CA2338385A1 - Human receptor-associated proteins - Google Patents

Abstract

The invention provides human receptor-associated proteins (HRAP) and polynucleotides which identify and encode HRAP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of HRAP.

Description

HUMAN RECEPTOR-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human receptor-associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, reproductive, cardiovascular, and gastrointestinal disorders.
BACKGROUND OF THE INVENTION
The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of the proteins termed receptors are cell surface proteins which, when they bind extracellular ligands, produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response.
Other receptors facilitate the specific transport of proteins out of the endoplasmic reticulum and localize enzymes to a particular compartment of the cell. The term may also be applied to proteins which act as receptors for ligands (with known or unknown chemical composition) which interact with other cellular components. For example, the steroid hormone receptors bind to and regulate transcription of genomic DNA.
Regulation of cell proliferation, differentiation, and migration is important for the formation and function of tissues. Secreted regulatory proteins such as growth factors coordinately control these cellular processes and act as mediators in cell-cell signaling pathways.
Growth factors are secreted from the cell, and bind to specific cell-surface receptors on target cells. The bound receptors trigger intracellular signal transduction pathways which activate various downstream effectors. Such processes regulate many cell functions including cell proliferation, differentiation, gene transcription, cell motility, and oncogenic transformation.
Cell. surface receptors are typically integral membrane proteins of the plasma membrane.
These receptors recognize hormones including the catecholamines, such as epinephrine, norepinephrine, and histamine; peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, parathyroid hormone, and vasopressin; growth and differentiation factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, platelet-derived growth factor, nerve growth factor, colony-stimulating factors, and erythropoietin; cytokines such as chemokines, interleukins, interferons, and tumor necrosis factor; small peptide factors such as bombesin, oxytocin, endothelin, angiotensin II, vasoactive intestinal peptide, and bradykinin; neurotransmitters such as neuropeptide Y, neurotensin, neuromedin N, melanocortins, somatostatin, galanin, tachykinins;
opioids such as enkephalins, endorphins and dynorphins; and circulatory system-borne signaling molecules such as angiotensin, complement, calcitonin, endothelins, and formyl-methionyl peptides. Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptide. Other cell surface receptors bind ligands to be internalized by the cell. This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transfenrin, glucose- or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin (Lodish, H. et al. (1995) Molecular Cell Biolo~v, Scientific American Books, New York, NY, p. ?23; Mikhailenko, I. et al. (1997) J. Biol.
Chem. 272:6784-6791 ).
Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates immune regulation and inflammatory responses. The cellular responses triggered by TNF are initiated through its interaction with two distinct cell surface receptors, TNF-R1 and TNF-R2 (Tartaglia, L.A. and Goeddel, D.V. (1992) Immunol. Today 13:151-153). Both TNF receptors are part of the TNF
receptor (TNFR) superfamily, whose members include the Fas antigen, the p75 subunit of the NGF receptor, the TRAIL receptor, TRUNND, Sa1F19R, CD27, CD30, and CD40.
Members of the TNFR superfamily share the TNFR/NGFR family cysteine-rich region signature, which consists of cysteine-rich pseudo-repeats in the extracellular domains (ExPASy PROSITE
document PDOC00561; Pan, G. et al. (1998) FEBS Lett 424:41-45; Bairoch, A. et al. (1997) Nucl.
Acids. Res. 25:217-221; and Smith, C.A. et al. (1994) Cell 76:959-962).
Many growth factor receptors, including epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor, contain intrinsic protein kinase activities. When the polypeptide growth factor binds to the receptor, it triggers the autophosphorylation of a tyrosine residue on the receptor. It is believed that these phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins in the signaling pathway that eventually links the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule. These signaling proteins contain a common domain referred to as a SRC homology 2 (SH2) domain. SH2 domains are found in a variety of signaling molecules and oncogenic proteins such as phospholipase C-y, Ras GTPase activating protein, and pp60''sxc (Lowenstein, E.J. et al.
(1992) Cell 70:431-42).
G protein coupled receptors (GPCR) are integral membrane proteins containing seven transmembrane regions, an extracellular N-terminus that binds ligand, and a cytoplasmic C-terminus that interacts with G proteins (Strosberg, A.D. (1991) Eur. J.
