in infectious agent recognition. It can function as an independent immune network or in conjunction with other humoral immune responses. The complement system is comprised of numerous plasma and membrane proteins that act in a cascade of reaction sequences whereby one component activates the next. The result is a rapid and amplified response to infection through either an inflammatory response or increased phagocytosis.

[0031] The complement system has more than 30 protein components which can be divided into functional groupings including modified serine proteases, membrane-binding proteins, and regulators of complement activation. Activation occurs through two different pathways, the classical and the alternative. Both pathways serve to destroy infectious agents through distinct triggering mechanisms that eventually merge with the involvement of the component C3.

[0032] The anaphylatoxin C5a is a proinflammatory peptide produced during activation of the complement system. The structure of C5a includes a core region consisting of four antiparallel alpha-helices held together by three disulfide linkages and a structured C-terminal tail. The C5a receptor belongs to the large class of seven transmembrane, G-protein-linked receptors. C5a receptors are concentrated on blood granulocytes (neutrophils, eosinophils, and basophils) and tissue inflammatory cells (macrophages, mast cells, microglia). C5a receptors are also present in lower concentrations, on non-myeloid cells including endothelial and smooth muscle cells, where they may further influence inflammatory reactions such as blood cell emigration and tissue edema. C5a has been implicated in many acute and chronic disorders (Pellas T C, Wennogle L P. (1999) Curr. Pharm. Des. 5:737-755). Peptide agonists derived from human C5a anaphylatoxin are of interest for development of peptide/peptidomimetic modulators of C5a receptor-mediated function. Response-selective C5a agonists capable of generating antigen-specific humoral and cellular immune responses are of therapeutic interest (Taylor, S. M. et al. (2001) Curr. Med. Chem. 8:675-684).

[0033] The classical pathway requires antibody binding to infectious agent antigens. The antibodies serve to define the target and initiate the complement system cascade, culminating in the destruction of the infectious agent. In this pathway, since the antibody guides initiation of the process, the complement system can be seen as an effector arm of the humoral immune system.

[0034] The alternative pathway of the complement system does not require the presence of pre-existing antibodies for targeting infectious agent destruction. Rather, this pathway, through low levels of an activated component, remains constantly primed and provides surveillance in the non-immune host to enable targeting and destruction of infectious agents. In this case foreign material triggers the cascade, thereby facilitating phagocytosis or lysis (Paul, supra pp.918-919).

[0035] Another important component of host defense is the process of inflamation. Inflammatory responses are divided into four categories on the basis of pathology and include allergic inflammation, cytotoxic antibody mediated inflammation, immune complex mediated inflammation, and monocyte mediated inflammation. Inflammation manifests as a combination of each of these forms with one predominating.

[0036] Allergic acute inflamation is observed in individuals wherein specific antigens stimulate IgE antibody production. Mast cells and basophils are subsequently activated by the attachment of antigen-IgE complexes, resulting in the release of cytoplasmic granule contents such as histamine. The products of activated mast cells can increase vascular permeability and constrict the smooth muscle of breathing passages, resulting in anaphylaxis or asthma.

[0037] Acute inflamation is also mediated by cytotoxic antibodies and can result in the destruction of tissue through the binding of complement-fixing antibodies to cells. In this case the antibodies responsible are of the IgG or IgM types and resultant clinical disorders including autoimmune hemolytic anemia and thrombocytopenia as associated with systemic lupus erythematosis.

[0038] Immune complex mediated acute inflammation involves the IgG or IgM antibody types which combine with antigen to activate the complement cascade. When such immune complexes bind to neutrophils and macrophages they activate the respiratory burst to form protein and vessel damaging agents such as hydrogen peroxide, hydroxyl radical, hypochlorous acid, and chloramines. Clinical manifestations include rheumatoid arthritis and systemic lupus erythematosus.

[0039] In chronic inflammation or delayed-type hypersensitivity, macrophages are activated and process antigen for presentation to T cells that subsequently produce lymphokines and monokines. This type of inflammatory response is likely important for defense against intracellular parasites and certain viruses. Clinical associations include granulomatous disease, tuberculosis, leprosy, and sarcoidosis (Paul, supra pp. 1017-1018).

[0040] Most cell surface and soluble molecules that mediate functions such as recognition, adhesion or binding have evolved from a common evolutionary precursor (i.e., these proteins have structural homology). A number of molecules outside the immune system that have similar functions are also derived from this same evolutionary precursor. These molecules are classified as members of the immunoglobulin (Ig) superfamily. The criteria for a protein to be a member of the Ig superfamily is to have one or more Ig domains, which are regions of 70-110 amino acid residues in length homologous to either Ig variable-like (V) or Ig constant-like (C) domains. Members of the Ig superfamily include antibodies (Ab), T cell receptors (TCRs), class I and II major histocompatibility (MHC) proteins, CD2, CD3, CD4, CD8, poly-Ig receptors, Fc receptors, neural cell-adhesion molecule (NCAM) and platelet-derived growth factor receptor (PDGFR).

[0041] Ig domains (V and C) are regions of conserved amino acid residues that give a polypeptide a globular tertiary structure called an immunoglobulin (or antibody) fold, which consists of two approximately parallel layers of β-sheets. Conserved cysteine residues form an intrachain disulfide-bonded loop, 55-75 amino acid residues in length, which connects the two layers of the β-sheets. Each β-sheet has three or four anti-parallel β-strands of 5-10 amino acid residues. Hydrophobic and hydrophilic interactions of amino acid residues within the β-strands stabilize the Ig fold (hydrophobic on inward facing amino acid residues and hydrophilic on the amino acid residues in the outward facing portion of the strands). A V domain consists of a longer polypeptide than a C domain, with an additional pair of β-strands in the Ig fold.

[0042] A consistent feature of Ig superfamily genes is that each sequence of an Ig domain is encoded by a single exon. It is possible that the superfamily evolved from a gene coding for a single Ig domain involved in mediating cell-cell interactions. New members of the superfamily then arose by exon and gene duplications. Modern Ig superfamily proteins contain different numbers of V and/or C domains. Another evolutionary feature of this superfamily is the ability to undergo DNA rearrangements, a unique feature retained by the antigen receptor members of the family.

[0043] Many members of the Ig superfamily are integral plasma membrane proteins with extracellular Ig domains. The hydrophobic amino acid residues of their transmembrane domains and their cytoplasmic tails are very diverse, with little or no homology among Ig family members or to known signal-transducing structures. There are exceptions to this general superfamily description. For example, the cytoplasmic tail of PDGFR has tyrosine kinase activity. In addition Thy-i is a glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic tail, but is instead attached to the plasma membrane by a covalent glycophosphatidylinositol linkage.

[0044] Another common feature of many Ig superfamily proteins is the interactions between Ig domains which are essential for the function of these molecules. Interactions between Ig domains of a multimeric protein can be either homophilic or heterophilic (i.e., between the same or different Ig domains). Antibodies are multimeric proteins which have both homophilic and heterophilic interactions between Ig domains. Pairing of constant regions of heavy chains forms the Fc region of an antibody and pairing of variable regions of light and heavy chains form the antigen binding site of an antibody. Heterophilic interactions also occur between Ig domains of different molecules. These interactions provide adhesion between cells for significant cell-cell interactions in the immune system and in the developing and mature nervous system. (Reviewed in Abbas, A. K. et al. (1991) Cellular and Molecular Immunology, W. B. Saunders Company, Philadelphia, Pa., pp.142-145.)

[0045] Neural Cell Adhesion Proteins

[0046] Neural cell adhesion proteins (NCAPs) play roles in the establishment of neural networks during development and regeneration of the nervous system (Uyemura et al. (1996) Essays Biochem. 31:37-48; Brummendorf and Rathjen (1996) Curr. Opin. Neurobiol. 6:584-593). NCAP participates in neuronal cell migration, cell adhesion, neurite outgrowth, axonal fasciculation, pathfinding, synaptic target-recognition, synaptic formation, myelination and regeneration. NCAPs are expressed on the surfaces of neurons associated with learning and memory. Mutations in genes encoding NCAPS are linked with neurological diseases, including Charcot-Marie-Tooth disease (a hereditary neuropathy), Dejerine-Sottas disease, X-linked hydrocephalus, MASA syndrome (mental retardation, aphasia, shuffling gait and adducted thumbs), and spastic paraplegia type I. In some cases, expression of NCAP is not restricted to the nervous system. L1, for example, is expressed in melanoma cells and hematopoietic tumor cells where it is implicated in cell spreading and migration, and may play a role in tumor progression (Montgomery et al. (1996) J. Cell Biol. 132:475-485).

[0047] NCAPs have at least one immunoglobulin constant or variable domain (Uyemura et al., supra). They are generally linked to the plasma membrane through a transmembrane domain and/or a glycosyl-phosphatidylinositol (GPI) anchor. The GPI linkage can be cleaved by GPI phospholipase C. Most NCAPs consist of an extracellular region made up of one or more immunoglobulin domains, a membrane spanning domain, and an intracellular region. Many NCAPs contain post-translational modifications including covalently attached oligosaccharide, glucuronic acid, and sulfate. NCAPs fall into three subgroups: simple-type, complex-type, and mixed-type. Simple-type NCAPs contain one or more variable or constant immunoglobulin domains, but lack other types of domains. Members of the simple-type subgroup include Schwann cell myelin protein (SMP), limbic system-associated membrane protein (LAMP) and opiate-binding cell-adhesion molecule (OBCAM). The complex-type NCAPs contain fibronectin type III domains in addition to the immunoglobulin domains. The complex-type subgroup includes neural cell-adhesion molecule (NCAM), axonin-1, F11, Bravo, and L1. Mixed-type NCAPs contain a combination of immunoglobulin domains and other motifs such as tyrosine kinase, epidermal growth factor-like, sema, and PSI (plexins, semaphorins, and integrins) domains. This subgroup includes Trk receptors of nerve growth factors such as nerve growth factor (NGF) and neurotropin 4 (NT4), Neu differentiation factors such as glial growth factor II (GGFII) and acetylcholine receptor-inducing factor (ARIA), the semaphorin/collapsin family such as semaphorin B and collapsin, and receptors for members of the semaphorin/collapsin family such as plexin (for plexin, see below).

[0048] An NCAP subfamily, the NCAP-LON subgroup, includes cell adhesion proteins expressed on distinct subpopulations of brain neurons. Members of the NCAP-LON subgroup possess three immunoglobulin domains and bind to cell membranes through GPI anchors. Kilon (a kindred of NCAP-LON), for example, is expressed in the brain cerebral cortex and hippocampus (Funatsu et al. (1999) J. Biol. Chem. 274:8224-8230). Immunostaining localizes Kilon to the dendrites and soma of pyramidal neurons. Kilon has three C2 type immunoglobulin-like domains, six predicted glycosylation sites, and a GPI anchor. Expression of Kilon is developmentally regulated. It is expressed at higher levels in adult brain in comparison to embryonic and early postnatal brains. Confocal microscopy shows the presence of Kilon in dendrites of hypothalamic magnocellular neurons secreting neuropeptides, oxytocin, or arginine vasopressin (Miyata et al. (2000) J. Comp. Neurol. 424:74-85). Arginine vasopressin regulates body fluid homeostasis, extracellular osmolarity and intravascular volume. Oxytocin induces contractions of uterine smooth muscle during child birth and of myoepithelial cells in mammary glands during lactation. In magnocellular neurons, Kilon is proposed to play roles in the reorganization of dendritic connections during neuropeptide secretion.

[0049] Sidekick (SDK) is a member of the NCAP family. The extracellular region of SDK contains six immunoglobulin domains and thirteen fibronectin type III domains. SDK is involved in cell-cell interaction during eye development in Drosophila (Nguyen, D. N. T. et al. (1997) Development 124: 3303).

[0050] Synaptic Membrane Glycoproteins

[0051] Specialized cell junctions can occur at points of cell-cell contact. Among these cell junctions are communicating junctions which mediate the passage of chemical and electrical signals between cells. In the central nervous system, communicating junctions between neurons are known as synaptic junctions. They are composed of the membranes and cytoskeletons of the pre- and post-synaptic neurons. Some glycoproteins, found in biochemically isolated synaptic subfractions such as the synaptic membrane (SM) and postsynaptic density (PSD) fractions, have been identified and their functions established. An example is the SM glycoprotein, gp50, identified as the β2 subunit of the Na+/K+-ATPase.

[0052] Two glycoproteins, gp65 and gp55, are major components of synaptic membranes prepared from rat forebrain. They are members of the Ig superfamily containing three and two Ig domains, respectively. As members of the Ig superfamily, it is proposed that a possible function of these proteins is to mediate adhesive interactions at the synaptic junction. (Langnaese, K. et al. (1997) J. Biol. Chem.272:821-827.)

[0053] Lectins

[0054] Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K. and Taylor, M. E. (1993) Annu. Rev. Cell Biol. 9:237-264). This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L. A. (1991) J. Cell. Biochem. 45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).

[0055] Sialic acid binding Ig-like lectins (SIGLECs) are members of the Ig superfamily that bind to sialic acids in glycoproteins and glycolipids. SIGLECs include sialoadhesin, CD22, CD33, myelin-associated glycoprotein (MAG), SIGLEC-5, SIGLEC6, SIGLEC-7, and SIGLEC-8. The extracellular region of SIGLEC has a membrane distal V-set domain followed by varying numbers of C2-set domains. The sialic acid binding domain is mapped to the V-set domain. Except for MAG which is expressed exclusively in the nervous system, most SIGLECs are expressed on distinct subsets of hemopoietic cells. For example, SIGLEC-8 is expressed exclusively in eosinophils, one form of polymorphonuclear leucocyte (granulocyte) (Floyd, H. et al. (2000) J. Biol. Chem. 275: 861-866).

[0056] Leucine-Rich Repeat Proteins

[0057] Leucine-rich repeat proteins (LRRPs) are involved in protein-protein interactions. LRRPs such as mammalian neuronal leucine-rich repeat proteins (NLLR-1 and NLLR-2), Drosophila connectin, slit, chaopin, and toll all play roles in neuronal development. The extracellular region of LRRPs contains varying numbers of leucine-rich repeats, immunoglobulin-like domains, and fibronectin type III domains (Taguchi, A. et al. (1996) Brain Res. Mol. Brain Res. 35:3140).

[0058] In addition to the V and C2 sets of immunoglobulin-like domains, there is a D set immunoglobulin-like domain, named IPT/TIG (for immunoglobulin-like fold shared by plexins and transcription factors). IPT/TIG containing proteins include plexins, MET/RON/SEA (hepatocyte growth factor receptor family), and the transcription factor XCoe2, a transcription factor of the CoV/Olf-1/EBF family involved in the specification of primary neurons in Xenopus (Bork, P. et al. (1999) Trends in Biochem. 24:261-263; Santoro, N. M. et al. (1996) Mol. Cell Biol. 16:7072-7083; Dubois L. et al. (1998) Curr. Biol.8: 199-209). Plexins such as plexin A and VESPR have been shown to be neuronal semaphorin receptors that control axon guidance (Winberg M. L. et al. (1998) Cell 95:903-916).

[0059] Sushi domains, also known as complement control protein (CCP) modules, or short consensus repeats (SCR), are found in a wide variety of complement and adhesion proteins. CD21 (also called C3d receptor, CR2, Epstein Barr virus receptor or EBV-R) is the receptor for EBV and for C3d, C3dg and iC3b. Complement components may activate B cells through CD21. CD21 is part of a large signal-transduction complex that also involves CD19, CD81, and Leu13. Some of the proteins in this group are responsible for the molecular basis of the blood group antigens, surface markers on the outside of the red blood cell membrane. Most of these markers are proteins, but some are carbohydrates attached to lipids or proteins (for a review see Reid, M. E. and C. Lomas-Francis (1977) The Blood Group Antigen Facts Book Academic Press, San Diego, Calif.). Complement decay-accelerating factor (Antigen CD55) belongs to the Cromer blood group system and is associated with Cr(a); Dr(a), Es(a), Tc(a/b/c), Wd(a), WES(a/b), IFC and UMC antigens. Complement receptor type 1 (C3b/C4b receptor) (Antigen CD35) belongs to the Knops blood group system and is associated with Kn(a/b), McC(a), Sl(a) and Yk(a) antigens.

[0060] Human leukocyte-specific transcript 1 (LST1) is a small protein that modulates immune responses and cellular morphogenesis. LST1 is expressed at high levels in dendritic cells. A DNA-binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA (Yu, X. and Weissman, S. M. (2000) J. Biol. Chem. 275:34597-34608).

[0061] Spalpha is a member of the scavenger receptor cysteine-rich (SRCR) family of proteins. Spalpha is expressed only in lymphoid tissues, where it is implicated in monocyte activity (Gebe, J. A. (1997) J. Biol. Chem. 272:6151-6158). Such a domain is also found once in the C-terminal section of mammalian macrophage scavenger receptor type I, a membrane glycoprotein implicated in the pathologic deposition of cholesterol in arterial walls during atherogenesis (Freeman, M. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8810-8814).

[0062] Parkinson's Disease

[0063] Parkinson's disease is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway, and the presence of Lewy bodies. Genetic linkages to chromosomes 2p4, 4p5, and three loci on 1q6-8 have been identified (Gwinn-Hardy K. (2002) Mov. Disord. 17:645-656). Clinical disorders classified as parkinsonism include PD, dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), and essential tremor. Several neurodegenerative diseases share share pathogenic mechanisms involving tau or synuclein aggregation. These disorders include Alzheimer's disease, and Pick's disease as well as PD and progressive supranuclear palsy (Hardy, J. (2001) J. Alzheimers Dis. 3:109-116). Several genetically distinct forms of PD can be caused by mutations in single genes. Genes for monogenically inherited forms of Parkinson's disease (PD) have been mapped and/or cloned. In some families with autosomal dominant inheritance and typical Lewy-body pathology, mutations have been identified in the gene for alpha-synuclein. Aggregation of this protein in Lewy-bodies may be a crucial step in the molecular pathogenesis of familial and sporadic PD. On the other hand, mutations in the parkin gene cause early-onset autosomal recessive parkinsonism in which nigral degeneration is not accompanied by Lewy-body formation. Parkin-mutations appear to be a common cause of PD in patients with very early onset. Parkin has been implicated in the cellular protein degradation pathways, as it has been shown that it functions as a ubiquitin ligase. A mutation in the gene for ubiquitin C-terminal hydrolase L1in this pathway has been identified in another small family with PD. Other loci have been mapped to chromosome 2p and 4p, respectively, in families with dominantly inherited PD. These early-onset forms differ from the common sporadic form of PD. It is widely believed that a combination of interacting genetic and environmental causes may be responsible in this majority of PD-cases Gasser, T. (2001) J. Neurol. 2001 248:833-840).

[0064] Expression Profiling

[0065] 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.

[0066] 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.

[0067] Cancer

[0068] Colorectal cancer is the second leading cause of cancer death in the United States, and is considered a disease of aging since 90% of cases occur in individuals over the age of 55. A widely accepted hypothesis is that several mutations must accumulate over time before an individual develops the disease. 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 generally applied. 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 the disease. 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.

[0069] Breast cancer is the most frequently diagnosed type of cancer in American women and the second most frequent cause of cancer death. 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. A number of risk factors have been identified, including hormonal and genetic factors. 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. 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.

[0070] Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologically distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-small cell lung cancers (NSCLCs). The fourth group of cancers is referred to as small cell lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K-ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease.

[0071] Osteosarcoma is the most common malignant bone tumor in children. Approximately 80% of patients present with non-metastatic disease. After the diagnosis is made by an initial biopsy, treatment involves the use of 34 courses of neoadjuvant chemotherapy before definitive surgery, followed by post-operative chemotherapy. The most significant prognostic factor predicting the outcome in a patient with non-metastatic osteosarcoma is the histopathologic response of the primary tumor resected at the time of definitive surgery.

[0072] Adipocytes

[0073] Adipose tissue stores and releases fat during periods of feeding and fasting. White adipose tissue is the major energy reserve in periods of excess energy use, and its primary purpose is mobilization during energy deprivation. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance.

[0074] Thiazolidinedione, a family of peroxisome proliferator-activated receptor (PPAR) agonist drugs that increase sensitivity to insulin, are able to induce preadipocytes to differentiate into mature fat cells. The majority of research in adipocyte biology to date has been done using a transformed mouse preadipocyte cell line. Culture conditions that stimulate mouse preadipocyte differentiation are different from those inducing primary preadipocyte differentiation in human cells. Thiazolidinediones, or PPAR-γ agonists, are a new class of antidiabetic agents that improve insulin sensitivity and reduce plasma glucose and blood pressure in patients with type II diabetes. These agents can bind and activate an orphan nuclear receptor, (PPAR-γ) and some of them have been proven to induce human adipocyte differentiation.

[0075] Phorbol Myristate Acetate

[0076] Jurkat is an acute T cell leukemia cell line that grows actively in the absence of external stimuli. Jurkat has been extensively used to study signaling in human T cells. Phorbol myristate acetate (PMA) is a broad activator of the protein kinase C-dependent pathways. Ionomycin is a calcium ionophore that permits the entry of calcium in the cell, hence increasing the cytosolic calcium concentration. 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.

[0077] There is a need in the art for new compositions, including nucleic acids and proteins, for the diagnosis, prevention, and treatment of immune system, neurological, developmental, muscle, and cell proliferative disorders.

SUMMARY OF THE INVENTION

[0078] Various embodiments of the invention provide purified polypeptides, immune response associated proteins, referred to collectively as ‘IRAP’ and individually as ‘IRAP-1,’ ‘IRAP-2,’ ‘IRAP-3,’ ‘IRAP-4,’ ‘IRAP-5,’ ‘IRAP-6,’ ‘IRAP-7,’ ‘IRAP-8,’ ‘IRAP-9,’ ‘IRAP-10,’ ‘IRAP-11,’ ‘IRAP-12,’ ‘IRAP-13,’ ‘IRAP-14,’ ‘IRAP-15,’ ‘IRAP-16,’ ‘IRAP-17,’ ‘RAP-18,’ ‘IRAP-19,’ ‘IRAP-20,’ ‘IRAP-21,’ ‘IRAP-22,’ ‘IRAP-23,’ ‘IRAP-24,’ ‘IRAP-25,’ ‘IRAP-26,’ ‘IRAP-27,’ ‘IRAP-28,’ ‘IRAP-29,’ ‘IRAP-30,’ ‘IRAP-31,’ ‘IRAP-32,’ ‘IRAP-33,’ ‘IRAP-34,’ and ‘IRAP-35’ 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 immune response 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 immune response associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.

[0079] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO:1-35.

[0080] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. In another embodiment, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-35. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:36-70.

[0081] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide.

[0082] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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.

[0083] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35.

[0084] 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:36-70, 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:36-70, 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.

[0085] 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:36-70, 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:36-70, 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.

[0086] Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:36-70, 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:36-70, 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.

[0087] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, 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:1-35. Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional TRAP, comprising administering to a patient in need of such treatment the composition.

[0088] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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 IRAP, comprising administering to a patient in need of such treatment the composition.

[0089] Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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 IRAP, comprising administering to a patient in need of such treatment the composition.

[0090] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID) NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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.

[0091] 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:1-35, 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:1-35, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D) NO:1-35, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-35. 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.

[0092] Still yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:36-70, 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.

[0093] 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:36-70, 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:36-70, 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:36-70, 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:36-70, 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.

BRIEF DESCRIPTION OF THE TABLES

[0094] Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.

[0095] 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.

[0096] 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.

[0097] Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.

[0098] Table 5 shows representative cDNA libraries for polynucleotide embodiments.

[0099] Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.

[0100] Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with applicable descriptions, references, and threshold parameters.

[0101] 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

[0102] 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.

[0103] 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.

[0104] 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.

[0105] Definitions

[0106] “IRAP” refers to the amino acid sequences of substantially purified IRAP 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.

[0107] The term “agonist” refers to a molecule which intensifies or mimics the biological activity of IRAP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of IRAP either by directly interacting with IRAP or by acting on components of the biological pathway in which IRAP participates.

[0108] An “allelic variant” is an alternative form of the gene encoding IRAP. 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.

[0109] “Altered” nucleic acid sequences encoding IRAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as IRAP or a polypeptide with at least one functional characteristic of IRAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding IRAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding IRAP. 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 IRAP. 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 IRAP 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.

[0110] 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.

[0111] “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.

[0112] The term “antagonist” refers to a molecule which inhibits or attenuates the biological activity of IRAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of IRAP either by directly interacting with IRAP or by acting on components of the biological pathway in which IRAP participates.

[0113] 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 IRAP 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.

[0114] 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 immnunize 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.

[0115] 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).

[0116] 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).

[0117] 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.

[0118] 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.

[0119] 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 IRAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

[0120] “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′.

[0121] 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 IAAP or fragments of IRAP 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.).

[0122] “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 (GCG, Madison Wis.) or Phrap (University of Washington, Seattle Wash.). Some sequences have been both extended and assembled to produce the consensus sequence.

[0123] “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.

1Original ResidueConservative SubstitutionAlaGly, SerArgHis, LysAsnAsp, Gln, HisAspAsn, GluCysAla, SerGlnAsn, Glu, HisGluAsp, Gln, HisGlyAlaHisAsn, Arg, Gln, GluIleLeu, ValLeuIle, ValLysArg, Gln, GluMetLeu, IlePheHis, Met, Leu, Trp, TyrSerCys, ThrThrSer, ValTrpPhe, TyrTyrHis, Phe, TrpValIle, Leu, Thr

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] “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.

[0129] “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.

[0130] A “fragment” is a unique portion of IRAP or a polynucleotide encoding IRAP 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.

[0131] A fragment of SEQ ID NO:36-70 can comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:36-70, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:36-70 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:36-70 from related polynucleotides. The precise length of a fragment of SEQ ID NO:36-70 and the region of SEQ ID NO:36-70 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

[0132] A fragment of SEQ ID NO:1-35 is encoded by a fragment of SEQ ID NO:36-70. A fragment of SEQ ID NO:1-35 can comprise a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-35. For example, a fragment of SEQ ID NO:1-35 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ D NO:1-35. The precise length of a fragment of SEQ ID NO:1-35 and the region of SEQ ID NO:1-35 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.

[0133] 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.

[0134] “Homology” refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

[0135] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of identical residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

[0136] 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.

[0137] 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. 215:403410), which is available from several sources, including the NCBI, Bethesda, Md., and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. 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 at http://www.ncbi.nlm.nih.gov/gorf/b12.html. 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:

[0138] Matrix: BLOSUM62

[0139] Reward for match: 1

[0140] Penalty for mismatch: −2

[0141] Open Gap: 5 and Extension Gap: 2 penalties

[0142] Gap x drop-off: 50

[0143] Expect: 10

[0144] Word Size: 11

[0145] Filter: on

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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=l, gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix is selected as the default residue weight table.

[0150] 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:

[0151] Matrix: BLOSUM62

[0152] Open Gap: 11 and Extension Gap: 1 penalties

[0153] Gap x drop-off: 50

[0154] Expect: 10

[0155] Word Size: 3

[0156] Filter: on

[0157] 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.

[0158] “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.

[0159] 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.

[0160] “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, denatured salmon sperm DNA.

[0161] 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. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.; specifically see volume 2, chapter 9.

[0162] 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.

[0163] 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., C0 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).

[0164] 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.

[0165] “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.

[0166] An “immunogenic fragment” is a polypeptide or oligopeptide fragment of IRAP 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 IRAP which is useful in any of the antibody production methods disclosed herein or known in the art.

[0167] The term “microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.

[0168] 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.

[0169] The term “modulate” refers to a change in the activity of IRAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of IRAP.

[0170] 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.

[0171] “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.

[0172] “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.

[0173] “Post-translational modification” of an IRAP 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 IRAP.

[0174] “Probe” refers to nucleic acids encoding IRAP, 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).

[0175] 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 the references, for example Sambrook, J. et al. (1989; Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview 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.).

[0176] 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.

[0177] 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, 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.

[0178] 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.

[0179] 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.

[0180] “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.

[0181] 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.

[0182] The term “sample” is used in its broadest sense. A sample suspected of containing IRAP, nucleic acids encoding IRAP, 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.

[0183] 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.

[0184] 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.

[0185] A “substitution” refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.

[0186] “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.

[0187] 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.

[0188] “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.

[0189] 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 et al. (1989), supra.

[0190] 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 of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotides that vary from one species to another. The resulting polypeptides 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.

[0191] 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 least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.

[0192] The Invention

[0193] Various embodiments of the invention include new human immune response associated proteins (IRAP), the polynucleotides encoding IRAP, and the use of these compositions for the diagnosis, treatment, or prevention of immune system, neurological, developmental, muscle, and cell proliferative disorders.

[0194] Table 1 sumamarizes 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.

[0195] Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ 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.

[0196] 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 (Genetics Computer Group, Madison Wis.). 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.

[0197] Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are immune response associated proteins.

[0198] For example, SEQ ID NO:2 is 100% identical, from residue E88 to residue K306, to human complement-clq tumor necrosis factor-related protein (GenBank ID g13274520) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.1e-137, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:2 also contains a Clq domain, and a collagen triple helix repeat domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, BLAST, and MOTIFS analyses provide further corroborative evidence that SEQ ID NO:2 is an immune response associated protein.

[0199] As another example, SEQ ID NO:3 is 99% identical, from residue D45 to residue S408, to human T-cell receptor alpha chain-c6.1A fusion protein (GenBank ID g7717235) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.5e-193, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:3 also contains a Mov34/MPN/PAD-1 family domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST-DOMO and BLAST-PRODOM analyses provide further corroborative evidence that SEQ ID NO:3 is an immune response associated protein.

[0200] As another example, SEQ ID NO:5 is 100% identical, from residue M25 to residue E593, to human complement factor H-related protein 5 (GenBank ID g13195239) 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:5 also contains a Sushi domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.j Data from BLAST-DOMO and BLAST-PRODOM analyses provide further corroborative evidence that SEQ ID NO:5 is an immune response associated protein.

[0201] As another example, SEQ ID NO:6 is 95% identical, from residue M1 to residue L41, to human C5a anaphylatoxin receptor (GenBank ID g179700) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 1.4e-16, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:6 is a plasma membrane G-protein coupled receptor that mediates anaphylaxis and the migration and activation of neutrophils and macrophages, as determined by BLAST analysis using the PROTEOME database. (See Table 3.) Data from BLAST analyses using the PRODOM database provide further corroborative evidence that SEQ ID NO:6 is a G-protein coupled receptor.

[0202] As another example, SEQ ID NO:8 is 100% identical, from residue P21 to residue W277, and from residue M1 to A19, to human CD1E antigen, isoform 2 (GenBank ID g8249471) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 3.4e-140, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:8 is a member of the CD1 family of non classical major histocompatibility complex class I molecules, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:8 also contains an immunoglobulin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from further BLAST analyses provide corroborative evidence that SEQ ID NO:8 is a CD1E molecule.

[0203] As another example, SEQ ID NO:13 is 59% identical, from residue Q34 to residue A217, to Mus musculus Fca/m receptor (GenBank ID g11071950) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.5e-56, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:13 is related to the Fca/m receptor, which is localized to the plasma membrane, mediates endocytosis of IgM-coated microbes, and is an Fc receptor involved in the immune response to microbes, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:13 also contains an immunoglobulin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLAST analysis of the DOMO database provides further corroborative evidence that SEQ ID NO:13 is an immune response-associated protein.

[0204] As another example, SEQ ID NO:15 is 99% identical, from residue D19 to residue G240, to human SP alpha (GenBank ID g2702314) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.2e-129, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:15 also has homology to extracellular proteins that are scavenger receptors, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:15 also contains a scavenger receptor cysteine-rich domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:15 shares homology with scavenger receptors.

[0205] As another example, SEQ ID NO:23 is 99% identical, from residue M1 to residue S208, to a human RING 7 protein (GenBank ID g313002) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.1e-124, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:23 also has homology to proteins that are localized to the plasma membranes, have HLA gene function, and are RING 7 HLA proteins, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:23 also contains a class II histocompatibility antigen, beta, domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:23 is a histocompatibility protein.

[0206] As another example, SEQ ID NO:27 is 100% identical, from residue P21 to residue K80, to human CD1E antigen (GenBank ID g8249469) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 5.9e-147, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:27 also has homology to proteins that are localized to the cytoplasmic membrane, have antigen presentation function, and are CD1E antigens, as determined by BLAST analysis using the PROTEOME database. SEQ ID NO:27 also contains an immunoglobulin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from other BLAST analyses provide further corroborative evidence that SEQ ID NO:27 is a cell-surface antigen.

[0207] SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9-12, SEQ ID NO:14, SEQ ID NO:16-22, SEQ ID NO:24-26, and SEQ ID NO:28-35 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-35 are described in Table 7.

[0208] 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:36-70 or that distinguish between SEQ ID NO:36-70 and related polynucleotides.

[0209] 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 FLXXXXXX_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).

[0210] 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).

2PrefixType of analysis and/or examples of programsGNN, GFG,Exon prediction from genomic sequences using, forENSTexample, GENSCAN (Stanford University, CA, USA)or FGENES (Computer Genomics Group, The SangerCentre, Cambridge, UK).GBIHand-edited analysis of genomic sequences.FLStitched or stretched genomic sequences(see Example V).INCYFull length transcript and exon prediction frommapping of EST sequences to the genome. Genomiclocation and EST composition data are combinedto predict the exons and resulting transcript.

[0211] 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.

[0212] 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.

[0213] 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 (CB 1 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 nid (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.

[0214] The invention also encompasses IRAP variants. A preferred RAP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the IRAP amino acid sequence, and which contains at least one functional or structural characteristic of IRAP.

[0215] Various embodiments also encompass polynucleotides which encode IRA?. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:36-70, which encodes IRAP. The polynucleotide sequences of SEQ ID NO:36-70, 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.

[0216] The invention also encompasses variants of a polynucleotide encoding IRAP. 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 IRAP. A particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:36-70 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:36-70. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of IRAP.

[0217] In addition, or in the alternative, a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding IRAP. A splice variant may have portions which have significant sequence identity to a polynucleotide encoding IRAP, but will generally have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate splicing of exons during mRNA processing. A 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 IAAP 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 IRAP. For example, a polynucleotide comprising a sequence of SEQ ID NO:64, a polynucleotide comprising a sequence of SEQ ID NO:43, and a polynucleotide comprising a sequence of SEQ ID NO:62 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:49 and a polynucleotide comprising a sequence of SEQ ID NO:65 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:59 and a polynucleotide comprising a sequence of SEQ ID NO:66 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:60, a polynucleotide comprising a sequence of SEQ ID NO:67, a polynucleotide comprising a sequence of SEQ ID NO:68, a polynucleotide comprising a sequence of SEQ ID NO:69 and a polynucleotide comprising a sequence of SEQ ID NO:70 are splice variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:51 and a polynucleotide comprising a sequence of SEQ ID NO:57 are splice variants of each other; and a polynucleotide comprising a sequence of SEQ ID NO:54 and a polynucleotide comprising a sequence of SEQ ID NO:55 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 IRAP.

[0218] 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 IRAP, 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 IRAP, and all such variations are to be considered as being specifically disclosed.

[0219] Although polynucleotides which encode IRAP and its variants are generally capable of hybridizing to polynucleotides encoding naturally occurring IRAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding IRAP 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 IRAP 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.

[0220] The invention also encompasses production of polynucleotides which encode IRAP and IRAP 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 IRAP or any fragment thereof.

[0221] 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:36-70 and fragments thereof, under various conditions of stringency (Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in “Definitions.”

[0222] 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 1, 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).

[0223] The nucleic acids encoding IRAP 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.

[0224] 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.

[0225] 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 IRAP may be cloned in recombinant DNA molecules that direct expression of IRAP, 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 IRAP.

[0226] The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter IRAP-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.

[0227] 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 IRAP, 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.

[0228] In another embodiment, polynucleotides encoding IRAP 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, IRAP 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, W H 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 IRAP, 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.

[0229] The peptide may be substantially purified by preparative high performance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392421). The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).

[0230] In order to express a biologically active IRAP, the polynucleotides encoding IRAP 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 IRAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding IRAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where a polynucleotide sequence encoding IRAP 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).

[0231] Methods which are well known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding IRAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel et al., supra, ch. 1, 3, and 15).

[0232] A variety of expression vector/host systems may be utilized to contain and express polynucleotides encoding IRAP. 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, 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 (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.

[0233] In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding IRAP. For example, routine cloning, subcloning, and propagation of polynucleotides encoding TRAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Invitrogen). Ligation of polynucleotides encoding IRAP 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 IRAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of IRAP may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.

[0234] Yeast expression systems may be used for production of IRAP. 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).

[0235] Plant systems may also be used for expression of IRAP. Transcription of polynucleotides encoding IRAP 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. 30 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).

[0236] 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 IRAP 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 IRAP 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.

[0237] 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).

[0238] For long term production of recombinant proteins in mammalian systems, stable expression of IRAP in cell lines is preferred. For example, polynucleotides encoding IRAP 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.

[0239] 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 (Hartman, 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).

[0240] 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 TRAP is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding IRAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding IRAP 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.

[0241] In general, host cells that contain the polynucleotide encoding IRAP and that express IRAP 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.

[0242] Immunological methods for detecting and measuring the expression of IAAP 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 RAP 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.).

[0243] 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 IRAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, polynucleotides encoding TRAP, 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 in 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 IRAP 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 IRAP may be designed to contain signal sequences which direct secretion of IRAP through a prokaryotic or eukaryotic cell membrane.

[0244] 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 W138) 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.

[0245] In another embodiment of the invention, natural, modified, or recombinant polynucleotides encoding IRAP 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 IRAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of IRAP 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-myc, 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 IRAP encoding sequence and the heterologous protein sequence, so that IRAP 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.

[0246] In another embodiment, synthesis of radiolabeled IRAP 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.

[0247] IRAP, fragments of IRAP, or variants of [RAP may be used to screen for compounds that specifically bind to IRAP. One or more test compounds may be screened for specific binding to IRAP. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to IRAP. Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.

[0248] In related embodiments, variants of IRAP can be used to screen for binding of test compounds, such as antibodies, to IRAP, a variant of IRAP, or a combination of IRAP and/or one or more variants IRAP. In an embodiment, a variant of IRAP can be used to screen for compounds that bind to a variant of IRAP, but not to IRAP having the exact sequence of a sequence of SEQ ID NO:1-35. IRAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to IRAP, with various embodiments having 60%, 70%, 75%, 80%, 85%,90%, and 95% sequence identity.

[0249] In an embodiment, a compound identified in a screen for specific binding to IRAP can be closely related to the natural ligand of IRAP, 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 IRAP (Howard, A. D. et al. (2001) Trends Pharmacol. Sci.22: 132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

[0250] In other embodiments, a compound identified in a screen for specific binding to IRAP can be closely related to the natural receptor to which IRAP 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 IRAP which is capable of propagating a signal, or a decoy receptor for IRAP 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 (TNF) receptor dimer linked to the Fc portion of human IgG1 (Taylor, P. C. et al. (2001) Curr. Opin. Immunol. 13:611-616).

[0251] In one embodiment, two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to IRAP, fragments of IRAP, or variants of IRAP. The binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of IRAP. In one embodiment, an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of IRAP. 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 IRAP.

[0252] In an embodiment, anticalins can be screened for specific binding to IRAP, fragments of IRAP, or variants of IRAP. 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) 1. 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.

[0253] In one embodiment, screening for compounds which specifically bind to, stimulate, or inhibit IRAP involves producing appropriate cells which express IRAP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing IRAP or cell membrane fractions which contain IRAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either IRAP or the compound is analyzed.

[0254] 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 IRAP, either in solution or affixed to a solid support, and detecting the binding of IRAP 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.

[0255] 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).

[0256] IRAP, fragments of IRAP, or variants of IRAP may be used to screen for compounds that modulate the activity of IRAP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for IRAP activity, wherein IRAP is combined with at least one test compound, and the activity of IRAP in the presence of a test compound is compared with the activity of IRAP in the absence of the test compound. A change in the activity of IRAP in the presence of the test compound is indicative of a compound that modulates the activity of IRAP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising IRAP under conditions suitable for IRAP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of IRAP 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.

[0257] In another embodiment, polynucleotides encoding IRAP 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. 25:43234330). 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.

[0258] Polynucleotides encoding IRAP 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).

[0259] Polynucleotides encoding RAP 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 IRAP 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 IRAP, e.g., by secreting IRAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

[0260] Therapeutics

[0261] Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of IRAP and immune response associated proteins. In addition, the expression of IRAP is closely associated with breast tumor, esophageal, fetal spleen, knee cartilage, liver, prostate tumor, thymus, and tumor-associated ovarian tissues, pineal gland tissue from a patient with Alzheimer's disease, and hNT2 cells derived from a human teratocarcinoma. In addition, examples of tissues expressing IRAP can be found in Table 6 and can also be found in Example XI. Therefore, IRAP appears to play a role in immune system, neurological, developmental, muscle, and cell proliferative disorders. In the treatment of disorders associated with increased IRAP expression or activity, it is desirable to decrease the expression or activity of IRAP. In the treatment of disorders associated with decreased IRAP expression or activity, it is desirable to increase the expression or activity of IRAP. Therefore, in one embodiment, TRAP 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 IRAP. Examples of such disorders include, but are not limited to, an immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing 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 including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, 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 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, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; and 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, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

[0262] In another embodiment, a vector capable of expressing IRAP 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 IRAP including, but not limited to, those described above.

[0263] In a further embodiment, a composition comprising a substantially purified IRAP 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 IRAP including, but not limited to, those provided above.

[0264] In still another embodiment, an agonist which modulates the activity of IRAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IRAP including, but not limited to, those listed above.

[0265] In a further embodiment, an antagonist of IRAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IRAP. Examples of such disorders include, but are not limited to, those immune system, neurological, developmental, muscle, and cell proliferative disorders described above. In one aspect, an antibody which specifically binds IRAP 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 IRAP.

[0266] In an additional embodiment, a vector expressing the complement of the polynucleotide encoding IRAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IRAP 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 IRAP may be produced using methods which are generally known in the art. In particular, purified IRAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind IRAP. Antibodies to IRAP 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. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use. Single chain antibodies (e.g., from camels or llamas) may be potent enzyme inhibitors and may have advantages in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyidermans, S. (2001) J. Biotechnol. 74:277-302).

[0267] For the production of antibodies, various hosts including goats, rabbits, rats, mice, camels, dromedaries, llamas, humans, and others may be immunized by injection with IRAP 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 Corynebactenum parvum are especially preferable. It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to IRAP 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 identical to a portion of the amino acid sequence of the natural protein. Short stretches of IRAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.

[0268] Monoclonal antibodies to RAP 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-cell hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:3142; 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).

[0269] 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 314:452454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce IRAP-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. Nat). Acad. Sci. USA 88:10134-10137).

[0270] 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).

[0271] Antibody fragments which contain specific binding sites for IRAP may also be generated. For example, such fragments include, but are not lui ted 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).

[0272] 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 IRAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering IRAP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).

[0273] Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for IRAP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of RAP-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 IRAP epitopes, represents the average affinity, or avidity, of the antibodies for IRAP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular IRAP 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 IRAP-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 IRAP, 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.).

[0274] 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 IRAP-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 IRAP, 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 IRAP. 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 RAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa N.J.).

[0275] 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. 102:469475; 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 at; (1997) Nucleic Acids Res. 25:2730-2736).

[0276] In another embodiment of the invention, polynucleotides encoding IRAP 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 at. (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 270:404410; 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 (HIV) (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 IRAP expression or regulation causes disease, the expression of IRAP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.

[0277] In a further embodiment of the invention, diseases or disorders caused by deficiencies in IRAP are treated by constructing mammalian expression vectors encoding IRAP and introducing these vectors by mechanical means into RP-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. Récipon (1998) Curr. Opin. Biotechnol. 9:445-450).

[0278] Expression vectors that may be effective for the expression of IRAP 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.). IRAP 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. 9:451456), 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 IRAP from a normal individual.

[0279] Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION 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.

[0280] In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to IRAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding RAP 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, I. 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 retrovirat 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. 71:47074716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

[0281] In an embodiment, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding IRAP to cells which have one or more genetic abnormalities with respect to the expression of IRAP. 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).

[0282] In another embodiment, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding IRAP to target cells which have one or more genetic abnormalities with respect to the expression of IRAP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing IRAP 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.

[0283] In another embodiment, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding IRAP to target cells. The biology of the prototypic alphavirus, Semliki 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. 9:464469). 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 IRAP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of IRAP-coding RNAs and the synthesis of high levels of IRAP 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 IRAP 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.

[0284] 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.

[0285] 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 IRAP.

[0286] 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 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.

[0287] 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 IRAP. 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.

[0288] 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.

[0289] An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding IRAP. 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 IRAP expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding IRAP may be therapeutically useful, and in the treatment of disorders associated with decreased IRAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding IRAP may be therapeutically useful.

[0290] At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, 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 IRAP 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 IRAP 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 IRAP. 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).

[0291] 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).

[0292] 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.

[0293] 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 IRAP, antibodies to IRAP, and mimetics, agonists, antagonists, or inhibitors of IRAP.

[0294] The compositions utilized in this invention 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.

[0295] 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 has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.

[0296] 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.

[0297] Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising IRAP or fragments thereof. For example, liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, IRAP 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).

[0298] 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.

[0299] A therapeutically effective dose refers to that amount of active ingredient, for example IRAP or fragments thereof, antibodies of RAP, and agonists, antagonists or inhibitors of IRAP, 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.

[0300] 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.

[0301] 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.