Biochem. 196:1-10). The seven hydrophobic transmembrane domains form a bundle of antiparallel alpha helices which account for structural and functional features of the receptor. In most cases, the bundle of helices forms a binding pocket; however, when the binding site must accommodate bulky molecules, the extracellular N-terminal segment or one or more of the three extracellular loops participate in ligand binding which induces a conformational change in intracellular portions of the receptor.
The activated receptor interacts with an intracellular heterotrimeric G-protein complex which triggers further intracellular signaling activities. These activities include interactions with guanine nucleotide binding (G) proteins and the production of second messengers such as cyclic AMP
(cAMP), phospholipase C, inositol triphosphate, or interactions with ion channel proteins (Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6:180-190).
The amino-terminus of the GPCR is extracellular, of variable length, and often glycosylated; the carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular IS loops of the GPCR alternate with intracellular loops and link the transmembrane domains. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. GPCRs range in size from under 400 to over 1000 amino acids (Coughlin, S.R. ( 1994) Curr. Opin. Cell Biol. 6:191-197).
GPCRs respond to a diverse array of ligands including lipid analogs, amino acids and their derivatives, peptides, cytokines, and specialized stimuli such as light, taste, and odor. GPCRs function in physiological processes including vision (the rhodopsins), smell (the olfactory receptors), neurotransmission (muscarinic acetylcholine, dopamine, and adrenergic receptors), and hormonal response (luteinizing hormone and thyroid-stimulating hormone receptors).
GPCR mutations, which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene. Parma, J. et al. ( 1993, Nature 365:649-65 I ) report that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggest that certain G-protein-coupled receptors susceptible to constitutive activation may behave as proto-oncogenes.
The frizzled cell surface receptor, originally identified in Drosophila melanogaster, is important for proper bristle and hair polarity on the wing, leg, thorax, abdomen, and eye of the developing insect (Wang, Y. et al. (1996) J. Biol. Chem. 271:4468-4476). The frizzled gene encodes a 587 amino acid protein which contains an N-terminal signal sequence and seven putative transmembrane regions. The cysteine-rich N-terminus is probably extracellular and the C-terminus is probably cytosolic. Multiple frizzled gene homologs have been found in rat, mouse, and human. The frizzled receptors are not homologous to other seven-transmembrane-region receptors and their ligands are still unknown.
T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells. Although a population of T cells can recognize a wide range of different antigens, an individual T cell can only recognize a single antigen and only when it is presented to the T cell receptor (TCR) as a peptide complexed with a major histocompatibility molecule (MHC) on the surface of an antigen presenting cell. The TCR on most T
cells consists of immunoglobulin-like integral membrane glycoproteins containing two polypeptide subunits, a and ~, of similar molecular weight. The TCR ~ subunit has an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. ( 1984) Nature 309:757-762). The genes for the TCR subunits are constructed through somatic rearrangement of different gene segments.
Interaction of antigen in the proper MHC context with the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components of the immune system (Weiss, A. (1991) Annu. Rev. Genet. 25: 487-510). Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas, leukemias, autoimmune disorders, and immunodeficiency disorders (Aisenberg, A.C. et al. (1985) N. Engl. J. Med.
313:529-533; Weiss, su ra .
The immunoglobulin E (IgE) receptor is another receptor important in regulating the immune response. The IgE receptor is responsible for initiating the allergic response which begins with the binding of an allergen to receptor-bound IgE. This binding leads to cell activation and the release of mediators such as histamine that are responsible for the manifestations of allergy (Kuester, H. et al. (1992) J. Biol. Chern. 2b7:12782-12787). The high affinity IgE receptor (FceRI) is a tetrameric hetero-oligomer composed of an a chain, a p chain, and two disulfide-linked y chains. The (3 chain contains four transmembrane segments and long cytoplasmic domains thought to play an important role in intracellular signaling.
Visual excitation and the phototransmission of light signals is another form of signaling cascade in which receptors play an important role. The process begins in retinal rod cells with the absorption of light by the photoreceptor rhodopsin, a seven-transmembrane containing protein composed of a 40-kDa protein, opsin, and a chromophore, 11-cis-retinal. The photoisomerization of the retinal chromophore initiates a biochemical cascade that leads to a reduction in cyclic-GMP
and closure of cyclic-GMP regulated, Ca2+-specific channels in the plasma membrane of the rod.