[0302] Diagnostics

[0303] In another embodiment, antibodies which specifically bind IRAP may be used for the diagnosis of disorders characterized by expression of IRAP, or in assays to monitor patients being treated with IRAP or agonists, antagonists, or inhibitors of IRAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for IRAP include methods which utilize the antibody and a label to detect IRAP 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.

[0304] A variety of protocols for measuring IRAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of IRAP expression. Normal or standard values for IRAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to IRAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of ]RAP 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.

[0305] In another embodiment of the invention, polynucleotides encoding IRAP 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 IRAP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of IRAP, and to monitor regulation of IRAP levels during therapeutic intervention.

[0306] In one aspect, hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding IRAP or closely related molecules may be used to identify nucleic acid sequences which encode IRAP. 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 IRAP, allelic variants, or related sequences.

[0307] Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the IRAP 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:36-70 or from genomic sequences including promoters, enhancers, and introns of the IRAP gene.

[0308] Means for producing specific hybridization probes for polynucleotides encoding IRAP include the cloning of polynucleotides encoding IRAP or RAP 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.

[0309] Polynucleotides encoding IRAP may be used for the diagnosis of disorders associated with expression of IRAP. Examples of such disorders include, but are not limited to, an immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Bruton, common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak-Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, allergies, ankylosing 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 including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, 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 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, seizure disorders such as Syndenham's chorea and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, cataract, and sensorineural hearing loss; a muscle disorder such as cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; and 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, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. Polynucleotides encoding IRAP 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 IRAP expression. Such qualitative or quantitative methods are well known in the art.

[0310] In a particular aspect, polynucleotides encoding IRAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above. Polynucleotides complementary to sequences encoding IRAP 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 IRAP 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.

[0311] In order to provide a basis for the diagnosis of a disorder associated with expression of IRAP, 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 RAP, 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.

[0312] 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.

[0313] 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.

[0314] Additional diagnostic uses for oligonucleotides designed from the sequences encoding IRAP 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 IRAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding IRAP, 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.

[0315] In a particular aspect, oligonucleotide primers derived from polynucleotides encoding IRAP 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 IRAP 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.).

[0316] 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) Cuff. Opin. Neurobiol. 11:637-641).

[0317] Methods which may also be used to quantify the expression of IRAP 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 colorimetric response gives rapid quantitation.

[0318] 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.

[0319] In another embodiment, IRAP, fragments of IRAP, or antibodies specific for IRAP 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.

[0320] 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.

[0321] 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.

[0322] 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. 112-113:467471). 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 at http://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.

[0323] 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.

[0324] A proteomic profile may also be generated using antibodies specific for IRA? to quantify the levels of IRAP 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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 WO95/251116; 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).

[0329] In another embodiment of the invention, nucleic acid sequences encoding IRAP 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. 7:149-154Once 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).

[0330] 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 IRAP 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.

[0331] 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.

[0332] In another embodiment of the invention, IRAP, 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 IRAP and the agent being tested may be measured.

[0333] 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 WO84/03564). In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with IRAP, or fragments thereof, and washed. Bound IRAP is then detected by methods well known in the art. Purified IRAP 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.

[0334] In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding IRAP specifically compete with a test compound for binding IRAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with IRAP.

[0335] In additional embodiments, the nucleotide sequences which encode IRAP 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.

[0336] 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 preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0337] The disclosures of all patents, applications, and publications mentioned above and below, including U.S. Ser. No. 60/324,034, U.S. Ser. No. 60/327,395, U.S. Ser. No. 60/328,923, U.S. Ser. No. 60/342,810, U.S. Ser. No. 60/344,468, U.S. Ser. No. 60/332,140, U.S. Ser. No. 60/340,282, U.S. Ser. No. 60/347,693, U.S. Ser. No. 60/361,088, U.S. Ser. No. 60/358,279, U.S. Ser. No. 60/364,494, U.S. Ser. No. 60/379,876, and U.S. Ser. No. 60/388,180 are hereby expressly incorporated by reference.

EXAMPLES

[0338] I. Construction of cDNA Libraries

[0339] 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.).

[0340] 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 CL4B 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), PCDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), 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.

[0341] II. Isolation of cDNA Clones

[0342] 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.

[0343] 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).

[0344] III. Sequencing and Analysis

[0345] 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.

[0346] 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 (HMM)-based protein family databases such as PFAM, INCY, and TIGRFAM (Haft, D. H. et a]. (2001) Nucleic Acids Res. 29:4143); 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). (HMM 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 (HMM)-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 alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

[0347] 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).

[0348] 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:36-70. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 2.

[0349] IV. Identification and Editing of Coding Sequences from Genomic DNA

[0350] Putative immune response 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 immune response associated proteins, the encoded polypeptides were analyzed by querying against PFAM models for immune response associated proteins. Potential immune response associated proteins were also identified by homology to Incyte cDNA sequences that had been annotated as immune response 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.

[0351] V. Assembly of Genomic Sequence Data with cDNA Sequence Data

[0352] “Stitched” Sequences

[0353] 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 mi 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.

[0354] “Stretched” Sequences

[0355] 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.

[0356] VI. Chromosomal Mapping of IRAP Encoding Polynucleotides

[0357] The sequences which were used to assemble SEQ ID NO:36-70 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:36-70 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.

[0358] 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 (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.

[0359] Association of IRAP polynucleotides with Parkinson's Disease

[0360] Several genes have been identified as showing linkage to autosomal dominant forms of Parkinson's Disease (PD). PD is a common neurodegenerative disorder causing bradykinesia, resting tremor, muscular rigidity, and postural instability. Cytoplasmic eosinophilic inclusions called Lewy bodies, and neuronal loss especially in the substantia nigra pars compacta, are pathological hallmarks of PD (Valente, E. M. et al (2001) Am. J. Hum. Genet. 68:895-900). Lewy body Parkinson disease has been thought to be a specific autosomal dominant disorder (Wakabayashi, K. et al. (1998) Acta Neuropath. 96:207-210). Juvenile parkinsonism may be a specific autosomal recessive disorder (Matsumine, H. et al. (1997) Am. J. Hum. Genet. 60: 588-596, 1997). (Online Mendelian Inheritance in Man, OMIM. Johns Hopkins University, Baltimore, Md. MIM Number: 168600: Sep. 9, 2002:. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/)

[0361] Association of a disease with a chromosomal locus can be determined by Lod score. Lod score is a statistical method used to test the linkage of two or more loci within families having a genetic disease. The Lod score is the logarithm to base 10 of the odds in favor of linkage. Linkage is defined as the tendency of two genes located on the same chromosome to be inherited together through meiosis (Genetics in Medicine, Fifth Edition, (1991) Thompson, M. W. Et al. W. B. Saunders Co. Philadelphia). A lod score of +3 or greater indicates a probability of 1 in 1000 that a particular marker was found solely by chance in affected individuals, which is strong evidence that two genetic loci are linked.

[0362] One such gene implicated in PD is PARK3, which maps to 2p13 (Gasser, T. et al. (1998) Nature Genet. 18:262-265). A marker at chromosomal position D2S441 was found to have a Lod score of 3.2 in the region of PARK3. This marker supported the disease association of PARK3 in the chromosomal interval from D2S134 to D2S286 (Gasser et al., supra). Markers located within chromosomal intervals D2S134 and D2S286, which map between 83.88 to 94.05 centiMorgans on the short arm of chromosome 2, were used to identify genes that map in the region between D2S134 and D2S286.

[0363] A second PD gene, implicated in early-onset recessive parkinsonism, is PARK6, located on chromosome 1 at p35-p36. Several markers were obtained with lod scores greater than 3, including D1S199, D1S2732, D1S2828, D1S478, D1S2702, D1S2734, D1S2674 (Valente, E. M. et al. supra). These markers were used to determine the PD-relevant range of chromosome loci and identify sequences that map to chromosome 1 between D1S199 and D1S2885. IRAP polynucleotides were found to map within the chromosomal region in which markers associated with disease or other physiological processes of interest were located. Genomic contigs available from NCBI were used to identify IRAP polynucleotides which map to a disease locus. Contigs longer than 1 Mb were broken into subcontigs of 1 Mb in length with overlapping sections of 100 kb. A preliminary step used an algorithm, similar to MEGABLAST (NCBI), to identify mRNA sequence/masked genomic DNA contig pairings. SIM4 (Florea, L. et al. (1998) Genome Res. 8:967-74, version May 2000, was optimized for high throughput and strand assignment confidence, and used to further select cDNA/genomic pairings. The SIM4-selected mRNA sequence/genomic contig pairs were further processed to determine the correct location of the IRAP polynucleotides on the genomic contig and their strand identity.

[0364] SEQ ID NO:56 was mapped to a region of contig GBI:NT—004359—001.8 from the February 2002 NCBI release, localizing SEQ ID NO:56 to within 14.8 Mb of the Parkinson's disease locus at p35-p36 on chromosome 1. Therefore, SEQ ID NO:56 is in proximity with loci shown to consistently associate with Parkinson's disease.

[0365] VII. Analysis of Polynucleotide Expression

[0366] 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, supra, ch. 7; Ausubel et al., supra, ch. 4).

[0367] 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:

\frac{BLAST Score \times Percent Identity}{5 \times minimum {length (Seq . 1), length (Seq . 2)}}

[0368] 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.

[0369] Alternatively, polynucleotides encoding RAP are analyzed with respect to the tissue sources from which they were derived. For example, some full 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 IRAP. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

[0370] VIII. Extension of IRAP Encoding Polynucleotides

[0371] 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.

[0372] 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.

[0373] 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.

[0374] The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X 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 (Labsystems 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.

[0375] 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 37° C. in 384-well plates in LB/2× carb liquid media.

[0376] 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, 3and 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).

[0377] 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.

[0378] IX. Identification of Single Nucleotide Polymorphisms in IRAP Encoding Polynucleotides

[0379] Common DNA sequence variants known as single nucleotide polymorphisms (SNPs) were identified in SEQ ID NO:36-70 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 9f 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 immunoglobulins or T-cell receptors.

[0380] 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.

[0381] X. Labeling and Use of Individual Hybridization Probes

[0382] Hybridization probes derived from SEQ D NO:36-70 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-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: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).

[0383] 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.

[0384] XI. Microarrays

[0385] 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 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).

[0386] 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.

[0387] Tissue or Cell Sample Preparation

[0388] 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 μM 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.

[0389] Microarray Preparation

[0390] 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 SEPHACRYL-400 (Amersham Biosciences).

[0391] 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% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

[0392] 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 100ng/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.

[0393] Microarrays are UV-crosslinked using a STRATALINKER 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.

[0394] Hybridization

[0395] 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.

[0396] Detection

[0397] 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.

[0398] 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.

[0399] 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.

[0400] 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.

[0401] 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 exhibited at least about a two-fold change in expression, a signal-to-background ratio of at least 2.5, and an element spot size of at least 40% were identified as differentially expressed.

[0402] Expression

[0403] Adipose tissue stores and releases fat. Adipose tissue is also one of the important target tissues for insulin. Adipogenesis and insulin resistance in type II diabetes are linked. Most patients with type II diabetes are obese and obesity in turn causes insulin resistance. For these RNA expression experiments, human primary subcutaneous preadipocytes were isolated from adipose tissue of a 40-year old healthy female with a body mass index (BMI) of 32.47. The preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in the differentiation medium containing active components PPAR-γ agonist and human insulin. Thiazolidinediones or PPAR-γ agonists are a new class of antidiabetic agents that improve insulin sensitivity and reduce plasma glucose and blood pressure in subjects with type II diabetes. These agents can bind and activate an orphan nuclear receptor and some of them have been proven to be able to induce human adipocyte differentiation.

[0404] For these experiments, human preadipocytes were treated with human insulin and PPAR-γ agonist for 3 days and subsequently were switched to medium containing insulin alone. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. The expression of SEQ ID NO:37 was decreased by at least two-fold. These experiments indicate that SEQ ID NO:37 exhibited significant differential expression patterns using microarray techniques. Therefore, in various embodiments, SEQ ID NO:37 can be used for one or more of the following: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and iii) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.

[0405] As another example, in an attempt to understand the molecular pathways involved in colon cancer progression, gene expression patterns in normal colon tissue and colon tumors from the same donor were compared. SEQ ID NO:45 was found to be upregulated at least two fold in one out of seven donors. Therefore, in various embodiments, SEQ ID NO:45 can be used for one or more of the following: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.

[0406] As another example, SEQ ID NO:50 showed decreased expression in preadipocytes treated with a differentiation-inducing medium versus untreated preadipocytes, as determined by microarray analysis. Human primary preadipocytes were isolated from adipose tissue of a 36-year-old female with body mass index (BMI) 27.7 (overweight, but otherwise healthy). The preadipocytes were cultured and induced to differentiate into adipocytes by culturing them in a proprietary differentiation medium containing an active component such as peroxisome proliferator-activated receptor (PPAR)-γ agonist and human insulin (Zen-Bio). Human preadipocytes were treated with human insulin and PPAR agonist for 3 days and subsequently switched to medium containing insulin only for 5, 9, and 12 more days. Differentiated adipocytes were compared to untreated preadipocytes maintained in culture in the absence of inducing agents. An overall differentiation rate of more than 60% was observed after 15 days in culture. Therefore, in various embodiments, SEQ ID NO:50 can be used for one or more of the following: i) monitoring treatment of diabetes, ii) diagnostic assays for diabetes, and iii) developing therapeutics and/or other treatments for diabetes.

[0407] As another example, SEQ ID NO:50 showed decreased expression in bone tissue affected by osteosarcoma versus normal osteoblasts, as determined by microarray analysis. Messenger RNA from normal human osteoblast was compared with mRNA from biopsy specimens, osteosarcoma tissues, or primary cultures or metastasized tissues. A normal osteoblast primary culture, NHOst 5488 served as the reference. The comparison of mRNA from biopsy specimen was compared with that of definitive surgical specimen from the same patient. Extended study of this basic set included mRNA from primary cell cultures of the definitive surgical specimen, muscle, or cartilage tissue from the same patient, as well as biopsy specimens, definitive surgical specimens, or lung metastatic tissues from different individuals. Therefore, in various embodiments, SEQ ID NO:50 can be used for one or more of the following: i) monitoring treatment of osteosarcoma, ii) diagnostic assays for osteosarcoma, and iii) developing therapeutics and/or other treatments for osteosarcoma.

[0408] As another example, SEQ ID NO:50 showed decreased expression in Jurkat cells activated by treatment with phorbol myristate acetate (PMA) and ionomycin versus untreated Jurkat cells, as determined by microarray analysis. Jurkat is an acute T-cell leukemia cell line. Jurkat cells were treated with combinations of graded doses of phorbol myristate acetate (PMA) and ionomycin and collected at a 1 hour time point. In T cells, the combination of PMA and ionomycin mimics the type of secondary signaling events elicited during optimal B cell activation. The treated cells were compared to untreated Jurkat cells kept in culture in the absence of stimuli. Therefore, in various embodiments, SEQ ID NO:50 can be used for one or more of the following: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and iii) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.

[0409] As another example, SEQ ID NO:56 was differentially expressed in human colon tumor tissue as compared to normal colon tissue. Colon cancer develops through a multi-step process in which pre-malignant colonocytes undergo a relatively defined sequence of events that lead to tumor formation. Factors that contribute to the process of tumor progression and malignant transformation include genetics, mutations, and selection. Despite efforts to characterize the molecular events leading to colon cancer, the process of tumor development and progression has not been delineated. To identify genes differentially expressed in colon cancer, we compared gene expression patterns in normal and tumor tissues. Matched normal and tumor samples from the same individual were compared by competitive hybridization. This process eliminates some of the individual variation due to genetic background, and enhances differences due to the disease process. Therefore, in various embodiments, SEQ ID NO:56 can be used for one or more of the following: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and iii) developing therapeutics and/or other treatments for colon cancer.

[0410] As another example, SEQ ID NO:67 showed differential expression in breast tumor tissue as compared to normal breast tissue from the same donor as determined by microarray analysis. Tumor from the right breast was compared to grossly uninvolved breast tissue from the same donor, a 43 year old female diagnosed with invasive lobular carcinoma in situ. The expression of SEQ ID NO:67 was decreased by at least two-fold in the tumor tissue as compared to the matched non-tumor tissue. Therefore, in various embodiments, SEQ ID NO:67 can be used for one or more of the following: i) monitoring treatment of breast cancer, ii) diagnostic assays for breast cancer, and iii) developing therapeutics and/or other treatments for breast cancer.

[0411] As another example, SEQ ID NO:67 showed differential expression in lung tumor tissues compared to normal lung tissue from the same donor as determined by microarray analysis. Samples of normal lung were compared to lung tumor from the same donor for four different donors (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). The expression of SEQ ID NO:67 was decreased by at least two-fold in tumor tissue as compared to the matched normal lung. Therefore, in various embodiments, SEQ ID NO:67 can be used for one or more of the following: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and iii) developing therapeutics and/or other treatments for lung cancer.

[0412] XII. Complementary Polynucleotides

[0413] Sequences complementary to the IRAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring IRAP. 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 IRA”. 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 IRAP-encoding transcript.

[0414] XIII. Expression of IRAP

[0415] Expression and purification of IRAP is achieved using bacterial or virus-based expression systems. For expression of RAP 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 IRAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of IRAP 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 IRAP 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).

[0416] In most expression systems, RAP 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 IRAP 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 IRAP obtained by these methods can be used directly in the assays shown in Examples XVII and XVIII, where applicable.

[0417] XIV. Functional Assays

[0418] IRAP function is assessed by expressing the sequences encoding IRAP 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.).

[0419] The influence of IRAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding IRAP 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 IRAP and other genes of interest can be analyzed by northern analysis or microarray techniques.

[0420] XV. Production of IRAP Specific Antibodies

[0421] IRAP substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488495), or other purification techniques, is used to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies using standard protocols.

[0422] Alternatively, the IRAP 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).

[0423] 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-IRAP activity by, for example, binding the peptide or IRAP to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

[0424] XVI. Purification of Naturally Occurring IRAP Using Specific Antibodies

[0425] Naturally occurring or recombinant IRAP is substantially purified by immunoaffinity chromatography using antibodies specific for IRAP. An immunoaffinity column is constructed by covalently coupling anti-IRAP 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.

[0426] Media containing IRAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of IRAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/IRAP 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 IRAP is collected.

[0427] XVII. Identification of Molecules Which Interact with IRAP

[0428] IRAP, or biologically active fragments thereof, are labeled with 251I 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 IRAP, washed, and any wells with labeled IRAP complex are assayed. Data obtained using different concentrations of IRAP are used to calculate values for the number, affinity, and association of TRAP with the candidate molecules.

[0429] Alternatively, molecules interacting with IRAP 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).

[0430] IRAP 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).

[0431] XVIII. Demonstration of IRAP Activity

[0432] An assay for IRAP activity measures the ability of IRAP to recognize and precipitate antigens from serum. This activity can be measured by the quantitative precipitin reaction (Golub, E. S. et al. (1987) Immunology: A Synthesis, Sinauer Associates, Sunderland, Mass., pages 113-115). IRAP is isotopically labeled using methods known in the art. Various serum concentrations are added to constant amounts of labeled IRAP. IRAP-antigen complexes precipitate out of solution and are collected by centrifugation. The amount of precipitable IRAP-antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitable IRAP-antigen complex is plotted against the serum concentration. For various serum concentrations, a characteristic precipitin curve is obtained, in which the amount of precipitable IRAP-antigen complex initially increases proportionately with increasing serum concentration, peaks at the equivalence point, and then decreases proportionately with further increases in serum concentration. Thus, the amount of precipitable IRAP-antigen complex is a measure of IRAP activity which is characterized by sensitivity to both limiting and excess quantities of antigen.

[0433] Alternatively, an assay for IRAP activity measures the expression of IRAP on the cell surface. cDNA encoding IRAP is transfected into a non-leukocytic cell line. Cell surface proteins are labeled with biotin (de la Fuente, M. A. et al. (1997) Blood 90:2398-2405). Immunoprecipitations are performed using IRAP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of IRAP expressed on the cell surface.

[0434] Alternatively, an assay for IRAP activity measures the amount of cell aggregation induced by overexpression of IRAP. In this assay, cultured cells such as NIH3T3 are transfected with cDNA encoding IRAP contained within a suitable mammalian expression vector under control of a strong promoter. Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH), is useful for identifying stable transfectants. The amount of cell agglutination, or clumping, associated with transfected cells is compared with that associated with untransfected cells. The amount of cell agglutination is a direct measure of IRAP activity.

[0435] Alternatively, an assay for IRAP activity measures binding of IRAP to bacteria (Liu, C et al., supra). IRAP is incubated with bacteria, and bacterial-bound proteins are isolated by centrifugation, washed, and detected by Western blots Various modifications and variations of the described compositions, methods, and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. It will be appreciated that the invention provides novel and useful proteins, and their encoding polynucleotides, which can be used in the drug discovery process, as well as methods for using these compositions for the detection, diagnosis, and treatment of diseases and conditions. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Nor should the description of such embodiments be considered exhaustive or limit the invention to the precise forms disclosed. Furthermore, elements from one embodiment can be readily recombined with elements from one or more other embodiments. Such combinations can form a number of embodiments within the scope of the invention. It is intended that the scope of the invention be defined by the following claims and their equivalents.