The resultant membrane hyperpolarization generates a nerve signal. Recovery of the dark state of the rod involves another receptor, recoverin. Recoverin is a Ca2'-binding protein that detects the lowering of cytosolic Ca2+ and subsequently binds to and activates guanylate cyclase. Activation of guanylate cyclase leads to increased cyclic-GMP levels and the reopening of Caz+-specific channels (Stryer, L. (1991) J. Biol. Chem. 266:1071 I-10714).
Abnormal receptor activity is associated with a variety of diseases and disorders.
Abnormal hormonal secretion is linked to disorders including diabetes insipidus, hyper- and hypoglycemia, Grave's disease and goiter, and Cushing's and Addison's diseases. Cancer cells secrete excessive amounts of hormones or other biologically active peptides.
Disorders related to excessive secretion of biologically active peptides by tumor cells include fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors;
hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal cramps, diarrhea, and valvular heart disease, caused by excessive amounts of vasoactive substances secreted from IS intestinal tumors. Tumors may exhibit ectopic synthesis and secretion of biologically active peptides, including ACTH and vasopressin in lung and pancreatic cancers;
parathyroid hormone in lung and bladder cancers; calcitonin in lung and breast cancers; and thyroid-stimulating hormone in medullary thyroid carcinoma.
Inflammation is a molecular, cellular, and tissue program during which foreign substances and pathogens are destroyed, and injured tissue is repaired through a variety of biochemical, biophysical, and cellular mechanisms. The principal cellular mediators of inflammation are leukocytes, particularly granulocytes and the monocytes/macrophages.
Macrophages recognize, internalize, and destroy a variety of foreign (non-self) and endogenous substances and pathogens, including bacteria, parasites, and viruses. The exact recognition mechanism for non-self pathogens is unknown, but it has been proposed that receptors with broad binding specificity are used to discriminate between self and non-self antigens. Macrophages are also thought to play an important role in the immune response by presenting foreign antigens to lymphocytes.
The discovery of new human receptor-associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, reproductive, cardiovascular, and gastrointestinal disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human receptor-associated proteins, referred to collectively as "HRAP". In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the atriino acid sequences selected from the group consisting of SEQ
ID NO:1-16, and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:I-16, and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof.
The invention also provides an isolated and purified poiynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32, and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucieotide sequence selected from the group consisting of SEQ ID N0:17-32, and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:17-32, and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ
ID NO:I-16, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO:1-16, and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of HRAP, the method comprising administering to a subject in need of such treatment an effective amount of a phanmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of HRAP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-16, and fragments thereof.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows nucleotide and polypeptide sequence identification numbers (SEQ
ID NO), clone identification numbers (clone ID), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding HRAP.
Table 2 shows features of each polypeptide sequence including potential motifs, homologous sequences, and methods and algorithms used for identification of HRAP.
Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis, diseases or disorders associated with these tissues, selected fragments of the nucleotide sequences encoding HItAP which are useful as hybridization probes, and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which Incyte cDNA clones encoding HRAP were isolated.
Table 5 shows the programs, their descriptions, references, and threshold parameters used to analyze HRAP.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, 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 present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"HRAP" refers to the amino acid sequences of substantially purified HRAP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which, when bound to HR.AP, increases or prolongs the duration of the effect of HRAP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HRAP.
An "allelic variant" is an alternative form of the gene encoding HRAP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mltNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational _g_ changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding I-IRAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as HRAP or a polypeptide with at least one functional characteristic of HRAP.
Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HRAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HItAP. 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 HRAP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of I~iRAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
The terms "amino acid" or "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. In this context, "fragments," "immunogenic fragments," or "antigenic fragments" refer to fragments of HRAP which are preferably at least 5 to about I S amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of HRAP. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence"
and like tenors are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The team "antagonist" refers to a molecule which, when bound to HRAP, decreases the amount or the duration of the effect of the biological or immunological activity of I-IRAP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules _g_ which decrease the effect of HRAP.
The term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab'~, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind HRAP 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 (ICLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen I S used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation "negative" can refer to the antisense strand, and the designation "positive" can refer to the sense strand.
The term "biologically active," refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic HRAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms "complementary" or "complementarily" refer to the natural binding of polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'." Complementarily between two single-stranded molecules may be "partial," such that only some of the nucleic acids bind, or it may be "complete," such that total complementarily exists between the single stranded molecules. The degree of complementarily between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the WU UU~08155 PCT/US99~17777 design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding.
HRAP or fragments of HRAP 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., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence"refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW Fragment Assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding HRAP, by northern analysis is indicative of the presence of nucleic acids encoding HRAP
in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding HRAP.
A "deletion"refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, 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.
The term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity" may substitute for the word "similarity." A
partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction.
The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30%
similarity or identity).
In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Madison WI). The MEGALIGN program can create alignments between two or more sequences IS according to different methods, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A
and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
"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 stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.