3TABLE 1IncyteIncyteIncytePolypeptidePolypeptidePolynucleotidePolynucleotideIncyte FullProject IDSEQ ID NO:IDSEQ ID NO:IDLength Clones749945317499453CD1367499453CB1749981527499815CD1377499815CB195017273CA2316534633165346CD1383165346CB1509295445092954CD1395092954CB11859004CA2,4209127CA2,90133145CA2,90133229CA2,90133245CA2749956057499560CD1407499560CB170243658670243658CD4170243658CB160210458CA2750019677500196CD1427500196CB190027016CA2,90027024CA2,90027032CA2,90027124CA2,90027132CA2,90027148CA2750035187500351CD1437500351CB190206067CA2,90206435CA2,90215217CA2750092397500923CD1447500923CB12258292102258292CD1452258292CB17500283117500283CD1467500283CB17600263127600263CD1477600263CB190196025CA27503686137503686CD1487503686CB190034204CA2,90034220CA2,90034236CA2,90034244CA2,90034284CA2,90173787CA27504791147504791CD1497504791CB16571458CA27504885157504885CD1507504885CB13796648CA27504915167504915CD1517504915CB17504926177504926CD1527504926CB17505049187505049CD1537505049CB190208355CA2,95084027CA2900342121990034212CD5490034212CB190034212CA27503683207503683CD1557503683CB190034268CA2716163652171616365CD5671616365CB17505047227505047CD1577505047CB13377315CA27505779237505779CD1587505779CB190179707CA2,90179916CA27505782247505782CD1597505782CB190051960CA2,90052052CA2,90052057CA27500207257500207CD1607500207CB190056836CA2,90056928CA2,90057028CA27500208267500208CD1617500208CB190056736CA27500313277500313CD1627500313CB190206203CA21436493281436493CD1631436493CB13402768CA27501101297501101CD1647501101CB190206165CA2,90206390CA27504972307504972CD1657504972CB17511788317511788CD1667511788CB17504642327504642CD1677504642CB190056744CA2,90056920CA2,90056936CA2,90057004CA2,90057020CA2,90066705CA2,90066729CA27504643337504643CD1687504643CB17504745347504745CD1697504745CB190056744CA2,90056920CA2,90056936CA2,90057004CA2,90057020CA2,90066705CA2,90066729CA27504746357504746CD1707504746CB1

[0436]

4

TABLE 2

Incyte
GenBank ID NO:

Polypeptide
Polypeptide
or PROTEOME
Probability

SEQ ID NO:
ID
ID NO:
Score
Annotation

1
7499453CD1
g8117977
5.8E−191
[Homo sapiens] killer-cell immunoglobulin-like receptor KIR2DL5.1

Vilches, C. et al. (2000) J. Immunol. 164: 5797-5804

2
7499815CD1
g13274520
1.1E−137
[Homo sapiens] complement-c1q tumor necrosis factor-related protein

3
3165346CD1
g7717235
3.5E−193
[Homo sapiens] T-cell receptor alpha chain-c6.1A fusion protein

Thick, J. et al. (1994) Leukemia 8: 564-573

4
5092954CD1
g188479
8.4E−41
[Homo sapiens] HLA-DPB1

Korioth, F. et al. (1992) Tissue Antigens 39: 216-219

5
7499560CD1
g13195239
0.0
[Homo sapiens] complement factor H-related protein 5

McRae, J. L. (2001) J. Biol. Chem. 276: 6747-6754

6
70243658CD1
g179700
1.4E−16
[Homo sapiens] C5a anaphylatoxin receptor

Boulay, F. et al. Expression cloning of a receptor for C5a anaphylatoxin on

differentiated HL-60 cells. Biochemistry 30 (12), 2993-2999 (1991)

334408|C5R1
1.3E−17
[Homo sapiens][Receptor (signalling)][Plasma membrane] C5a chemoattractant

(anaphylatoxin) receptor, a G protein-coupled receptor that mediates anaphylaxis

and the migration and activation of neutrophils and macrophages

7
7500196CD1
g7406952
7.4E−77
[Homo sapiens] 8D6 antigen

Li, L. et al. Identification of a human follicular dendritic cell molecule that

stimulates germinal center B cell growth. J. Exp. Med. 191 (6), 1077-1084 (2000)

476035|8D6A
6.6E−78
[Homo sapiens] Antigen expressed by follicular dendritic cells, stimulates

germinal center B cell growth

608328|425O18-1
4.1E−29
[Mus musculus][Small molecule-binding protein] Protein containing two low-

density lipoprotein receptor class A domains, has a region of high similarity to a

region of very low density lipoprotein receptor (human VLDLR), which may be

associated with susceptibility to Alzheimer's disease

8
7500351CD1
g8249471
3.4E−140
[Homo sapiens] CD1E antigen, isoform 2

Angenieux, C. et al. (2000) J. Biol. Chem. 275 (48), 37757-37764

697353|CD1E
1.0E−115
[Homo sapiens][Golgi; Endosome/Endosomal vesicles; Cytoplasmic]CD1E

antigen, e polypeptide, member of the CD1 family of non classical major

histocompatibility complex class I molecules

347492|CD1A
1.4E−70
[Homo sapiens][Small molecule-binding protein][Plasma membrane]Member of

the CD1 family that is involved in antigen presentation, expressed as a beta 2-

microglobulin-associated heterodimer on cortical thymocytes and T cell

leukemias, associates with CD1b, CD1c and CD8

334518|CD1D
3.8E−70
[Homo sapiens][Ligand][Plasma membrane] Member of the CD1 family that is

involved in antigen presentation, expressed on the cell surface as a beta 2-

microglobulin-associated heterodimer

334514|CD1B
1.6E−64
[Homo sapiens][Small molecule-binding protein][Endosome/Endosomal

vesicles; Cytoplasmic; Plasma membrane]Member of the CD1 family that is

involved in antigen presentation of bacterial lipids and self glycosphingolipids to

T cells, expressed as a beta 2-microglobulin-associated heterodimer on cortical

thymocytes and T cell leukemias, associates with CD1b, CD1c and CD8

334516|CD1C
3.8E−63
[Homo sapiens][Endosome/Endosomal vesicles; Cytoplasmic; Plasma membrane]

Member of the CD1 family that is involved in antigen presentation, expressed as a

beta 2-microglobulin-associated heterodimer on cortical thymocytes and T cell

leukemias, associates with CD1b, CD1c and CD8

9
7500923CD1
g16506269
1.0E−129
[f1][Homo sapiens] FCRLc1

g4973116
2.1E−23
[Mus musculus] high affinity immunoglobulin gamma Fc receptor I (Gavin, A.L.

et al. (2000) Immunogenetics 51 (3), 206-211)

584771|Fcgr1
5.4E−24
[Mus musculus][Receptor (signalling)][Plasma membrane] Fc gamma RI, a

member of the immunoglobulin superfamily and a receptor for the Fc domain of

IgG, binds to immune complexes of IgG with high affinity and is expressed in

cells of the myeloid lineage

618340|FCGR1A
1.8E−22
[Homo sapiens][Receptor (signalling)][Plasma membrane] Fcgamma RI, a

member of the immunoglobulin superfamily that is a receptor for the Fc domain of

IgG and is expressed only in cells of the myeloid lineage, induced by gamma-

interferon (IFN-gamma) and has a role in immune response

10
2258292CD1
g13898390
2.5E−89
[Mus musculus] TARPP Kisielow, J. et al. (2001) Eur. J. Immunol. 31 (4), 1141-

1149

424812|KIAA0029
9.2E−166
[Homo sapiens] Protein containing a R3H domain, which may mediate binding of

ssDNA

11
7500283CD1
g7406952
1.4E−75
[Homo sapiens] 8D6 antigen Li, L. et al. (2000) J. Exp. Med. 191 (6), 1077-1084

476035|8D6A
1.2E−76
[Homo sapiens] Antigen expressed by follicular dendritic cells, stimulates

germinal center B cell growth

608328|425O18-1
1.1E−28
[Mus musculus][Small molecule-binding protein] Protein containing two low-

density lipoprotein receptor class A domains, has a region of high similarity to a

region of very low density lipoprotein receptor (human VLDLR), which may be

associated with susceptibility to Alzheimer's disease

12
7600263CD1
g15590684
8.0E−180
[Homo sapiens] (AY035376) peptidoglycan recognition protein-I-alpha precursor

Liu, C. et al. Peptidoglycan recognition proteins: a novel family of four human

innate immunity pattern recognition molecules. J. Biol. Chem. 276, 34686-34694

(2001)

610930|LOC57115
5.7E−115
[Homo sapiens] Protein has a region of moderate similarity to murine Pglyrp,

which is a cytokine involved in innate immunity and which triggers apoptosis via

an NF-kappaB independent mechanism

341898|PGLYRP
2.8E−42
[Homo sapiens][Receptor (signalling)] Protein with an affinity for peptidoglycans

that plays a role in innate immunity and is expressed mostly in the bone marrow

and spleen

Kang, D. et al. A peptidoglycan recognition protein in innate immunity conserved

from insects to humans. Proc Natl Acad Sci USA 95, 10078-82 (1998).

582443|Pglyrp
2.4E−38
[Mus musculus][Ligand] Cytokine with an affinity for peptidoglycans, involved in

innate immunity and triggers apoptosis via an NF-kappaB independent mechanism

13
7503686CD1
g16580799
3.0E−43
[5′ incom][Mus musculus] Fca/m receptor

g11071950
5.5E−56
[Mus musculus] Fca/m receptor

Shibuya, A. et al. Fcalpha/mu receptor mediates endocytosis of IgM-coated

microbes. Nat. Immunol. 1, 441-446 (2000)

629070|Fcamr
4.8E−57
[Mus musculus][Receptor (signalling)][Plasma membrane] Fcalpha/mu receptor,

binds both IgA and IgM with intermediate or high affinity, expressed on most B

lymphocytes and macrophages, and mediates endocytosis of IgM-coated microbes

Shibuya, A. et al. (supra)

623902|PIGR
8.0E−21
[Homo sapiens][Receptor (protein translocation); Transporter][Plasma membrane]

Polymeric immunoglobulin receptor (transmembrane secretory component),

protein that transports J chain-containing polymeric IgA and pentameric IgM

across the mucosal epithelia into external fluids, may be involved in

pneumococcal invasion

Blanch, V. J. et al. Cutting edge: coordinate regulation of IFN regulatory factor-1

and the polymeric Ig receptor by proinflammatory cytokines. J Immunol 162,

1232-5 (1999).

Piskurich, J. F. et al. Interferon-gamma induces polymeric immunoglobulin

receptor mRNA in human intestinal epithelial cells by a protein synthesis

dependent mechanism. Mol. Immunol. 30, 413-21 (1993).

329908|Pigr
1.0E−20
[Rattus norvegicus][Receptor (protein translocation)] [Extracellular (excluding cell

wall); Plasma membrane] Polymeric immunoglobulin receptor (secretory

component), protein that transports dimeric J chain-containing polymeric IgA and

IgM across the mucosal epithelia into external fluids, may be involved in the

antimicrobial humoral response

14
7504791CD1
g2145066
3.5E−37
[Homo sapiens] cL232ST1/A splice variant

de Baey, A. et al. (1997) Genomics 45: 591-600 Complex expression pattern of

the TNF region gene LST1 through differential regulation, initiation, and

alternative splicing.

568854|LY117
9.8E−17
[Homo sapiens] [Unspecified membrane] Protein that is expressed in leukocytes

and induced by IFN-gamma, possibly functions in the immune response of

monocytes and T cells

325492|Lst1
5.8E−12
[Mus musculus] Protein expressed in monocytes, upregulated by interferon-

gamma, present as many splice variants

15
7504885CD1
g2702314
2.2E−129
[Homo sapiens] Sp alpha

Gebe, J. A. et al. (1997) J. Biol. Chem. 272: 6151-6158

Molecular cloning, mapping to human chromosome 1 q21-q23, and cell binding

characteristics of Sp alpha, a new member of the scavenger receptor cysteine-rich

(SRCR) family of proteins.

342958|CD5L
1.9E−130
[Homo sapiens] [Extracellular (excluding cell wall)] CD5 antigen-like protein (Sp-

alpha), a member of the group B scavenger receptor cysteine-rich family, may

regulate monocyte activation, functions and survival

584265|Api6
5.1E−91
[Mus musculus] [Receptor (protein translocation)] Protein with high similarity to

lymphoid sp alpha (human CD5L), which may be involved in the regulation of

monocyte activation, function, and survival, contains scavenger receptor cysteine

rich domains, which may mediate protein-protein interactions

340878|CD163
2.8E−51
[Homo sapiens] [Receptor (protein translocation); Receptor (signalling)] [Plasma

membrane] Macrophage-associated antigen, putative member of the scavenger

receptor superfamily, which are membrane glycoproteins implicated in the

pathologic deposition of cholesterol in arterial walls

743982|M160
7.2E−49
[Homo sapiens] Member of the scavenger receptor cysteine-rich superfamily,

expressed by cells within the monocyte-macrophage lineage

568386|DMBT1
3.1E−47
[Homo sapiens] [Inhibitor or repressor; Receptor (protein translocation); Receptor

(signalling)] Deleted in malignant brain tumors 1, a member of the scavenger

receptor cysteine-rich superfamily, may function as an opsonin receptor for

surfactant protein D (SFTPD); a potential tumor suppressor protein that may

modulate the immune response to cancer

16
7504915CD1
g183763
6.5E−139
[Homo sapiens] factor H homologue

Estaller, C. et al. (1991) J. Immunol. 146: 3190-3196

Cloning of the 1.4-kb mRNA species of human complement factor H reveals a

novel member of the short consensus repeat family related to the carboxy terminal

of the classical 150-kDa molecule.

623836|HFL1
5.6E−140
[Homo sapiens] [Structural protein] [Extracellular (excluding cell wall)] Protein

with similarity to complement factor H, contains short consensus repeats (SCRs)

335768|HF1
2.0E−112
[Homo sapiens] [Extracellular (excluding cell wall)] Factor H (complement factor

H), a protein with short consensus repeats (SCRs); mutations in the corresponding

gene cause factor H deficiency

Ault, B. H. et al. (1997) J. Biol. Chem. 272: 25168-25175

Human factor H deficiency. Mutations in framework cysteine residues and block

in H protein secretion and intracellular catabolism.

477286|CFHRB
7.0E−78
[Mus musculus] Protein with similarity to complement factor H, contains short

consensus repeats (SCRs) and is a member of the superfamily of C3b/C4b binding

proteins

343468|HFL3
2.6E−71
[Homo sapiens] [Extracellular (excluding cell wall)] H factor (complement)-like

3, a putative complement component that contains short consensus repeats (SCRs)

422126|Cfh
3.9E−68
[Mus musculus] [Inhibitor or repressor] Complement factor H, may function as a

repressor of complement activation, expression is upregulated by dexamethasone

and interferon gamma

17
7504926CD1
g3342533
2.0E−38
[Homo sapiens] peptidoglycan recognition protein precursor

Kang, D. (1998) Proc. Natl. Acad. Sci. U.S.A. 95: 10078-10082

A peptidoglycan recognition protein in innate immunity conserved from insects to

humans.

341898|PGLYRP
1.7E−39
[Homo sapiens] [Receptor (signalling)] Protein with an affinity for peptidoglycans

that plays a role in innate immunity and is expressed mostly in the bone marrow

and spleen

582443|Pglyrp
4.0E−11
[Mus musculus] [Ligand] Cytokine with an affinity for peptidoglycans, involved

in innate immunity and triggers apoptosis via an NF-kappa B independent

18
7505049CD1
g180056
5.0E−132
[Homo sapiens] CD1b antigen precursor

Aruffo, A. and Seed, B. (1989) J. Immunol. 143: 1723-1730 Expression of cDNA

clones encoding the thymocyte antigens CD1a, b, c demonstrates a hierarchy of

exclusion in fibroblasts.

334514|CD1B
4.3E−133
[Homo sapiens] [Small molecule-binding protein] [Endosome/Endosomal

vesicles; Cytoplasmic; Plasma membrane] Member of the CD1 family that is

involved in antigen presentation of bacterial lipids and self glycosphingolipids to

T cells, expressed as a beta 2-microglobulin-associated heterodimer on cortical

thymocytes and T cell leukemias, associates with CD1b, CD1c and CD8

334516|CD1C
1.3E−83
[Homo sapiens] [Endosome/Endosomal vesicles; Cytoplasmic; Plasma membrane

Member of the CD1 family that is involved in antigen presentation, expressed as a

beta 2-microglobulin-associated heterodimer on cortical thymocytes and T cell

leukemias, associates with CD1b, CD1c and CD8

697353|CD1E
1.9E−68
[Homo sapiens] [Golgi; Endosome/Endosomal vesicles; Cytoplasmic] CD1E

antigen, e polypeptide, member of the CD1 family of nonclassical major

histocompatibility complex class I molecules

347492|CD1A
1.4E−63
[Homo sapiens] [Small molecule-binding protein] [Plasma membrane] Member of

the CD1 family that is involved in antigen presentation, expressed as a beta 2-

microglobulin-associated heterodimer on cortical thymocytes and T cell

leukemias, associates with CD1b, CD1c and CD8

334518|CD1D
2.0E−57
[Homo sapiens] [Ligand] [Plasma membrane] Member of the CD1 family that is

involved in antigen presentation, expressed on the cell surface as a beta 2-

microglobulin-associated heterodimer

19
90034212CD1
g16580799
4.0E−43
[5′ incom][Mus musculus] Fca/m receptor

g11071950
4.0E−65
[Mus musculus] Fca/m receptor

Shibuya, A. (2000) Nat. Immunol. 1: 441-446 Fcalpha/mu receptor mediates

endocytosis of IgM-coated microbes.

629070|Fcamr
3.5E−66
[Mus musculus] [Receptor (signalling)] [Plasma membrane] Fc alpha/mu receptor,

binds both IgA and IgM with intermediate or high affinity, expressed on most B

lymphocytes and macrophages, and mediates endocytosis of IgM-coated microbes

623902|PIGR
8.0E−21
[Homo sapiens] [Receptor (protein translocation); Transporter] (Plasma

membrane] Polymeric immunoglobulin receptor (transmembrane secretory

component), protein that transports J chain-containing polymeric IgA and

pentameric IgM across the mucosal epithelia into external fluids, may be involved

in pneumococcal invasion

329908|Pigr
1.0E−20
[Rattus norvegicus] [Receptor (protein translocation)] [Extracellular (excluding

cell wall); Plasma membrane] Polymeric immunoglobulin receptor (secretory

component), protein that transports dimeric J chain-containing polymeric IgA and

IgM across the mucosal epithelia into external fluids, may be involved in the

antimicrobial humoral response

586491|Pigr
1.7E−20
[Mus musculus] [Receptor (protein translocation)] [Plasma membrane] Polymeric

immunoglobulin receptor (transmembrane secretory component), protein that

transports dimeric J chain-containing polymeric IgA and IgM across the mucosal

epithelia into external fluids

342258|TOSO
2.8E−10
[Homo sapiens] [Inhibitor or repressor] Toso, inhibits Fas(TNFRSF6) -mediated

apoptosis in lymphoid cells, may function through the activation of cFLIP,

resulting in the inhibition of caspase-8 (CASP8) activity

20
7503683CD1
g11071950
1.6E−44
[Mus musculus] Fca/m receptor.

Shibuya, A. et al. (2000) (supra)

629070|Fcamr
1.4E−45
[Mus musculus][Receptor (signalling)][Plasma membrane] Fcalpha/mu receptor,

binds both IgA and IgM with intermediate or high affinity, expressed on most B

lymphocytes and macrophages, and mediates endocytosis of IgM-coated

microbes.

Shibuya, A. (2000) Fc alpha/mu receptor mediates endocytosis of IgM-coated

microbes. Nat. Immunol. 1: 441-446.

21
71616365CD1
g14290438
8.1E−116
[Homo sapiens] complement component 1, q subcomponent, beta polypeptide.

623754|C1QB
1.9E−116
[Homo sapiens][Extracellular (excluding cell wall)] B-chain of complement

subcomponent Clq, has a collagen-like region.

Sellar, G. C. et al. (1991) Characterization and organization of the genes encoding

the A-, B- and C-chains of human complement subcomponent C1q. The complete

derived amino acid sequence of human C1q. Biochem. J. 481-490.

22
7505047CD1
g180056
3.0E−130
[Homo sapiens] CD1b antigen precursor.

Aruffo, A. and Seed, B. (1989) Expression of cDNA clones encoding the

thymocyte antigens CD1a, b, c demonstrates a hierarchy of exclusion in

fibroblasts. J. Immunol. 143: 1723-1730.

334514|CD1B
2.6E−131
[Homo sapiens][Small molecule-binding protein][Endosome/Endosomal vesicles;

Cytoplasmic; Plasma membrane] CD1B antigen b polypeptide, binds and presents

lipid and glycolipidantigens to T cells, expressed as a beta 2-microglobulin (B2M)-

associated heterodimer; may play a role in the development of multiple sclerosis

and other autoimmune diseases.

Balk, S. P. et al. (1991) Isolation and expression of cDNA encoding the murine

homologues of CD1. J. Immunol. 146: 768-774.

23
7505779CD1
g313002
5.1E−124
[Homo sapiens] RING 7.

Kelly, A. P. et al. (1991) A new human HLA class II-related locus, DM. Nature

353: 571-573.

335790|HLA-DMB
4.5E−125
[Homosapiens][Chaperones][Lysosome/vacuole; Endosome/Endosomal vesicles;

Cytoplasmic; Plasma membrane] Beta chain of a heterodimer that facilitates the

binding of peptides to MHC class II molecules.

Doebele, R. C. et al. (2000) Determination of the HLA-DM Interaction Site on

HLA-DR Molecules. Immunity 13: 517-527.

24
7505782CD1
g1000997
5.3E−174
[Homo sapiens] N ramp.

Kishi, F. and Nobumoto, M. (1995) Identification of natural resistance-associated

macrophage protein in peripheral blood lymphocytes. Immunol. Lett. 47: 93-96.

346248|SLC11A2
2.7E−93
[Homo sapiens][Active transporter, secondary; Transporter][Unspecified

membrane; Plasma membrane] Iron transporter protein, essential both for normal

intestinal iron absorption and for transport of iron out of endosomes within the

transferrin cycle.

Tabuchi, M. et al. (2000) Human NRAMP2/DMT1, which mediates iron transport

across endosomal membranes, is localized to late endosomes and lysosomes in

HEp-2 cells. J. Biol. Chem. 275: 22220-22228.

25
7500207CD1
g3323609
3.1E−94
[Homo sapiens] KE04p.

343494|KEO4
2.7E−95
[Homo sapiens][Unspecified membrane] Protein containing an SPFH

domain/Band 7 family, which are implicated in regulating targeted turnover of

membrane proteins.

26
7500208CD1
g3323609
1.1E−90
[Homo sapiens] KE04p.

343494|KEO4
9.5E−92
[Homo sapiens][Unspecified membrane] Protein containing an SPFH

domain/Band 7 family, which are implicated in regulating targeted turnover of

membrane proteins.

27
7500313CD1
g21655223
1.0E−146
[fl][Macaca mulatta] (AY094979) CD1e

g8249469
5.9E−147
[Homo sapiens] CD1E antigen, isoform 1.

Angenieux, C. et al. (2000) J. Biol. Chem. 275: 37757-37764.

697353|CD1E
1.1E−115
[Homo sapiens][Golgi; Endosome/Endosomal vesicles; Cytoplasmic] Cluster of

differentiation 1 antigen pombe polypeptide, member of the CD1 family of

nonclassical MHC class I glycoproteins, involved in nonpeptide antigen

processing; distinguished from CD1 group by cell localization and antigen

presentation properties.

Woolfson, A. and Milstein, C. (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 6683-

6687.

334518|CD1D
3.4E−71
[Homo sapiens][Ligand] [Plasma membrane] Member of the CD1 family that is

involved in antigen presentation, expressed on the cell surface as a beta 2-

microglobulin-associated heterodimer.

Jenkinson, H. J. et al. (1999) Immunology 96: 649-655.

28
1436493CD1
g1262852
2.9E−40
[Mus musculus] M17 protein.

Christoph, T. et al. (1994) Int. Immunol. 6: 1203-1211.

322342|Gcet
2.6E−41
[Mus musculus][Small molecule-binding protein][Cytoplasmic] Germinal center

expressed transcript, a putative lipid-binding protein that may be involved in

signal transduction in germinal center B cells.

Christoph, T. et al. (1994) supra.

29
7501101CD1
g8249475
3.6E−172
[Homo sapiens] CD1E antigen, isoform 4.

Angenieux, C. et al. (2000) J. Biol. Chem. 275: 37757-37764.

703661|CD1E
1.1E−140
[Homo sapiens][Golgi; Endosome/Endosomal vesicles; Cytoplasmic] Cluster of

differentiation 1 antigen pombe polypeptide, member of the CD1 family of

nonclassical MHC class I glycoproteins, involved in nonpeptide antigen

processing; distinguished from CD1 group by cell localization and antigen

presentation properties.

Woolfson, A. and Milstein, C. (1994) Proc. Natl. Acad. Sci. U.S.A. 91: 6683-6687

30
7504972CD1
g1127546
2.3E−32
[Homo sapiens] Lst-1 gene product

Holzinger, I. et al. (1995) Cloning and genomic characterization of LST1: a new

gene in the human TNF region. Immunogenetics 42: 315-322.

568854|LY117
1.9E−33
[Homo sapiens][Unspecified membrane] Lymphocyte antigen 117 (leukocyte-

specific transcript 1), cell surface antigen, alternative forms may localize to

various sites, inhibits lymphocyte proliferation, may be involved in immune

response and cellular morphogenesis, induces filopodia formation

de Baey, A. et al. (1997) Complex expression pattern of the TNF region gene

LST1 through differential regulation, initiation, and alternative splicing.

Genomics 45: 591-600.

Rollinger-Holzinger, I. et al. (2000) LST1: a gene with extensive alternative

splicing and immunomodulatory function. J. Immunol. 164: 3169-3176.

Raghunathan, A. et al. (2001) Functional analysis of B144/LST1: a gene in the

tumor necrosis factor cluster that induces formation of long filopodia in eukaryotic

cells. Exp. Cell Res. 268: 230-244.

31
7511788CD1
g1000999
3.9E−227
[Homo sapiens] Nramp

Kishi, F. et al. Identification of natural resistance-associated macrophage protein

in peripheral blood lymphocytes. Immunol. Lett. 47, 93-96 (1995).

618358|SLC11A1
2.6E−224
[Homo sapiens][Transporter][Unspecified membrane; Plasma membrane] Solute

carrier family 11 (proton-coupled divalent metal ion transporters) member 1, a

hydrogen ion-divalent cation antiporter that functions as a cytoskeletal anchoring

protein; mutations in mouse Slc11a1 result in susceptibility to infection.

Goswami, T. et al. Natural-resistance-associated macrophage protein 1 is an

H+/bivalent cation antiporter. Biochem J 354, 511-9. (2001).

609268|Slc11a1
2.1E−196
[Mus musculus][Transporter][Unspecified membrane; Plasma membrane] Solute

carrier family 11 (proton-coupled divalent metal ion transporters) member 1, a

hydrogen ion-divalent cation antiporter that may confer resistance to intracellular

macrophage parasites; mutations result in susceptibility to infection.

Vidal, S. et al. Natural resistance to infection with intracellular parasites:

molecular genetics identifies Nramp1 as the Bcg/Ity/Lsh locus. J Leukoc Biol 58,

382-90 (1995).

32
7504642CD1
g3323609
7.8E−28
[Homo sapiens] KE04p

343494|KEO4
6.6E−29
[Homo sapiens][Unspecified membrane] Protein containing an SPFH

domain/Band 7 family, which are implicated in regulating targeted turnover of

membrane proteins

33
7504643CD1
g3323609
5.1E−95
[Homo sapiens] KE04p

343494|KEO4
4.3E−96
[Homo sapiens][Unspecified membrane] Protein containing an SPFH

domain/Band 7 family, which are implicated in regulating targeted turnover of

membrane proteins

34
7504745CD1
343494|KEO4
6.6E−29
[Homo sapiens] Protein containing an SPFH domain/Band 7 family, which are

implicated in regulating targeted turnover of membrane proteins

35
7504746CD1
343494|KEO4
4.3E−96
[Homo sapiens] Protein containing an SPFH domain/Band 7 family, which are

implicated in regulating targeted turnover of membrane proteins

[0437]

5

TABLE 3

SEQ
Incyte
Amino

Analytical

ID
Polypeptide
Acid

Methods

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

1
7499453CD1
375
Signal_cleavage: M1-T21
SPSCAN

Signal Peptide: M4-T21; M1-T21; M4-D27; M4-Q26
HMMER

Immunoglobulin domain: G42-G97, G137-G195
HMMER_PFAM

Cytosolic domain: C264-I375; Transmembrane
TMHMMER

domain: A241-W263; Non-cytosolic domain: M1-

H240

RECEPTOR CELL NK GLYCOPR PD01652:
BLIMPS_PRODOM

G119-H154, P109-E160, W204-A248, F252-Y298

RECEPTOR NK CELL KILLER PRECURSOR
BLAST_PRODOM

SIGNAL LEUCOCYTE IMMUNOGLOBULIN-

LIKE NATURAL INHIBITORY PD000659:

P109-P277

RECEPTOR NK CELL KILLER NATURAL MHC
BLAST_PRODOM

CLASS I PRECURSOR SIGNAL PD001851:

A278-S338

RECEPTOR NK CELL KILLER INHIBITORY
BLAST_PRODOM

MHC NATURAL CLASS I PRECURSOR

PD002456: G24-I75

RECEPTOR NK MHC CELL NATURAL KILLER
BLAST_PRODOM

INHIBITORY CLASS I PRECURSOR

PD003172: K232-P277

IMMUNOGLOBULIN DM00001|P43627|131-208:
BLAST_DOMO

LI26-W204|P43629|226-303: L126-

W204|P43629|31-105: L31-W106S53115|132-211:

L126-Y202

Potential Phosphorylation Sites: S102 S145 S148
MOTIFS

S200 S225 S230 S265 S315 S353 T92 T133 T186

T190

Potential Glycosylation Sites: N139 N173
MOTIFS

2
7499815CD1
306
Signal_cleavage: M1-C22
SPSCAN

Signal Peptide: M1-C22; M1-D24
HMMER

C1q domain: A179-L302
HMMER_PFAM

Collagen triple helix repeat (20 copies): G114-P173
HMMER_PFAM

C1q domain proteins BL01113: G194-M229,
BLIMPS_BLOCKS

D262-R281, S295-E304, G114-E140

Complement C1Q domain signature PR00007:
BLIMPS_PRINTS

F188-R214, F215-D234, D262-G283, R293-F303

PRECURSOR SIGNAL COLLAGEN ALPHA
BLAST_PRODOM

3IX CHAIN EXTRACELLULAR MATRIX

CONNECTIVE TISSUE PD028299: G105-G171

SIMILAR TO CUTICULAR COLLAGEN
BLAST_PRODOM

PD067228: P115-E175

PRECOLLAGEN P PRECURSOR SIGNAL
BLAST_PRODOM

PD072959: G111-G171

PRECURSOR SIGNAL COLLAGEN REPEAT
BLAST_PRODOM

HYDROXYLATION GLYCOPROTEIN CHAIN

PLASMA EXTRACELLULAR MATRIX

PD002992: N192-L302

C1Q DOMAIN DM00777|P23206|477-673:
BLAST_DOMO

R110-L302|S23297|465-674: R110-L301|P98085|222-

418: G123-K306|P27658|551-743: G111-F303

Potential Phosphorylation Sites: S30 S52 S78 S108
MOTIFS

S198 T33 T47 T72 T137 T212

Potential Glycosylation Sites: F11N70 N71
MOTIFS

3
3165346CDI
408
Mov34/MPN/PAD-1 family: Q7-G149
HMMER_PFAM

C6.1A PROTEIN PROTOONCOGENE
BLAST_PRODOM

CHROMOSOMAL TRANSLOCATION PD004392:

M1-S267

ALPHA; T-CELL; IMMUNOGLOBULIN;
BLAST_DOMO

HISTOCOMPATIBILITY; DM01841|S57494|109-269:

D268-S407|S18893|113-275: D268-S407|S25117|93-267:

D268-S407|S03715|112-269: T261-S407

Potential Phosphorylation Sites: S47 S84 S133
MOTIFS

S232 S285 S333 S360 T29 T46 T56 T62 T103 T112

T166 T255 T293 T312 T315 T402

Potential Glycosylation Sites: F20N265 N300
MOTIFS

N334 N345 N381

4
5092954CD1
157
Signal_cleavage: M1-A31
SPSCAN

Signal Peptide: M2-S29; M1-A31; M1-S29
HMMER

Class II histocompatibility antigen, beta: Y59-D103
HMMER_PFAM

Cytosolic domain: Q28-S157 Transmembrane
TMHMMER

domain: P10-V27 Non-cytosolic domain: M1-G9

Class II histocompatibil PF00969: T12-V54,
BLIMPS_PFAM

G56-Q91, M95-K144, R30-Y64

MHC CLASS II ANTIGEN CHAIN PRECURSOR
BLAST_PRODOM

SIGNAL HISTOCOMPATIBILITY

TRANSMEMBRANE GLYCOPROTEIN

PD009130: M2-I58

MHC II CLASS PRECURSOR SIGNAL CHAIN
BLAST_PRODOM

BETA ANTIGEN HISTOCOMPATIBILITY

TRANSMEMBRANE PD000328: Y59-D103

CLASS II HISTOCOMPATIBILITY ANTIGEN
BLAST_DOMO

DM00134|P04440|4-121: L4-R12I|P15982|7-128:

L4-R121|B60404|7-128: L4-R121|P15983|4-125:

L4-R121

Cell attachment sequence: R121-D123
MOTIFS

Potential Phosphorylation Sites: S90 T50 Y38 Y59
MOTIFS

Potential Glycosylation Sites: N48
MOTIFS

5
7499560CD1
593
Signal_cleavage: M1-G42
SPSCAN

Signal Peptide: M25-T39; M25-G42
HMMER

Sushi domain (SCR repeat): C111-C164, C171-C225,
HMMER_PFAM

C47-C107, C355-C405, C232-C286, C473-

C527, C413-C466, C293-C346

Cytosolic domain: M1-R19 Transmembrane domain:
TMHMMER

L20-G42 Non-cytosolic domain: E43-E593

COMPLEMENT FACTOR H-RELATED PROTEIN
BLAST_PRODOM

PD012214: E170-S231

FACTOR COMPLEMENT PRECURSOR SIGNAL
BLAST_PRODOM

PROTEIN GLYCOPROTEIN REPEAT

SUSHI H-RELATED PLASMA PD004248: C47-F115

COMPLEMENT FACTOR H PRECURSOR
BLAST_PRODOM

ALTERNATE PATHWAY PLASMA

GLYCOPROTEIN REPEAT SUSHI PD020831:

V347-K408

PROTEIN F36H2.3A F36H2.3B PD004794: F373-S522
BLAST_PRODOM

COMPLEMENT FACTOR H REPEAT
BLAST_DOMO

DM00010|I56100|21-88: T45-F113|Q03591|21-86: T45-

C111|G35070|25-91: T45-C111|Q03591|88-144:

F113-T167

Potential Phosphorylation Sites: S152 S188 S198
MOTIFS

S239 S369 T45 T95 T104 T167 T224 T285 T406

T499 T515 Y389 Y472 Y554

Potential Glycosylation Sites: N150 N424
MOTIFS

6
70243658CD1
58
Inorganic pyrophosphatase signature: M1-L41
PROFILESCAN

C5A ANAPHYLATOXIN CHEMOTACTIC
BLAST_PRODOM

RECEPTOR C5AR CD88 ANTIGEN GPROTEIN

COUPLED TRANSMEMBRANE GLYCOPROTEIN

CHEMOTAXIS PD051119: M1-K28

Potential Phosphorylation Sites: T7 T24 T32
MOTIFS

Potential Glycosylation Sites: N5
MOTIFS

7
7500196CD1
162
Signal_cleavage: M1-G30
SPSCAN

Signal Peptide: M6-G28, M6-G30, M6-A35
HMMER

Cytosolic domains: M1-Q8, R134-P162
TMHMMER

Transmembrane domains: V9-L31, V111-L133

Non-cytosolic domain: E32-G110

Leucine zipper pattern: L17-L38
MOTIFS

Potential Phosphorylation Sites: S72 S98
MOTIFS

Potential Glycosylation Sites: N75 N93
MOTIFS

8
7500351CD1
277
Signal_cleavage: M1-N17
SPSCAN

Signal Peptide: M1-A19
HMMER

Immunoglobulin domain: P124-V188
HMMER_PFAM

Cytosolic domain: V225-W277; Transmembrane
TMHMMER

domain: W202-V224; Non-cytosolic domain: M1-

H201

PRECURSOR SIGNAL T-CELL GLYCOPROTEIN
BLAST_PRODOM

SURFACE IMMUNOGLOBULIN FOLD

ANTIGEN TRANSMEMBRANE MULTIGENE

PD004615: P21-K107

IMMUNOGLOBULIN DM00001|P15812|202-285:
BLAST_DOMO

E113-D197

CLASS I HISTOCOMPATIBILITY ANTIGEN
BLAST_DOMO

DM00083|P15812|2-192: P21-S104

IMMUNOGLOBULIN DM00001|P29016|206-289:
BLAST_DOMO

E113-D197

IMMUNOGLOBULIN DM00001|P15813|206-289:
BLAST_DOMO

E113-D197

Potential Phosphorylation Sites: S185 S234 T155
MOTIFS

9
7500923CD1
242
Signal_cleavage: M1-A49
SPSCAN

Signal Peptide: M25-A44, M25-G46, M25-A49
HMMER

Immunoglobulin domain: G68-A125
HMMER_PFAM

Cytosolic domain: M1-K19 Transmembrane domain:
TMHMMER

L20-L42 Non-cytosolic domain: L43-E242

CELL SURFACE GLYCOPROTEIN GP42
BLAST_PRODOM

PRECURSOR SIGNAL GPI ANCHOR MEMBRANE

PD116497: V35-L155

IG-LIKE C2-TYPE DOMAIN
BLAST_DOMO

DM03427|P12314|189-331: F52-G145

IG-LIKE C2-TYPE DOMAIN
BLAST_DOMO

DM03427|I48471|199-336: E50-A150

IG-LIKE C2-TYPE DOMAIN
BLAST_DOMO

DM03427|P26151|198-339: F52-A150

IG-LIKE C2-TYPE DOMAIN
BLAST_DOMO

DM03427|P23505|16-198: E50-L155

Potential Phosphorylation Sites: S6 S183 T17
MOTIFS

T112 T167 T236

10
2258292CD1
1027
R3H domain: Q214-N264
HMMER_PFAM

PROTEIN REPEAT SIGNAL PRECURSOR
BLAST_PRODOM

PRION GLYCOPROTEIN NUCLEAR GPI

ANCHOR BRAIN MAJOR PD001091: G534-P770

Potential Phosphorylation Sites: S18 S54 S74 S86
MOTIFS

S96 S146 S153 S158 S179 S320 S361 S365 S380

S387 S390 S415 S445 S484 S639 S997 T43 T91

T187 T198 T257 T367 T382 T408 T930 T932

Potential Glycosylation Sites: N37 N42 N264
MOTIFS

N541 N711 N868 N995 N1001

11
7500283CD1
162
Signal_cleavage: M1-G30
SPSCAN

Signal Peptide: M6-G28, M6-G30, M6-A35
HMMER

Cytosolic domains: M1-R8, R134-P162
TMHMMER

Transmembrane domains: V9-L31, V111-L133 Non-

cytosolic domain: E32-G110

Leucine zipper pattern: L17-L38
MOTIFS

Potential Phosphorylation Sites: S72 S98
MOTIFS

N75 N93
MOTIFS

12
7600263CD1
339
PROTEIN PEPTIDOGLYCAN RECOGNITION
BLAST_PRODOM

PRECURSOR SIGNAL TUMOR-ASSOCIATED

CSP

Potential Phosphorylation Sites: S23 S133 S149
MOTIFS

S183 T162 Y240

Potential Glycosylation Sites: N111
MOTIFS

13
7503686CD1
265
signal_cleavage: M1-A61
SPSCAN

Signal Peptide: M46-A61
HMMER

Immunoglobulin domain: G120-I200 (E-value = 0.0013)
HMMER_PFAM

IMMUNOGLOBULIN DM00001: P01833|41-120:
BLAST_DOMO

H128-G201; P15083|41-120: H128-F208;

P01832|28-125: G120-G201; S48841|41-120:

H128-G201

Potential Phosphorylation Sites: S39 S108 S189
MOTIFS

S251 T6 T38 T88 Y24

Potential Glycosylation Sites: F89N212
MOTIFS

14
7504791CD1
82
Signal_cleavage: M1-C32
SPSCAN

Signal Peptide: M1-L34
HMMER

Cytosolic domain: W33-T82; Transmembrane
TMHMMER

domain: I10-C32; Non-cytosolic domain: M1-C9

LST1 (leukocyte-specific transcript 1) PD014831:
BLAST_PRODOM

R46-T82

Potential Phosphorylation Sites: S3 S44 T65 Y11
MOTIFS

Leucine zipper pattern: L20-L41
MOTIFS

15
7504885CD1
240
Signal_cleavage: M1-T13
SPSCAN

Signal Peptide: M1-L18, M1-P20
HMMER

Scavenger receptor cysteine-rich domain:
HMMER_PFAM

V140-S239, A34-E132

Speract receptor repeat proteins domain proteins
BLIMPS_BLOCKS

BL00420: C228-C238, D35-Y89

Speract (scavenger) receptor repeated domain
PROFILESCAN

signature: N122-W202, D19-W95

Speract receptor signature PR00258: V31-K47,
BLIMPS_PRINTS

G156-G167, A65-G75, D204-C218, D227-S239

ANTIGEN PRECURSOR SIGNAL M130
BLAST_PRODOM

TRANSMEMBRANE GLYCOPROTEIN REPEAT

VARIANT CYTOPLASMIC PROTEIN PD000767:

V140-S239, G36-C131

Precursor Signal Receptor Peptid Sperm-activating
BLAST_PRODOM

Speract Repeat glycoprotein I-crosslinked

C06B8.7 PD002499: D136-H220, S25-P134

SPERACT RECEPTOR AMINO-TERMINAL DM00148
BLAST_DOMO

P30205|1145-1256: T127-G240, E29-C131;

JC4361|452-565: L137-S239, E21-C131; P30205|926-

1031: D133-S239, V31-D133; P30205|371-476:

D133-S239, V31-D133

Potential Phosphorylation Sites: S61 S102 S147
MOTIFS

S160 S187 S209 T80 T107 T127 T229

Speract receptor repeated domain signature: G142-G179
MOTIFS

16
7504915CD1
265
Signal_cleavage: M1-S15
SPSCAN

Signal Peptide: M1-G18
HMMER

Sushi domain (SCR repeat): C143-C197, C82-C136,
HMMER_PFAM

C22-C75, C201-C262

FACTOR PRECURSOR SIGNAL COMPLEMENT
BLAST_PRODOM

GLYCOPROTEIN REPEAT SUSHI

PROTEIN PLASMA H-RELATED PD004223:

L198-C262

COMPLEMENT REGULATORY PLASMA
BLAST_PRODOM

PROTEIN PD101668: C49-W191

COMPLEMENT FACTOR H REPEAT DM00010
BLAST_DOMO

I56100|207-267: K142-I203; Q03591|207-267:

K142-I203; I56100|144-205: D79-

G141; Q03591|146-205: S81-G141

Potential Phosphorylation Sites: S63 S113 S166
MOTIFS

S242 T38 T140 T185 T218 T247 T251

Potential Glycosylation Sites: N61 N129
MOTIFS

17
7504926CD1
77
Signal_cleavage: M1-A21
SPSCAN

Signal Peptide: M6-A21, M6-E23, M1-A21, M1-T24,
HMMER

M1-C30, M1-E23

Potential Phosphorylation Sites: S2
MOTIFS

18
7505049CD1
278
Signal_cleavage: M1-S18
SPSCAN

Signal Peptide: M1-S18
HMMER

Cytosolic domain: M269-P278; Transmembrane
TMHMMER

domain: I246-Y268; Non-cytosolic domain: M1-

S245

PRECURSOR SIGNAL T-CELL GLYCOPROTEIN
BLAST_PRODOM

SURFACE IMMUNOGLOBULIN FOLD

ANTIGEN TRANSMEMBRANE MULTIGENE

PD004615: P14-Q200

CLASS I HISTOCOMPATIBILITY ANTIGEN DM00083
BLAST_DOMO

P29016|2-196: L2-A197; S47246|2-196: L2-A197;

P29017|2-197: L2-K196; P06126|2-195: L2-

Potential Phosphorylation Sites: S77 S143 S273 T175
MOTIFS

Potential Glycosylation Sites: N38 N75 N146
MOTIFS

19
90034212CD1
308
Signal_cleavage: M1-A61
SPSCAN

Signal Peptide: M46-A61
HMMER

IMMUNOGLOBULIN DM00001
BLAST_DOMO

P01833|41-120: H128-G201; P15083|41-120:

H128-F208; P01832|28-125: G120-G201; S48841|41-

120: H128-G201

Potential Phosphorylation Sites: S39 S108 S189
MOTIFS

S251 T6 T38 T88 T300 Y24

Potential Glycosylation Sites: N212
MOTIFS

20
7503683CD1
184
Signal_cleavage: M1-A61
SPSCAN

Signal Peptide: M46-A61, M46-P63
HMMER

IMMUNOGLOBULIN DM00001
BLAST_DOMO

|P01833|41-120: H128-V183; P01832|28-125:

G120-T184; S48841|41-120: H128-V183

Potential Phosphorylation Sites: S39 S108 T6 T38 T88 Y24
MOTIFS

21
71616365CD1
226
Signal_cleavage: M1-A27
SPSCAN

Signal Peptide: M3-I24, M3-A27, M3-L29, M1-A27
HMMER

C1q domain: A96-L220
HMMER_PFAM

Collagen triple helix repeat (20 copies): G33-T92
HMMER_PFAM

Cytosolic domain: M1-K4; Transmembrane domain:
TMHMMER

I5-A27; Non-cytosolic domain: Q28-A226

C1q domain proteins BL01113: A36-K62, T112-A147,
BLIMPS_BLOCKS

Q179-Q198, S213-P222

Complement C1Q domain signature PR00007:
BLIMPS_PRINTS

P106-K132, F133-N152, Q179-T200, A211-F221

PRECURSOR SIGNAL COLLAGEN REPEAT
BLAST_PRODOM

HYDROXYLATION GLYCOPROTEIN CHAIN

PLASMA EXTRACELLULAR MATRIX

PD002992: A96-L220

COLLAGEN ALPHA PRECURSOR CHAIN
BLAST_PRODOM

REPEAT SIGNAL CONNECTIVE TISSUE

EXTRACELLULAR MATRIX PD000007: G33-D88

PROCOLLAGEN ALPHA 3IV CHAIN PRECURSOR
BLAST_PRODOM

EXTRACELLULAR MATRIX CONNECTIVE TISSUE

REPEAT HYDROXYLATION GLYCOPROTEIN

BASEMENT MEMBRANE COLLAGEN SIGNAL CELL

ADHESION ALTERNATIVE SPLICING

POLYMORP PD051097: P34-P82

C1Q DOMAIN DM00777
BLAST_DOMO

P02746|70-250: G45-A226; S49158|70-253: G45-E225;

Q02105|71-245: G45-D223; P02747|104-

244: P80-D223

C1q domain signature: F115-Y145
MOTIFS

Potential Phosphorylation Sites: S130 S148 T92
MOTIFS

T112 T134 T169 T200

22
7505047CD1
240
Signal_cleavage: M1-S18
SPSCAN

Signal Peptide: M1-S18
HMMER

Cytosolic domain: M231-P240; Transmembrane
TMHMMER

domain: I208-Y230; Non-cytosolic domain: M1-S207

IG domain P124-V188
HMMER PFAM

Immunoglobulins L143-Q150, L184-Y201
BLIMPS_BLOCKS

PRECURSOR SIGNAL T CELL GLYCOPROTEIN
BLAST_PRODOM

SURFACE IMMUNOGLOBULIN FOLD

ANTIGEN TRANSMEMBRANE MULTIGENE

PD004615: P14-G119

CLASS I HISTOCOMPATIBILITY ANTIGEN DM00083
BLAST_DOMO

P29016|2-196: L2-K109; S47246|2-196: L2-K109

IMMUNOGLOBULIN DM00001
BLAST_DOMO

P29016|206-289: E113-D197; P15812|202-285: E113-D197

Potential Phosphorylation Sites: S77 S185 S191 S235
MOTIFS

Potential Glycosylation Sites: F93N38 N75 N165
MOTIFS

23
7505779CD1
224
Signal_cleavage: M1-G17
SPSCAN

Signal_Peptide: M1-G17, M1-G19, M1-G20, M1-A18, M1-A23
HMMER

Class II histocompatibility antigen, beta: E26-T109
HMMER_PFAM

Immunoglobulin domain: R128-V194
HMMER_PFAM

Immunoglobulins and major histocompatibility
BLIMPS_BLOCKS

complex proteins BL00290: M132-K154, Y190-W207

Immunoglobulins and major histocompatibility
PROFILESCAN

complex proteins signature: D171-W221

MHC CLASS II HISTOCOMPATIBILITY LOCUS
BLAST_PRODOM

ANTIGEN PRECURSOR SIGNAL CHAIN

BETA PD002846: C15-N110

MHC CLASS PRECURSOR SIGNAL ANTIGEN
BLAST_PRODOM

I CHAIN HISTOCOMPATIBILITY

GLYCOPROTEIN TRANSMEMBRANE PD000014:

R111-W207

COMPLEX; HISTOCOMPATIBILITY; MAJOR;
BLAST_DOMO

IMMUNOGLOBULIN; DM08805

P28068|1-114: M1-P115; P35737|1-114: L7-P115

IMMUNOGLOBULIN DM00001
BLAST_DOMO

|P28068|116-202: S116-I203; P35737|116-201: S116-P202

Immunoglobulins and major histocompatibility
MOTIFS

complex proteins signature: Y190-H196

Potential Phosphorylation Sites: S185 T36 T52 T109 T148
MOTIFS

Potential Glycosylation Sites: F170N110 N216
MOTIFS

24
7505782CD1
330
Natural resistance-associated macrophage pro: A84-L243
HMMER_PFAM

Cytosolic domains: G152-R180, M236-N241, T297-G330
TMHMMER

Transmembrane domains: G129-A151, V181-F198,

V213-L235, G242-V264, P274-W296

Non-cytosolic domains: M1-G128, R199-N212, V265-H273

Natural resistance-associated macrophage protein
BLIMPS_PRINTS

signature PR00447: G152-L171, R180-V197,

N212-S231

PROTEIN TRANSPORT TRANSMEMBRANE
BLAST_PRODOM

NATURAL MACROPHAGE RESISTANCE-

ASSOCIATED GLYCOPROTEIN N-RAMP

TRANSPORTER RESISTANCE PD001861: A97-

M167

NATURAL MACROPHAGE PROTEIN RESISTANCE-
BLAST_PRODOM

ASSOCIATED N-RAMP TRANSPORT

TRANSMEMBRANE GLYCOPROTEIN RESISTANCE

ASSOCIATED PD005040: L244-E321

NATURAL RESISTANCE-ASSOCIATED MACROPHAGE
BLAST_PRODOM

PROTEIN N-RAMP TRANSPORT

TRANSMEMBRANE GLYCOPROTEIN POLYMORPHISM

PD009944: M1-F53

PROTEIN TRANSPORT TRANSMEMBRANE
BLAST_PRODOM

NATURAL MACROPHAGE RESISTANCE-

ASSOCIATED GLYCOPROTEIN-RAMP

RESISTANCE ASSOCIATED PD002480: E168-L243

RESISTANCE; MALVOLIO; MACROPHAGE;
BLAST_DOMO

NATURAL; DM01594

P49279|49-492: M68-P272; I48693|46-489:

M68-H273; P51027|54-497: L82-L271; P49282|61-503:

A84-L271

Potential Phosphorylation Sites: S40 S54 S106
MOTIFS

Potential Glycosylation Sites: N104 N118
MOTIFS

25
7500207CD1
198
Signal_cleavage: M1-A23
SPSCAN

Signal Peptide: M3-A23, M1-A23, M3-K27
HMMER

Cytosolic domain: M1-A6
TMHMMER

Transmembrane domain: R7-H26

Non-cytosolic domain: K27-G198

Ribosomal protein L33 signature: A104-P154
PROFILESCAN

C42C1.9 PROTEIN KE04P PD156143: K27-F149,
BLAST_PRODOM

S24-R38

KE04P PD182878: G150-G198
BLAST_PRODOM

Potential Phosphorylation Sites: S95 S123 T61 T90
MOTIFS

T171 Y114

Potential Glycosylation Sites: N2
MOTIFS

26
7500208CD1
245
Signal_cleavage: M1-A66
SPSCAN

Signal Peptide: M3-A23, M1-A23, M3-K27
HMMER

Cytosolic domain: M1-A6; Transmembrane domain:
TMHMMER

R7-H26; Non-cytosolic domain: K27-G245

Ribosomal protein L33 signature: A151-P201
PROFILESCAN

C42C1.9 PROTEIN KE04P PD156143: V64-F196,
BLAST_PRODOM

S24-T70

KE04P PD182878: G197-G245
BLAST_PRODOM

S142 S170 T60 T108 T137 T218 Y161
MOTIFS

Potential Glycosylation Sites: F196N2
MOTIFS

27
7500313CD1
289
Signal_cleavage: M1-N17
SPSCAN

Signal Peptide: M1-A19
HMMER

Immunoglobulin domain: P124-V188
HMMER_PFAM

Cytosolic domain: D227-W289; Transmembrane
TMHMMER

domain: G204-V226; Non-cytosolic domain: M1-

G203

PRECURSOR SIGNAL T CELL GLYCOPROTEIN
BLAST_PRODOM

SURFACE IMMUNOGLOBULIN FOLD

ANTIGEN TRANSMEMBRANE MULTIGENE

PD004615: P21-K107

IMMUNOGLOBULIN DM00001
BLAST_DOMO

P15812|202-285: E113-D197; P29016|206-289:

E113-D197; P15813|206-289: E113-D197

CLASS I HISTOCOMPATIBILITY ANTIGEN
BLAST_DOMO

DM00083|P15812|2-192: P21-S104

Potential Phosphorylation Sites: D232S185 S234
MOTIFS

S235 T155

28
1436493CD1
178
GERMINAL CENTER EXPRESSED TRANSCRIPT
BLAST_PRODOM

M17 PROTEIN PD093901: Q29-H177

Potential Phosphorylation Sites: S26 S60 S102 S143
MOTIFS

T31 T79 T124 Y148

29
7501101CD1
333
Signal_cleavage: M1-L24
SPSCAN

Signal Peptide: M1-A19
HMMER

Cytosolic domain: D271-W333; Transmembrane
TMHMMER

domain: G248-V270; Non-cytosolic domain: M1-

G247

PRECURSOR SIGNAL T CELL GLYCOPROTEIN
BLAST_PRODOM

SURFACE IMMUNOGLOBULIN FOLD

ANTIGEN TRANSMEMBRANE MULTIGENE

PD004615: A30-K206

CLASS I HISTOCOMPATIBILITY ANTIGEN DM00083
BLAST_DOMO

D236P15812|2-192: L2-S203; P29017|2-197:

A30-E202; P29016|2-196: S26-S203; P06126|2-195:

E32-S203

Potential Phosphorylation Sites: S36 S86 S278 S279 T73
MOTIFS

Potential Glycosylation Sites: N47 N84
MOTIFS

30
7504972CD1
116
Sushi domain proteins (SCR repeat)
BLIMPS_PFAM

PF00084: W64-C73

B144 ISOFORM LST1 SPECIFIC LEUKOCYTE
BLAST_PRODOM

TRANSCRIPT LST-1

PD014831: E79-T116

LST-1 LST1 ISOFORM
BLAST_PRODOM

PD026827: 149-E79

Potential Phosphorylation Sites: F240S46 T99
MOTIFS

31
7511788CD1
427
Natural resistance-associated macrophage protein:
HMMER_PFAM

I78-K265, G266-L340

NRAMP family Mn2+/Fe2+ transporters: R56-M333
HMMER_TIGRFAM

Cytosolic domains: M1-K57, C106-R167,
TMHMMER

A217-R277, M333-N338, T394-G427

Transmembrane domains: L58-L73, Q83-L105,

I168-L187, A197-V216, V278-F295, V310-L332,

G339-V361, P371-W393

Non-cytosolic domains: D74-L82, D188-E196,

R296-N309, V362-H370

Natural resistance-associated macrophage protein
BLIMPS_PRINTS

signature PR00447: L137-L163, R167-F186, L192

E213, A244-F267, N309-S328

PROTEIN TRANSPORT TRANSMEMBRANE
BLAST_PRODOM

NATURAL MACROPHAGE

RESISTANCEASSOCIATED GLYCOPROTEIN

NRAMP TRANSPORTER RESISTANCE

PD001861: S54-K265

NATURAL MACROPHAGE PROTEIN RESISTANCE
BLAST_PRODOM

ASSOCIATED NRAMP TRANSPORT

TRANSMEMBRANE GLYCOPROTEIN RESISTANCE

ASSOCIATED PD005040: L341-E418

NATURAL RESISTANCEASSOCIATED MACROPHAGE
BLAST_PRODOM

PROTEIN NRAMP TRANSPORT

TRANSMEMBRANE GLYCOPROTEIN POLYMORPHISM

PD009944: M1-F53

PROTEIN TRANSPORT TRANSMEMBRANE
BLAST_PRODOM

NATURAL MACROPHAGE

RESISTANCEASSOCIATED GLYCOPROTEIN

NRAMP RESISTANCE ASSOCIATED

PD002480: K265-L340

NATURAL RESISTANCE, MACROPHAGE,
BLAST_DOMO

MALVOLIO DM01594

I48693|46-489: G51-K265 G235-H370;

P49282|61-503: F53-K265 G235-L368; P51027|54-497:

P50-F295 G235-L368; P49279|49-492: K49-K265

G235-P369

Potential Phosphorylation Sites: S40 S54 T117 T177
MOTIFS

32
7504642CD1
81
Signal_cleavage: M1-A23
SPSCAN

Signal Peptide: M1-1A23, M3-A23, M3-K27
HMMER

Cytosolic domains: M1-A6, V64-K81; Transmembrane
TMHMMER

domains: R7-H26, A41-S63; D258 Non-

cytosolic domain: K27-G40

C42C1.9 PROTEIN KE04P PD156143: S24-Q65
BLAST_PRODOM

Potential Phosphorylation Sites: S79 T60
MOTIFS

Potential Glycosylation Sites: F222N2
MOTIFS

33
7504643CD1
209
Signal_cleavage: M1-A23
SPSCAN

Signal Peptide: M1-A23, M3-A23, M3-K27
HMMER

Cytosolic domain: M1-A6; Transmembrane domain:
TMHMMER

R7-H26; Non-cytosolic domain: K27-N209

Prohibitin homologues domain: A23-S189
HMMER_SMART

C42C1.9 PROTEIN KE04P PD156143: S24-E191
BLAST_PRODOM

Protein secE/sec61-gamma signature: M161-S189
MOTIFS

Potential Phosphorylation Sites: F270S189 S195 T60 T134
MOTIFS

Potential Glycosylation Sites: N2 N108
MOTIFS

34
7504745CD1
81
Signal_cleavage: M1-A23
SPSCAN

Signal Peptide: M3-A23
HMMER

Signal Peptide: M1-A23
HMMER

Signal Peptide: M3-K27
HMMER

Cytosolic domains: M1-A6, V64-K81; Transmembrane
TMHMMER

domains: R7-H26, A41-S63; Non-cytosolic

domain: K27-G40

G-protein alpha subunit PR00442: K27-Y36
BLIMPS_PRINTS

Potential Phosphorylation Sites: S79 T60
MOTIFS

Potential Glycosylation Sites: F247N2
MOTIFS

35
7504746CD1
209
Signal_cleavage: M1-A23
SPSCAN

Signal Peptide: M3-A23
HMMER

Signal Peptide: M1-A23
HMMER

Signal Peptide: M3-K27
HMMER

prohibitin homologues: A23-S189
HMMER_SMRT

Cytosolic domain: M1-A6; Transmembrane domain:
TMHMMER

R7-H26; Non-cytosolic domain: K27-N209

G-protein alpha subunit PR00442: K27-Y36
BLIMPS_PRINTS

Protein secE/sec61-gamma signature: M161-S189
MOTIFS

Potential Phosphorylation Sites: S189 S195 T60 T134
MOTIFS

Potential Glycosylation Sites: N2 N108
MOTIFS

[0438]

6

TABLE 4

Polynucleotide

SEQ ID NO:/

Incyte ID/Sequence

Length
Sequence Fragments

36/7499453CB1/1596
1-1596, 255-766, 255-775, 255-776, 448-991

37/7499815CB1/1468
1-72, 1-149, 1-240, 3-309, 3-329, 181-333, 331-669, 331-725, 331-981, 334-705, 334-719, 348-618, 367-899, 373-

989, 378-1065, 397-1075, 405-861, 442-995, 444-737, 455-923, 479-1089, 488-986, 500-1020, 513-867, 517-841,

529-1176, 543-1197, 547-1073, 548-970, 554-1132, 557-733, 557-895, 563-1226, 599-734, 605-1089, 617-893, 617-

999, 636-1195, 637-887, 639-1138, 647-1268, 663-1130, 675-1336, 681-1058, 684-1280, 689-1193, 698-1148, 721-

1127, 742-1406, 758-1367, 762-1035, 776-1146, 781-1145, 787-1228, 793-1280, 818-1403, 820-1300, 822-1426,

839-1283, 866-1332, 879-1468, 882-1415, 897-1466, 909-1173, 941-1038

38/3165346CB1/1954
1-648, 78-218, 88-347, 90-218, 93-643, 98-202, 99-648, 237-1874, 291-382, 291-526, 329-501, 906-1029, 906-

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1078-1335, 1102-1246, 1122-1353, 1122-1872, 1127-1311, 1148-1427, 1168-1391, 1169-1399, 1191-1523, 1191-

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39/5092954CB1/1169
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40/7499560CB1/2830
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41/70243658CB1/685
1-193, 56-191, 56-193, 57-190, 58-193, 60-183, 60-685, 81-193, 126-193

42/7500196CB1/891
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43/7500351CB1/1049
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44/7500923CB1/1881
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947-1341,

958-1236, 963-1522, 972-1405, 974-1443, 977-1512, 977-1522, 980-1522, 989-1512, 990-1340, 990-1405, 992-

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1752, 1656-1723

45/2258292CB1/3829
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3554

46/7500283CB1/925
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606, 508-785, 687-925, 689-925

47/7600263CB1/1474
1-463, 1-1474, 396-1034, 705-808, 1176-1279

48/7503686CB1/1489
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789, 64-791, 64-795, 64-852, 64-883, 64-884, 64-885, 64-892, 64-894, 64-895, 64-913, 64-949, 64-954, 64-972, 64-

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1447, 631-1447, 633-1447, 639-1447, 660-1442, 693-1446, 736-882, 945-1447, 1031-1450, 1048-1219, 1215-1408

49/7504791CB1/672
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388-526, 389-525

50/7504885CB1/1567
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659, 491-1139, 516-1043, 523-1114, 528-1241, 536-825, 546-1343, 561-1230, 563-814, 594-1186, 603-1169, 620-

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51/7504915CB1/1136
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794, 395-653, 398-649, 399-958, 403-639, 406-943, 407-735, 408-685, 411-840, 415-682, 417-1010, 420-530, 422-

703, 424-683, 425-651, 425-694, 425-705, 428-671, 428-1083, 430-663, 431-705, 432-920, 437-1075, 439-711, 441-

693, 441-699, 451-976, 453-752, 455-1084, 460-702, 460-1056, 463-644, 463-746, 466-737, 467-715, 467-756, 469-

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1108, 607-

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1096, 894-1126, 899-1126, 899-1127, 911-1124, 913-1136, 914-1111, 930-1112, 947-1126, 966-1077, 969-1097,

1004-1126

52/7504926CB1/364
1-241, 28-358, 115-364

53/7505049CB1/1546
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54/90034212CB1/1376
1-850, 597-1376

55/7503683CB1/998
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56/71616365CB1/1061
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786, 568-823, 568-862, 569-1026, 571-991, 575-842, 582-817, 599-888, 602-837, 602-876, 605-825, 605-829, 606-

810, 609-983, 610-896, 625-844, 625-911, 627-861, 643-991, 658-885, 671-923, 701-922, 737-1008, 747-934, 773-

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57/7505047CB1/1435
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330, 138-668, 146-409, 146-415, 146-422, 151-397, 153-408, 160-677, 179-665, 240-792, 285-854, 285-914, 296-

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58/7505779CB1/1540
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1-375, 1-381, 1-387, 1-394, 1-405, 1-408, 1-411, 1-414, 1-440, 1-445, 1-456, 1-457, 1-464, 1-476, 1-488, 1-502, 1-

513, 1-539, 1-543, 1-548, 1-555, 1-624, 1-693, 1-1098, 2-427, 2-488, 3-446, 7-171, 7-446, 13-282, 23-275, 23-279,

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589, 159-589, 159-652, 163-462, 165-476, 174-436, 188-482, 189-490, 222-431, 223-463, 223-506, 223-547, 238-

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643, 384-678, 384-710, 395-682, 396-710, 400-577, 408-692, 421-705, 425-656, 425-696, 426-705, 448-710, 448-

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730-1098, 734-1109, 736-

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59/7505782CB1/1717
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60/7500207CB1/2730
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1124, 866-1319, 913-1371, 915-1156, 922-1178, 976-1143, 991-1112, 1001-1208, 1007-1522, 1025-1278, 1025-

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1573-2181, 1573-2243, 1591-2328, 1593-2091, 1601-1865, 1613-1806, 1613-2331, 1615-2329, 1616-2328, 1627-

1895, 1629-1819, 1629-1896, 1636-

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2668, 1806-2077, 1808-2643, 1811-2070, 1831-2311, 1837-2070, 1860-2386, 1870-2543, 1886-2640, 1891-2240,

1930-2141, 1930-2142, 1930-2173, 1935-2189, 1935-2219, 1936-2196, 1939-2199, 1939-2465, 1939-2689, 1941-

2195, 1941-2245, 1943-2371, 1946-2663, 1947-2190, 1947-2459, 1960-2265, 1965-2305, 1980-2226, 2014-2681,

2026-2467, 2029-2292, 2031-2261, 2042-2295, 2068-2249, 2069-2691, 2075-2257, 2075-2583, 2084-2632, 2108-

2667, 2117-2620, 2118-2704, 2152-2688, 2158-2723, 2160-2730, 2163-2707, 2164-2730, 2178-2687, 2185-2730,

2188-2730, 2195-2431, 2195-2700, 2221-2715, 2234-2516, 2241-2712, 2248-2720, 2250-2717, 2254-2730, 2257-

2428, 2257-2730, 2263-2715, 2263-2729, 2263-

2730, 2266-2719, 2274-2718, 2275-2714, 2278-2720, 2279-2688, 2280-2678, 2283-2715, 2284-2719, 2287-2730,

2288-2573, 2288-2729, 2293-2715, 2295-2664, 2297-2715, 2300-2715, 2303-2729, 2305-2455, 2307-2720, 2309-

2715, 2314-2730, 2317-2532, 2319-2682, 2323-2715, 2326-2715, 2329-2715, 2330-2715, 2335-2530, 2346-2719,

2347-2712, 2347-2715, 2350-2716, 2352-2679, 2360-2715, 2361-2715, 2365-2715, 2376-2715, 2386-2730, 2387-

2718, 2431-2715, 2433-2715, 2458-2710, 2475-2715, 2493-2617, 2570-2728, 2602-2730

61/7500208CB1/2871
1-279, 1-2857, 1-2871, 10-132, 11-279, 14-279, 15-279, 19-279, 20-424, 61-279, 63-781, 122-1031, 139-257, 277-

1031, 293-565, 293-871, 300-1146, 308-1112, 409-943, 458-1142, 460-664, 463-1053, 463-1054, 499-1121, 526-

1052, 615-1377, 623-1031, 664-1237, 680-1143, 724-1145, 739-1166, 743-1145, 762-1185, 796-1062, 818-1430,

832-1092, 839-933, 858-1145, 861-1297, 873-1466, 929-1169, 938-1179, 962-1413, 1007-1265, 1007-1460, 1054-

1512, 1056-1297, 1063-1319, 1117-1284, 1132-1253, 1142-1349, 1148-1663, 1166-1419, 1166-1484, 1219-1551,

1228-1780, 1276-1478, 1276-1542, 1302-1538, 1322-1868, 1332-1884, 1386-1891, 1417-1924, 1440-1608, 1466-

2150, 1484-1961, 1504-1938, 1510-1996, 1522-1780, 1552-2459, 1591-2146, 1604-2051, 1614-2245, 1630-2455,

1634-2269, 1659-2469, 1660-2472, 1683-1926, 1683-1948, 1690-1940, 1690-2072, 1695-2244, 1698-2009, 1714-

2322, 1714-2384, 1732-2469, 1734-

2232, 1742-2006, 1754-1947, 1754-2472, 1756-2470, 1757-2469, 1768-2036, 1770-1960, 1770-2037, 1777-2009,

1782-2244, 1790-2469, 1795-2469, 1799-2269, 1800-2468, 1803-2060, 1804-2276, 1804-2634, 1808-2446, 1821-

2086, 1835-1939, 1835-2106, 1852-2471, 1855-2266, 1855-2368, 1860-2388, 1877-2170, 1894-2401, 1898-2110,

1929-2125, 1931-2809, 1947-2218, 1949-2784, 1952-2211, 1972-2452, 1978-2211, 2001-2527, 2011-2684, 2027-

2781, 2032-2381, 2071-2282, 2071-2283, 2071-2314, 2076-2330, 2076-2360, 2077-2337, 2080-2340, 2080-2606,

2080-2830, 2082-2336, 2082-2386, 2084-2512, 2087-2514, 2087-2804, 2088-2331, 2088-2600, 2101-2406, 2106-

2446, 2121-2367, 2129-2501, 2155-2822, 2167-2608, 2170-2433, 2172-2402, 2183-2436, 2209-2390, 2210-2832,

2216-2398, 2216-2724, 2225-2773, 2249-2808, 2258-2761, 2259-2845, 2293-2829, 2299-2864, 2301-2871, 2304-

2848, 2305-2871, 2319-2828, 2326-2871, 2329-

2871, 2336-2572, 2336-2841, 2346-2507, 2362-2856, 2375-2657, 2382-2853, 2389-2861, 2391-2858, 2395-2871,

2398-2569, 2398-2871, 2404-2856, 2404-2870, 2404-2871, 2407-2860, 2415-2859, 2416-2855, 2419-2861, 2420-

2829, 2421-2819, 2424-2856, 2425-2860, 2428-2871, 2429-2714, 2429-2870, 2434-2856, 2436-2805, 2438-2856,

2441-2856, 2444-2870, 2446-2596, 2448-2861, 2450-2856, 2455-2871, 2458-2673, 2460-2823, 2464-2856, 2467-

2856, 2470-2856, 2471-2856, 2476-2671, 2487-2860, 2488-2853, 2488-2856, 2491-2857, 2493-2820, 2496-2856,

2501-2856, 2502-2856, 2506-2856, 2517-2856, 2527-2871, 2528-2859, 2572-2856, 2574-2856, 2599-2851, 2616-

2856, 2634-2758, 2711-2869, 2725-2853, 2743-2871

62/7500313CB1/1844
1-1465, 21-443, 31-481, 162-666, 164-873, 176-696, 180-752, 190-713, 191-731, 198-856, 205-818, 212-754, 219-

729, 231-476, 242-536, 253-820, 257-751, 264-807, 268-517, 269-546, 270-883, 307-483, 310-439, 310-546, 313-

907, 322-563, 334-840, 334-982, 337-856, 337-864, 348-619, 348-962, 353-923, 354-897, 360-997, 362-622, 370-

619, 386-939, 398-604, 401-656, 415-804, 417-703, 437-680, 448-1089, 450-1001, 454-1004, 456-629, 464-590,

492-1197, 507-1124, 515-1013, 529-839, 529-875, 546-836, 546-839, 564-808, 583-875, 592-1132, 595-H50, 607-

1150, 608-806, 618-1254, 657-1341, 665-1165, 666-1222, 714-1131, 726-1129, 728-1381, 742-1076, 798-1342, 813-

1301, 830-1113, 837-1603, 839-1247, 848-1465, 851-1084, 877-1171, 891-1129, 900-1066, 954-1191, 954-1207,

954-1222, 959-1389, 970-1237, 995-1248, 1031-1205, 1051-1279, 1067-1596, 1149-1332, 1163-1414, 1171-1844,

1313-1449

63/1436493CB1/732
1-622, 11-585, 17-732, 32-285, 32-719, 74-609, 92-732, 201-459

64/7501101CB1/1974
1-291, 1-434, 1-450, 1-454, 1-475, 1-499, 1-1595, 3-248, 12-253, 23-291, 28-247, 32-391, 46-456, 70-744, 118-416,

119-641, 120-635, 125-375, 151-408, 151-447, 161-624, 165-450, 171-329, 171-492, 179-357, 219-402, 228-715,

257-706, 260-845, 270-802, 277-749, 282-632, 286-378, 301-585, 305-603, 322-573, 377-637, 389-845, 391-508,

415-651, 460-935, 476-950, 528-773, 539-833, 565-814, 566-843, 604-780, 607-736, 607-843, 842-1090, 846-1263,

858-1261, 860-1513, 874-1208, 930-1474, 945-1433, 962-1245, 969-1733, 971-1379, 980-1595, 983-1216, 1009-

1303, 1023-1261, 1032-1198, 1086-1323, 1086-1339, 1086-1354, 1091-1521, 1102-1369, 1127-1380, 1163-1337,

1183-1411, 1199-1726, 1281-1464, 1295-1548, 1303-1974, 1445-1579

65/7504972CB1/818
1-768, 201-431, 350-818, 360-625, 430-626, 430-717, 431-656, 433-607, 467-713, 467-792, 468-761, 475-747, 488-

626, 489-625, 494-764, 502-754, 531-792, 608-780, 665-747, 665-801, 689-781

66/7511788CB1/1715
1-269, 1-476, 2-1689, 14-529, 16-691, 24-689, 28-265, 45-556, 56-350, 56-871, 59-599, 67-333, 68-562, 86-324, 90-

393, 97-731, 102-595, 102-839, 102-878, 102-898, 102-928, 102-930, 102-931, 102-932, 112-590, 120-359, 128-

397, 138-669, 149-843, 424-679, 424-783, 427-749, 450-576, 484-768, 536-737, 536-845, 542-807, 604-861, 636-

909, 716-915, 758-995, 774-932, 930-1530, 930-1664, 930-1667, 951-1664, 952-1664, 952-1667, 966-1667, 970-

1667, 999-1419, 1007-1260, 1032-1306, 1038-1231, 1039-1317, 1041-1677, 1052-1585, 1054-1663, 1063-1346,

1064-1311, 1064-1321, 1071-1288, 1071-1305, 1081-1114, 1081-1332, 1090-1329, 1116-1628, 1141-1394, 1141-

1395, 1156-1433, 1167-1583, 1180-1695, 1201-1503, 1203-1459, 1203-1689, 1208-1456, 1208-1480, 1238-1418,

1286-1703, 1304-1583, 1348-1715, 1357-1583

67/7504642CB1/2795
1-284, 5-2726, 14-136, 15-284, 18-284, 19-284, 23-284, 65-284, 67-493, 67-633, 67-816, 67-818, 67-835, 67-884,

143-261, 157-884, 159-884, 282-796, 299-465, 311-995, 313-517, 316-906, 316-907, 352-974, 379-905, 412-679,

412-755, 468-1230, 476-884, 489-798, 517-1090, 533-996, 577-998, 592-1019, 596-998, 615-1038, 649-915, 671-

1283, 685-945, 692-786, 711-998, 714-1150, 726-1319, 782-1022, 791-1032, 815-1266, 860-1118, 860-1313, 907-

1365, 909-1150, 916-1172, 970-1137, 985-1106, 995-1202, 1001-1517, 1019-1272, 1019-1337, 1072-1404, 1081-

1634, 1113-1347, 1113-1598, 1129-1331, 1129-1395, 1155-1391, 1175-1722, 1185-1738, 1239-1745, 1270-1778,

1293-1461, 1319-2005, 1337-1815, 1357-1792, 1363-1850, 1375-1634, 1405-2314, 1436-2100, 1444-2001, 1471-

1905, 1484-2310, 1488-2124, 1513-2324, 1514-2327, 1537-1780, 1537-1802, 1544-1794, 1544-1926, 1549-2099,

1552-1863, 1568-2177, 1568-

2239, 1586-2324, 1588-2087, 1596-1860, 1608-1801, 1608-2327, 1610-2325, 1611-2324, 1622-1890, 1624-1814,

1624-1891, 1631-1863, 1636-2099, 1644-2324, 1649-2324, 1653-2124, 1654-2323, 1657-1914, 1658-2131, 1658-

2489, 1662-2301, 1675-1940, 1689-1793, 1689-1960, 1706-2326, 1709-2121, 1709-2223, 1714-2243, 1731-2025,

1748-2256, 1752-1964, 1783-1979, 1785-2665, 1801-2073, 1803-2640, 1806-2066, 1826-2307, 1832-2066, 1855-

2382, 1865-2539, 1881-2637, 1886-2236, 1925-2137, 1925-2138, 1925-2169, 1930-2185, 1930-2215, 1931-2192,

1934-2195, 1934-2461, 1934-2686, 1936-2191, 1936-2241, 1938-2367, 1941-2369, 1941-2660, 1942-2186, 1942-

2455, 1955-2261, 1960-2301, 1977-2222, 1984-2356, 2010-2678, 2022-2463, 2025-2288, 2027-2257, 2038-2291,

2064-2245, 2065-2688, 2071-2253, 2071-2580, 2080-2629, 2104-2664, 2113-2617, 2114-2701, 2148-2685, 2154-

2720, 2156-2733, 2159-2704, 2160-2731, 2174-

2684, 2181-2727, 2184-2731, 2191-2427, 2191-2697, 2201-2362, 2217-2712, 2224-2703, 2229-2731, 2230-2512,

2237-2709, 2244-2717, 2246-2714, 2250-2735, 2253-2424, 2253-2731, 2259-2712, 2259-2726, 2259-2727, 2262-

2716, 2270-2713, 2270-2715, 2271-2711, 2274-2717, 2274-2730, 2275-2685, 2276-2675, 2279-2712, 2280-2716,

2281-2730, 2283-2735, 2284-2570, 2284-2726, 2289-2712, 2291-2661, 2293-2712, 2296-2712, 2299-2726, 2301-

2451, 2302-2557, 2302-2688, 2302-2725, 2303-2717, 2305-2712, 2310-2742, 2313-2528, 2315-2679, 2315-2723,

2318-2731, 2319-2712, 2322-2712, 2325-2712, 2326-2712, 2331-2526, 2338-2712, 2340-2712, 2342-2716, 2343-

2709, 2343-2712, 2345-2584, 2346-2713, 2348-2676, 2351-2712, 2356-2712, 2357-2712, 2360-2579, 2361-2712,

2362-2712, 2372-2712, 2375-2713, 2382-2764, 2383-2715, 2387-2711, 2394-2709, 2394-2714, 2420-2716, 2427-

2712, 2429-2712, 2434-2714, 2450-2714, 2450-2726, 2454-2707, 2471-2712, 2489-2614, 2489-2748, 2496-2712,

2534-2713, 2539-2725, 2567-2725, 2581-2709, 2599-2795

68/7504643CB1/3173
1-642, 13-296, 13-613, 13-652, 14-568, 15-3105, 16-482, 24-146, 25-313, 28-301, 29-306, 33-309, 39-592, 60-585,

70-659, 71-625, 75-302, 77-629, 77-645, 77-649, 77-661, 82-585, 88-622, 92-647, 109-387, 109-556, 153-271, 340-

591, 606-1262, 677-843, 689-1373, 694-1284, 694-1285, 730-1352, 757-1283, 846-1608, 854-1262, 895-1468, 911-

1374, 955-1376, 970-1397, 974-1376, 993-1416, 1027-1293, 1049-1661, 1063-1323, 1070-1164, 1089-1376, 1092-

1528, 1104-1697, 1160-1400, 1169-1410, 1193-1644, 1238-1496, 1238-1691, 1285-1743, 1287-1528, 1294-1550,

1348-1515, 1363-1484, 1373-1580, 1379-1895, 1397-1650, 1397-1715, 1450-1782, 1459-2012, 1491-1725, 1491-

1976, 1507-1709, 1507-1773, 1533-1769, 1553-2100, 1563-2116, 1617-2123, 1648-2156, 1671-1839, 1697-2383,

1715-2193, 1735-2170, 1741-2228, 1753-2012, 1783-2692, 1814-2478, 1822-2379, 1849-2283, 1862-2688, 1866-

2502, 1891-2702, 1892-2705,

1915-2158, 1915-2180, 1922-2172, 1922-2304, 1927-2477, 1930-2241, 1946-2555, 1946-2617, 1964-2702, 1966-

2465, 1974-2238, 1986-2179, 1986-2705, 1988-2703, 1989-2702, 2000-2268, 2002-2192, 2002-2269, 2009-2241,

2014-2477, 2022-2702, 2027-2702, 2031-2502, 2032-2701, 2035-2292, 2036-2509, 2036-2867, 2040-2679, 2053-

2318, 2067-2171, 2067-2338, 2084-2704, 2087-2499, 2087-2601, 2092-2621, 2109-2403, 2126-2634, 2130-2342,

2161-2357, 2163-3043, 2179-2451, 2181-3018, 2184-2444, 2204-2685, 2210-2444, 2233-2760, 2243-2917, 2259-

3015, 2264-2614, 2303-2515, 2303-2516, 2303-2547, 2308-2563, 2308-2593, 2309-2570, 2312-2573, 2312-2839,

2312-3064, 2314-2569, 2314-2619, 2316-2745, 2319-2747, 2319-3038, 2320-2564, 2320-2833, 2333-2639, 2338-

2679, 2355-2600, 2362-2734, 2388-3056, 2400-2841, 2403-2666, 2405-2635, 2416-2669, 2442-2623, 2443-3066,

2449-2631, 2449-2958, 2458-3007, 2482-3042,

2491-2995, 2492-3079, 2526-3063, 2532-3098, 2534-3111, 2537-3082, 2538-3109, 2552-3062, 2559-3105, 2562-

3109, 2569-2805, 2569-3075, 2579-2740, 2595-3090, 2602-3081, 2607-3109, 2608-2890, 2615-3087, 2622-3095,

2624-3092, 2628-3113, 2631-2802, 2631-3109, 2637-3090, 2637-3104, 2637-3105, 2640-3094, 2648-3091, 2648-

3093, 2649-3089, 2652-3095, 2652-3108, 2653-3063, 2654-3053, 2657-3090, 2658-3094, 2659-3108, 2661-3113,

2662-2948, 2662-3104, 2667-3090, 2669-3039, 2671-3090, 2674-3090, 2677-3104, 2679-2829, 2680-2935, 2680-

3066, 2680-3103, 2681-3095, 2683-3090, 2688-3120, 2691-2906, 2693-3057, 2693-3101, 2696-3109, 2697-3090,

2700-3090, 2703-3090, 2704-3090, 2709-2904, 2716-3090, 2718-3090, 2720-3094, 2721-3087, 2721-3090, 2723-

2962, 2724-3091, 2726-3054, 2729-3090, 2734-3090, 2735-3090, 2738-2957, 2739-3090, 2740-3090, 2750-3090,

2753-3091, 2760-3142, 2761-3093, 2765-3089, 2772-3087, 2772-3092, 2798-3094, 2805-3090, 2807-3090, 2812-

3092, 2828-3092, 2828-3104, 2832-3085, 2849-3090, 2867-2992, 2867-3126, 2874-3090, 2912-3091, 2917-3103,

2959-3087, 2977-3173

69/7504745CB1/1038
1-284, 5-1022, 14-136, 15-284, 18-284, 19-284, 23-284, 65-284, 67-493, 67-633, 67-816, 67-818, 67-835, 67-884,

143-261, 157-884, 159-884, 282-796, 299-465, 311-995, 313-517, 316-906, 316-907, 352-974, 379-905, 412-755,

476-884, 489-798, 533-996, 577-998, 592-1019, 596-998, 615-1038, 649-915, 685-945, 692-786, 711-998, 782-

1022, 791-1032

70/7504746CB1/1416
1-642, 13-296, 13-613, 13-652, 14-568, 15-1400, 16-482, 24-146, 25-313, 28-301, 29-306, 33-309, 39-592, 60-585,

70-659, 71-625, 75-302, 77-629, 77-645, 77-649, 77-661, 82-585, 88-622, 92-647, 109-387, 109-556, 153-271, 340-

591, 606-1262, 677-843, 689-1373, 694-1284, 694-1285, 730-1352, 757-1283, 854-1262, 911-1374, 955-1376, 970-

1397, 974-1376, 993-1416, 1027-1293, 1063-1323, 1070-1164, 1089-1376, 1160-1400, 1169-1410

[0439]

7

TABLE 5

Polynucleotide
Incyte
Representative

SEQ ID NO:
Project ID:
Library

37
7499815CB1
HNT2AGT01

38
3165346CB1
PROSTUT09

39
5092954CB1
BRSTTUT02

40
7499560CB1
LIVRNOT21

41
70243658CB1
MONOTXT01

42
7500196CB1
ADRETUT05

43
7500351CB1
THYMNOT03

44
7500923CB1
SPLNFET02

45
2258292CB1
OVARDIR01

46
7500283CB1
BRANDIT03

47
7600263CB1
ESOGTME01

48
7503686CB1
CARCTXT02

49
7504791CB1
MCLDTXN05

50
7504885CB1
SPLNNOT04

51
7504915CB1
LATRTUT02

53
7505049CB1
THYMDIT01

55
7503683CB1
CARCTXT02

56
71616365CB1
SYNORAB01

57
7505047CB1
THYMNOT03

58
7505779CB1
UCMCL5T01

59
7505782CB1
MONOTXS05

60
7500207CB1
BRSTNOT04

61
7500208CB1
BRSTNOT04

62
7500313CB1
THYMNOT03

63
1436493CB1
PANCNOT08

64
7501101CB1
THYMNOT05

65
7504972CB1
NEUTGMT01

66
7511788CB1
MONOTXS05

67
7504642CB1
BRSTNOT04

68
7504643CB1
UTRSDIC01

69
7504745CB1
PLACFER01

70
J7504746CB1
OSTEUNC01

[0440]

8

TABLE 6

Library
Vector
Library Description

ADRETUT05
pINCY
Library was constructed using RNA isolated from adrenal tumor tissue removed from a 52-year-old

Caucasian female during a unilateral adrenalectomy. Pathology indicated a pheochromocytoma.

BRANDIT03
pINCY
Library was constructed using RNA isolated from pineal gland tissue removed from a 79-year-old

Caucasian female who died from pneumonia. Neuropathology indicated severe Alzheimer Disease,

moderate to severe arteriolosclerosis of the intracranial blood vessels, moderate cerebral

amyloid angiopathy and infarctions involving the parieto-occipital lobes. There was atrophy of

all lobes, caudate, putamen, amygdala, hippocampus, vermis, optic nerve, and the cerebral

cortical white matter. There was cystic cavitation in the left medial occipital lobe, the right

posterior parietal region, the right side insular cortex, and the right occipital and inferior

parietal lobes. The ventricular system was severely dilated. Stains show numerous diffuse as

well as neuritic amyloid plaques throughout all neocortical areas examined. There were numerous

neurofibrillary tangles predominantly in the pyramidal cell neurons of layers 3 and 5, however,

small interneurons in layers 3, 4, and 6 also contain tangles. The caudate and putamen contain

large areas of mineralization and scattered neurofibrillary tangles. The amygdala was markedly

gliotic containing numerous neurofibrillary, argyrophilic and ghost type tangles; and scattered

cells with granulovacuolar degeneration and focal cells with Lewy-like body inclusions. The

hippocampus contains marked gliosis with complete loss of pyramidal cell neurons in the CA1

region. Silver stained sections show numerous neuritic plaques and scattered neurofibrillary

tangles within the dentate gyrus, CA2, and CA3 regions. The substantia nigra shows numerous

neurofibrillary tangles in the periaqueductal grey region. Patient history included gastritis

with bleeding, glaucoma, PVD, COPD, delayed onset tonic/clonic seizures, transient ischemic

attacks, pseudophakia, and allergies to aspirin and clindamycin. Family history included

Alzheimer disease.

BRSTNOT04
PSPORT1
Library was constructed using RNA isolated from breast tissue removed from a 62-year-old East

Indian female during a unilateral extended simple mastectomy. Pathology for the associated tumor

tissue indicated an invasive grade 3 ductal carcinoma. Patient history included benign

hypertension, hyperlipidemia, and hematuria. Family history included cerebrovascular and

cardiovascular disease, hyperlipidemia, and liver cancer.

BRSTTUT02
PSPORT1
Library was constructed using RNA isolated from breast tumor tissue removed from a 54-year-old

Caucasian female during a bilateral radical mastectomy with reconstruction. Pathology indicated

residual invasive grade 3 mammary ductal adenocarcinoma. The remaining breast parenchyma exhibited

proliferative fibrocystic changes without atypia. One of 10 axillary lymph nodes had metastatic

tumor as a microscopic intranodal focus. Patient history included kidney infection and condyloma

acuminatum. Family history included benign hypertension, hyperlipidemia, and a malignant colon

neoplasm.

CARCTXT02
PSPORT1
Library was constructed using RNA from chondrocytes that were isolated from pooled knee cartilage

obtained during total knee joint replacement. The cartilage was removed from the underlying bone,

chopped into smaller pieces, and stimulated with 5 ng/ml IL-1 for 18 hours.

ESOGTME01
PSPORT
This 5′ biased random primed library was constructed using RNA isolated from esophageal

tissue removed from a 53-year-old Caucasian male during a partial esophagectomy, proximal

gastrectomy, and regional lymph node biopsy. Pathology indicated no significant abnormality in

the non-neoplastic esophagus. Pathology for the matched tumor tissue indicated invasive grade 4

(of 4) adenocarcinoma, forming a sessile mass situated in the lower esophagus, 2 cm from the

gastroesophageal junction and 7 cm from the proximal margin. The tumor invaded through the

muscularis propria into the adventitial soft tissue. Metastatic carcinoma was identified in 2 of

5 paragastric lymph nodes with perinodal extension. The patient presented with dysphagia. Patient

history included membranous nephritis, hyperlipidemia, benign hypertension, and anxiety state.

Previous surgeries included an adenotonsillectomy, appendectomy, and inguinal hernia repair.

The patient was not taking any medications. Family history included atherosclerotic coronary

artery disease, alcoholic cirrhosis, alcohol abuse, and an abdominal aortic aneurysm rupture in

the father; breast cancer in the mother; a myocardial infarction and atherosclerotic coronary

artery disease in the sibling(s); and myocardial infarction and atherosclerotic coronary artery

disease in the grandparent(s).

HNT2AGT01
PBLUESCRIPT
Library was constructed at Stratagene (STR937233), using RNA isolated from the hNT2 cell line

derived from a human teratocarcinoma that exhibited properties characteristic of a committed

neuronal precursor. Cells were treated with retinoic acid for 5 weeks and with mitotic inhibitors

for two weeks and allowed to mature for an additional 4 weeks in conditioned medium.

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.

LIVRNOT21
pINCY
Library was constructed using RNA isolated from liver tissue removed from a 29-year-old Caucasian

male who died from massive head injury due to a motor vehicle accident. Serology was positive for

cytomegalovirus.

MCLDTXN05
pINCY
This normalized dendritic cell library was constructed from 1 million independent clones from a

pool of two derived dendritic cell libraries. Starting libraries were constructed using RNA

isolated from untreated and treated derived dendritic cells from umbilical cord blood CD34+

precursor cells removed from a male. The cells were derived with granulocyte/macrophage colony

stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF alpha), and stem cell factor (SCF).

The GM-CSF was added at time 0 at 100 ng/ml, the TNF alpha was added at time 0 at 2.5 ng/ml, and

the SCF was added at time 0 at 25 ng/ml. Incubation time was 13 days. The treated cells were then

exposed to phorbol myristate acetate (PMA), and Ionomycin. The PMA and Ionomycin were added at

13 days for five hours. The library was normalized in two rounds using conditions adapted from

Soares et al., PNAS (1994) 91: 9228 and Bonaldo et al., Genome Research 6 (1996): 791, except that

a significantly longer (48 hours/round) reannealing hybridization was used.

MONOTXS05
pINCY
Subtracted, treated monocyte tissue library was constructed using 7.5 million clones from a

treated monocyte library and were subjected to two rounds of subtraction hybridization with

1.03 × 1Oe7 clones from a second treated monocyte library. The starting library for

subtraction was constructed using treated monocytes from peripheral blood obtained from a

42-year-old female. The cells were treated with anti-interleukin-10 (anti-IL-10) and

lipopolysaccharide (LPS). The anti-IL-10 was added at time 0 at 10 ng/ml and LPS was added at

1 hour at 5 ng/ml. The monocytes were isolated from buffy coat by adherence to plastic.

Incubation time was 24 hours. The hybridization probe for subtraction was derived from a

similarly constructed library from RNA isolated from monocyte tissue, treated with

interleukin-10 (IL10) and lipopolysaccharide (LPS) 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.

MONOTXT01
pINCY
Library was constructed using RNA isolated from treated monocytes from peripheral blood obtained

from a 42-year-old female. The cells were treated with anti IL-10 and LPS.

NEUTGMT01
PSPORT1
Library was constructed using RNA isolated from peripheral blood granulocytes collected by

density gradient centrifugation through Ficoll-Hypaque. The cells were isolated from buffy coat

units obtained from 20 unrelated male and female donors. Cells were cultured in 10 nM GM-CSF for

1 hour before washing and harvesting for total RNA preparation.

OSTEUNC01
pINCY
This large size-fractionated library was constructed using RNA isolated from untreated osteoblast

tissue removed from the clavicle of a 40-year-old male.

OVARDIR01
PCDNA2.1
This random primed library was constructed using RNA isolated from right ovary tissue removed

from a 45-year-old Caucasian female during total abdominal hysterectomy, bilateral

salpingo-oophorectomy, vaginal suspension and fixation, and incidental appendectomy. Pathology

indicated stromal hyperthecosis of the right and left ovaries. Pathology for the matched tumor

tissue indicated a dermoid cyst (benign cystic teratoma) in the left ovary. Multiple (3)

intramural leiomyomata were identified. The cervix showed squamous metaplasia. Patient history

included metrorrhagia, female stress incontinence, alopecia, depressive disorder, pneumonia,

normal delivery, and deficiency anemia. Family history included benign hypertension,

atherosclerotic coronary artery disease, hyperlipidemia, and primary tuberculous complex.

PANCNOT08
pINCY
Library was constructed using RNA isolated from pancreatic tissue removed from a 65-year-old

Caucasian female during radical subtotal pancreatectomy. Pathology for the associated tumor

tissue indicated an invasive grade 2 adenocarcinoma. Patient history included type II diabetes,

osteoarthritis, cardiovascular disease, benign neoplasm in the large bowel, and a cataract.

Previous surgeries included a total splenectomy, cholecystectomy, and abdominal hysterectomy.

Family history included cardiovascular disease, type II diabetes, and stomach cancer.

PLACFER01
pINCY
The library was constructed using RNA isolated from placental tissue removed from a Caucasian

fetus, who died after 16 weeks' gestation from fetal demise and hydrocephalus. 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.

PROSTUT09
pINCY
Library was constructed using RNA isolated from prostate tumor tissue removed from a 66-year-old

Caucasian male during a radical prostatectomy, radical cystectomy, and urinary diversion.

Pathology indicated grade 3 transitional cell carcinoma. The patient presented with prostatic

inflammatory disease. Patient history included lung neoplasm, and benign hypertension. Family

history included a malignant breast neoplasm, tuberculosis, cerebrovascular disease,

atherosclerotic coronary artery disease and lung cancer.

SPLNFET02
pINCY
Library was constructed using RNA isolated from spleen tissue removed from a Caucasian male fetus,

who died at 23 weeks' gestation.

SPLNNOT04
pINCY
Library was constructed using RNA isolated from the spleen tissue of a 2-year-old Hispanic male,

who died from cerebral anoxia. Past medical history and serologies were negative.

SYNORAB01
PBLUESCRIPT
Library was constructed using RNA isolated from the synovial membrane tissue of a 68-year-old

Caucasian female with rheumatoid arthritis.

THYMDIT01
pINCY
The library was constructed using RNA isolated from diseased thymus tissue removed from a

16-year-old Caucasian female during a total excision of thymus and regional lymph node excision.

Pathology indicated thymic follicular hyperplasia. The right lateral thymus showed reactive lymph

nodes. A single reactive lymph node was also identified at the inferior thymus margin. The patient

presented with myasthenia gravis, malaise, fatigue, dysphagia, severe muscle weakness, and

prominent eyes. Patient history included frozen face muscles. Family history included depressive

disorder, hepatitis B, myocardial infarction, atherosclerotic coronary artery disease, leukemia,

multiple sclerosis, and lupus.

THYMNOT03
pINCY
Library was constructed using RNA isolated from thymus tissue removed from a 21-year-old

Caucasian male during a thymectomy. Pathology indicated an unremarkable thymus and a benign

parathyroid adenoma in the right inferior parathyroid. Patient history included atopic dermatitis,

a benign neoplasm of the parathyroid, and tobacco use. Previous surgeries included an operation

on the parathyroid gland. Patient medications included multivitamins. Family history included

atherosclerotic coronary artery disease in the father and benign hypertension in the grandparent(s).

THYMNOT05
pINCY
Library was constructed using RNA isolated from thymus tissue removed from a 3-year-old Hispanic

male during a thymectomy and closure of a patent ductus arteriosus. The patient presented with

severe pulmonary stenosis and cyanosis. Patient history included a cardiac catheterization and

echocardiogram. Previous surgeries included Blalock-Taussig shunt and pulmonary valvotomy. The

patient was not taking any medications. Family history included benign hypertension,

osteoarthritis, depressive disorder, and extrinsic asthma in the grandparent(s).

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.

UTRSDIC01
PSPORT1
This large size fractionated library was constructed using pooled cDNA from eight donors. cDNA

was generated using mRNA isolated from endometrial tissue removed from a 32-year-old female

(donor A); endometrial tissue removed from a 32-year-old Caucasian female (donor B) during

abdominal hysterectomy, bilateral salpingo-oophorectomy, and cystocele repair; from diseased

endometrium and myometrium tissue removed from a 38-year-old Caucasian female (donor C) during

abdominal hysterectomy, bilateral salpingo-oophorectomy, and exploratory laparotomy; from

endometrial tissue removed from a 41-year-old Caucasian female (donor D) during abdominal

hysterectomy with removal of a solitary ovary; from endometrial tissue removed from a 43-year-old

Caucasian female (donor E) during vaginal hysterectomy, dilation and curettage, cystocele repair,

rectocele repair and cystostomy; and from endometrial tissue removed from a

48-year-old Caucasian female (donor F) during a vaginal hysterectomy, rectocele repair, and

bilateral salpingo-oophorectomy. Pathology (A) indicated the endometrium was in secretory phase.

Pathology (B) indicated the endometrium was in the proliferative phase. Pathology (C) indicated

extensive adenomatous hyperplasia with squamous metaplasia and focal atypia, forming a polypoid

mass within the endometrial cavity. The cervix showed chronic cervicitis and squamous metaplasia.

Pathology (D, E) indicated the endometrium was secretory phase. Pathology (F) indicated the

endometrium was weakly proliferative.

[0441]

9

TABLE 7

Program
Description
Reference
Parameter Threshold

ABI FACTURA
A program that removes vector
Applied Biosystems, Foster City, CA.

sequences and masks

ambiguous bases in nucleic

acid sequences.

ABI/PARACEL FDF
A Fast Data Finder useful
Applied Biosystems, Foster City, CA;
Mismatch <50%

in comparing and
Paracel Inc., Pasadena, CA.

annotating amino acid or

nucleic acid sequences.

ABI AutoAssembler
A program that assembles
Applied Biosystems, Foster City, CA.

nucleic acid sequences.

BLAST
A Basic Local Alignment
Altschul, S. F. et al. (1990) J. Mol. Biol.
ESTs: Probability value = 1.0E−

Search Tool useful in
215: 403-410; Altschul, S. F. et al. (1997)
8 or less; Full Length sequences:

sequence similarity search
Nucleic Acids Res. 25: 3389-3402.
Probability value = 1.0E−10 or

for amino acid and nucleic

less

acid sequences. BLAST

includes five functions:

blastp, blastn, blastx,

tblastn, and tblastx.

FASTA
A Pearson and Lipman
Pearson, W. R. and D. J. Lipman (1988) Proc.
ESTs: fasta E value = 1.06E−6;

algorithm that searches for
Natl. Acad Sci. USA 85: 2444-2448; Pearson,
Assembled ESTs: fasta Identity =

similarity between a query
W. R. (1990) Methods Enzymol. 183: 63-98;
95% or greater and Match

sequence and a group of
and Smith, T. F. and M. S. Waterman (1981)
length = 200 bases or greater;

sequences of the same type.
Adv. Appl. Math. 2: 482-489.
fastx E value = 1.0E−8 or less;

FASTA comprises as

Full Length sequences: fastx

least five functions: fasta,

score = 100 or greater

tfasta, fastx, tfastx, and

ssearch.

BLIMPS
A BLocks IMProved Searcher
Henikoff, S. and J. G. Henikoff (1991)
Probability value = 1.0E−3 or

that matches a sequence
Nucleic Acids Res. 19: 6565-6572; Henikoff,
less

against those in BLOCKS,
J. G. and S. Henikoff (1996) Methods

PRINTS, DOMO, PRODOM,
Enzymol. 266: 88-105; and Attwood, T. K. et

and PFAM databases to search
al. (1997) J. Chem. Inf. Comput. Sci. 37: 417-

for gene families, sequence

homology, and structural

fingerprint regions.

HMMER
An algorithm for searching
Krogh, A. et al. (1994) J. Mol. Biol.
PFAM, INCY, SMART or

a query sequence against
235: 1501-1531; Sonnhammer, E. L. L. et al.
TIGRFAM hits: Probability

hidden Markov model
(1988) Nucleic Acids Res. 26: 320-322;
value = 1.0E−3 or less; Signal

(HMM)-based databases of
Durbin, R. et al. (1998) Our World View, in
peptide hits: Score = 0 or greater

protein family consensus
a Nutshell, Cambridge Univ. Press, pp. 1-

sequences, such as PFAM,

INCY, SMART and TIGRFAM.

ProfileScan
An algorithm that searches
Gribskov, M. et al. (1988) CABIOS 4: 61-66;
Normalized quality score ≧ GCG

for structural and
Gribskov, M. et al. (1989) Methods
specified “HIGH” value for that

sequence motifs in protein
Enzymol. 183: 146-159; Bairoch, A. et al.
particular Prosite motif.

sequences that match
(1997) Nucleic Acids Res. 25: 217-221.
Generally, score = 1.4-2.1.

sequence patterns

defined in Prosite.

Phred
A base-calling algorithm
Ewing, B. et al. (1998) Genome Res. 8: 175-

that examines automated
185; Ewing, B. and P. Green (1998) Genome

sequencer traces with high
Res. 8: 186-194.

sensitivity and probability.

Phrap
A Phils Revised Assembly
Smith, T. F. and M. S. Waterman (1981) Adv.
Score = 120 or greater; Match

Program including
Appl. Math. 2: 482-489; Smith, T. F. and
length = 56 or greater

SWAT and CrossMatch,
M. S. Waterman (1981) J. Mol. Biol. 147: 195-

programs based on efficient
197; and Green, P., University of

implementation of the
Washington, Seattle, WA.

Smith-Waterman algorithm,

useful in searching

sequence homology and

assembling DNA sequences.

Consed
A graphical tool for
Gordon, D. et al. (1998) Genome Res. 8: 195-

viewing and editing Phrap
202.

assemblies.

SPScan
A weight matrix analysis
Nielson, H. et al. (1997) Protein Engineering
Score = 3.5 or greater

program that scans protein
10: 1-6; Claverie, J. M. and S. Audic (1997)

sequences for the presence
CABIOS 12: 431-439.

of secretory signal

peptides.

TMAP
A program that uses weight
Persson, B. and P. Argos (1994) J. Mol. Biol.

matrices to delineate
237: 182-192; Persson, B. and P. Argos

transmembrane segments on
(1996) Protein Sci. 5: 363-371.

protein sequences and

determine orientation.

TMHMMER
A program that uses a
Sonnhammer, E. L. et al. (1998) Proc. Sixth

hidden Markov model (HMM)
Intl. Conf. On Intelligent Systems for Mol.

to delineate transmembrane
Biol., Glasgow et al., eds., The Am. Assoc.

segments on protein
for Artificial Intelligence (AAAI) Press,

sequences and determine
Menlo Park, CA, and MIT Press, Cambridge,

orientation.
MA, pp. 175-182.

Motifs
A program that searches
Bairoch, A. et al. (1997) Nucleic Acids Res.

amino acid sequences for
25: 217-221; Wisconsin Package Program

patterns that matched
Manual, version 9, page M51-59, Genetics

those defined in Prosite.
Computer Group, Madison, WI.

[0442]

10

TABLE 8

SEQ

All-

Caucasian
African
Asian
Hispanic

ID

EST
CB1
EST
Allele
ele
Amino
Allele 1
Allele 1
Allele 1
Allele 1

NO:
PID
EST ID
SNP ID
SNP
SNP
Allele
1
2
Acid
frequency
frequency
frequency
frequency

60
7500207
1400541H1
SNP00060974
42
2070
G
G
A
noncoding
nd
n/a
n/a
n/a

60
7500207
1970930H1
SNP00060973
205
1751
C
C
A
noncoding
n/a
n/a
n/a
n/a

60
7500207
2101935H1
SNP00107995
176
1294
C
T
C
noncoding
n/d
n/a
n/a
n/a

60
7500207
2183883H1
SNP00037213
187
1052
A
A
C
noncoding
n/a
n/a
n/a
n/a

60
7500207
2435336H1
SNP00136887
142
201
G
G
A
E39
n/a
n/a
n/a
n/a

60
7500207
4568395H1
SNP00037214
45
1427
C
C
T
noncoding
n/a
n/a
n/a
n/a

60
7500207
6453861H1
SNP00037212
184
505
A
A
G
I141
n/d
0.95
n/d
n/d

61
7500208
1400541H1
SNP00060974
42
2211
G
G
A
noncoding
n/d
n/a
n/a
n/a

61
7500208
1970930H1
SNP00060973
205
1892
C
C
A
noncoding
n/a
n/a
n/a
n/a

61
7500208
2101935H1
SNP00107995
176
1435
C
T
C
noncoding
n/d
n/a
n/a
n/a

61
7500208
2183883H1
SNP00037213
187
1193
A
A
C
noncoding
n/a
n/a
n/a
n/a

61
7500208
2435336H1
SNP00136887
142
202
G
G
A
G40
n/a
n/a
n/a
n/a

61
7500208
4568395H1
SNP00037214
45
1568
C
C
T
noncoding
n/a
n/a
n/a
n/a

61
7500208
6453861H1
SNP00037212
184
646
A
A
G
I188
n/d
0.95
n/d
n/d

62
7500313
2552626H1
SNP00104720
206
1096
G
G
A
noncoding
n/d
n/d
n/d
n/d

62
7500313
2556787H1
SNP00150901
136
147
G
G
A
G15
n/a
n/a
n/a
n/a

64
7501101
2552626H1
SNP00104720
206
1228
G
G
A
noncoding
n/d
n/d
n/d
n/d

64
7501101
2556787H1
SNP00150901
136
147
G
G
A
G15
n/a
n/a
n/a
n/a

64
7501101
2906994H1
SNP00014700
115
420
G
G
A
R106
0.37
0.81
0.68
0.45

66
7511788
2096346R6
SNP00114170
334
334
C
C
T
F66
0.51
0.65
0.79
0.53

66
7511788
2969420T6
SNP00059649
78
1506
A
A
G
noncoding
n/a
n/a
n/a
n/a

66
7511788
5752120H1
SNP00139376
47
1394
G
G
A
D420
n/a
n/a
n/a
n/a

67
7504642
1400541H1
SNP00060974
42
2066
G
G
A
noncoding
n/d
n/a
n/a
n/a

67
7504642
1970930H1
SNP00060973
205
1746
C
C
A
noncoding
n/a
n/a
n/a
n/a

67
7504642
1973850H1
SNP00060973
131
1745
C
C
A
noncoding
n/a
n/a
n/a
n/a

67
7504642
2101935H1
SNP00107995
176
1288
C
T
C
noncoding
n/d
n/a
n/a
n/a

67
7504642
2183883H1
SNP00037213
187
1046
A
A
C
noncoding
n/a
n/a
n/a
n/a

67
7504642
2435336H1
SNP00136887
142
206
G
G
A
G40
n/a
n/a
n/a
n/a

67
7504642
2812090H1
SNP00060974
127
2065
G
G
A
noncoding
n/d
n/a
n/a
n/a

67
7504642
2890774H1
SNP00136887
203
205
G
G
A
G39
n/a
n/a
n/a
n/a

67
7504642
3429243H1
SNP00060973
109
1734
C
C
A
noncoding
n/a
n/a
n/a
n/a

67
7504642
3719077H1
SNP00037213
74
1043
C
A
C
noncoding
n/a
n/a
n/a
n/a

67
7504642
4212865H1
SNP00060973
70
1744
C
C
A
noncoding
n/a
n/a
n/a
n/a

67
7504642
4568395H1
SNP00037214
45
1419
C
C
T
noncoding
n/a
n/a
n/a
n/a

67
7504642
5046625H1
SNP00136887
181
199
G
G
A
stop37
n/a
n/a
n/a
n/a

67
7504642
5954136H1
SNP00060974
105
2063
G
G
A
noncoding
n/d
n/a
n/a
n/a

67
7504642
6112222H1
SNP00060973
192
1743
C
C
A
noncoding
n/a
n/a
n/a
n/a

67
7504642
6453861H1
SNP00037212
184
499
A
A
G
noncoding
n/d
0.95
n/d
n/d

67
7504642
6493944H1
SNP00136887
172
193
G
G
A
V35
n/a
n/a
n/a
n/a

67
7504642
7732122J1
SNP00037214
420
1421
T
C
T
noncoding
n/a
n/a
n/a
n/a

68
7504643
1400541H1
SNP00060974
42
2444
G
G
A
noncoding
n/d
n/a
n/a
n/a

68
7504643
1970930H1
SNP00060973
205
2124
C
C
A
noncoding
n/a
n/a
n/a
n/a

68
7504643
1973850H1
SNP00060973
131
2123
C
C
A
noncoding
n/a
n/a
n/a
n/a

68
7504643
2101935H1
SNP00107995
176
1666
C
T
C
noncoding
n/d
n/a
n/a
n/a

68
7504643
2183883H1
SNP00037213
187
1424
A
A
C
noncoding
n/a
n/a
n/a
n/a

68
7504643
2435336H1
SNP00136887
142
216
G
G
A
G40
n/a
n/a
n/a
n/a

68
7504643
2812090H1
SNP00060974
127
2443
G
G
A
noncoding
n/d
n/a
n/a
n/a

68
7504643
2890774H1
SNP00136887
203
215
G
G
A
G39
n/a
n/a
n/a
n/a

68
7504643
3429243H1
SNP00060973
109
2112
C
C
A
noncoding
n/a
n/a
n/a
n/a

68
7504643
3614102H1
SNP00136887
114
214
G
G
A
G39
n/a
n/a
n/a
n/a

68
7504643
3719077H1
SNP00037213
74
1421
C
A
C
noncoding
n/a
n/a
n/a
n/a

68
7504643
4212865H1
SNP00060973
70
2122
C
C
A
noncoding
n/a
n/a
n/a
n/a

68
7504643
4523993H1
SNP00037213
26
1422
A
A
C
noncoding
n/a
n/a
n/a
n/a

68
7504643
4568395H1
SNP00037214
45
1797
C
C
T
noncoding
n/a
n/a
n/a
n/a

68
7504643
5046625H1
SNP00136887
181
209
G
G
A
stop37
n/a
n/a
n/a
n/a

68
7504643
5954136H1
SNP00060974
105
2441
G
G
A
noncoding
n/d
n/a
n/a
n/a

68
7504643
6112222H1
SNP00060973
192
2121
C
C
A
noncoding
n/a
n/a
n/a
n/a

68
7504643
6453861H1
SNP00037212
184
877
A
A
G
noncoding
n/d
0.95
n/d
n/d

68
7504643
6458841H2
SNP00136888
259
329
T
T
C
P77
n/a
n/a
n/a
n/a

68
7504643
6763225H1
SNP00107994
515
532
C
A
C
P145
n/a
n/a
n/a
n/a

68
7504643
7732122J1
SNP00037214
420
1799
T
C
T
noncoding
n/a
n/a
n/a
n/a

69
7504745
2435336H1
SNP00136887
142
206
G
G
A
G40
n/a
n/a
n/a
n/a

69
7504745
2890774H1
SNP00136887
203
205
G
G
A
G39
n/a
n/a
n/a
n/a

69
7504745
5046625H1
SNP00136887
181
199
G
G
A
stop37
n/a
n/a
n/a
n/a

69
7504745
6453861H1
SNP00037212
184
499
A
A
G
noncoding
n/d
0.95
n/d
n/d

69
7504745
6493944H1
SNP00136887
172
193
G
G
A
V35
n/a
n/a
n/a
n/a

70
7504746
2435336H1
SNP00136887
142
216
G
G
A
G40
n/a
n/a
n/a
n/a

70
7504746
2890774H1
SNP00136887
203
215
G
G
A
G39
n/a
n/a
n/a
n/a

70
7504746
3614102H1
SNP00136887
114
214
G
G
A
G39
n/a
n/a
n/a
n/a

70
7504746
5046625H1
SNP00136887
181
209
G
G
A
stop37
n/a
n/a
n/a
n/a

70
7504746
6453861H1
SNP00037212
184
877
A
A
G
noncoding
n/d
0.95
n/d
n/d

70
7504746
6458841H2
SNP00136888
259
329
T
T
C
P77
n/a
n/a
n/a
n/a

70
7504746
6763225H1
SNP00107994
515
532
C
A
C
P145
n/a
n/a
n/a
n/a

[0443]

Number	Date	Country
60324034	Sep 2001	US
60327395	Oct 2001	US
60342810	Oct 2001	US
60328923	Oct 2001	US
60344468	Nov 2001	US
60332140	Nov 2001	US
60340282	Dec 2001	US
60347693	Jan 2002	US
60358279	Feb 2002	US
60361088	Mar 2002	US
60364494	Mar 2002	US
60379876	May 2002	US
60388180	Jun 2002	US

Immune response associated proteins

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CPC

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Abstract

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Provisional Applications (13)