The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" or "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
"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.
The term "microarray" refers to an arrangement of distinct polynucleotides on a substrate.
The terms "element" or "array element" in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HRAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HRAP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, 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. In this context, "fragments" refers to those nucleic acid sequences which, comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:17-32, for example, as distinct from any other sequence in the same genome. For example, a fragment of SEQ ID N0:17-32 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:17-32 from related polynucleotide sequences. A fragment of SEQ ID N0:17-32 is at least about 15-20 nucleotides in length. The precise length of the fragment of SEQ ID N0:17-32 and the region of SEQ ID
N0:17-32 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. In some cases, a fragment, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.

The terms "operably associated" or "operably linked" refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
The tenor "oligonucleotide" refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. "Oligonucleotide" is substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"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.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding I-IRAP, or fragments thereof, or HRAP itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" or "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. 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 containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "stringent conditions" refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of fonmamide, or raising the hybridization temperature.
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 about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes 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, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "variant" of I-IRAP polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to HRAP. This definition may also include, for example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. 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 an absence of domains.
Species variants are poIynucleotide sequences that vary from one species to another. The resulting polypeptides generally will 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 base.
The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
THE INVENTION
The invention is based on the discovery of new human receptor-associated proteins (HRAP), the polynucleotides encoding HRAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, reproductive, cardiovascular, and gastrointestinal disorders.
Table I lists the Incyte Clones used to derive full length nucleotide sequences encoding HRAP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NO) of the amino acid and nucleic acid sequences, respectively. Column 3 shows the Clone ID of the Incyte Clone in which nucleic acids encoding each HRAP were identified, and column 4, the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones, their corresponding cDNA
libraries, and shotgun sequences useful as fragments in hybridization technologies, and which are part of the consensus nucleotide sequence of each HRAP.
The columns of Table 2 show various properties of the polypeptides of the invention:
column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3, potential phosphorylation sites; column 4, potential glycosylation sites;
column 5, the amino acid residues comprising signature sequences and motifs;
column 6, the identity of each protein; and column 7, analytical methods used to identify each protein through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and disease-association of nucleotide sequences encoding HItAP. The first column of Table 3 lists the polynucleotide sequence identifiers. The second column lists unique fragments of the nucleotide sequences encoding HRAP
which are useful as hybridization probes. The third column lists tissue categories which express HRAP as a fraction of total tissue categories expressing HRAP. The fourth column lists the disease classes associated with those tissues expressing HRAP. The fifth column lists the vectors used to subclone the cDNA library.
The invention also encompasses HRAP variants. A preferred HRAP variant is one which has at least about 80~/0, more preferably at least about 90%, and most preferably at least about 95%
amino acid sequence identity to the HRAP amino acid sequence, and which contains at least one functional or structural characteristic of HRAP.
The invention also encompasses polynucleotides which encode HRAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ 1D N0:17-32, which encodes HRAP.
The invention also encompasses a variant of a polynucleotide sequence encoding HRAP.
In particular, such a variant polynucleotide sequence will have at least about 70%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HRAP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:17-32 which has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:17-32. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HRAP.
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 HRAP, 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 colon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HRAP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode HRAP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HRAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HRAP or its derivatives possessing a substantially different colon usage, e.g., inclusion of non-naturally occurring colons. Colons 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 colons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding HRAP and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode HRAP
and wo oorosiss pc~.~us99im~»
I-IRAP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding HRAP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID
N0:17-32 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M.
and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM
NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at feast about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ~cg/ml denatured salmon sperm DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM
NaCI, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art.
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 polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (Perkin-Elmer), thermostable T7 polymerise (Amersham Phanmacia Biotech, Piscataway NJ), or combinations of polymerises and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence preparation is automated with machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno NV), Pettier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377 DNA sequencing systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA). The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular BioloQV, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding HRAP 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. (See, e.g., 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. (See, e.g., 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. (See, e.g., 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. (See, e.g., Parker, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon wo oorosiss Pc-~.~s99i1~~~~
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 MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), 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, polynucleotide sequences or fragments thereof which encode HRAP may be cloned in recombinant DNA molecules that direct expression of HRAP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HRAP-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 colon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding HRAP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucl.

Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232.) Alternatively, HRAP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the.ABI 431A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of HRAP, 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.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. ( 1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY.) In order to express a biologically active HRAP, the nucleotide sequences encoding HRAP
or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding HRAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HRAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding HRAP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcripdonal 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. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HRAP and appropriate transcriptional and transiational control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. ( 1989) Molecular Clonin~Laboratorv Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. el al. (1995) Current Protocols in Molecular Bioloev, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding HRAP. 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. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HRAP.
For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding HRAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding HRAP
into the vector's multiple cloning site disrupts the IacZ 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. (See, e.g., Van Heeke, G.
and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of HRAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of HRAP
may be used. For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HRAP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, 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 sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra;
Grant et al. ( 1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. ( 1994) Bio/Technology 12:181-184.) Piant systems may also be used for expression of HRAP. Transcription of sequences encoding HRAP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
( 1987) EMBO J. 6:307-311 ). Alternatively, plant promoters such as the small subunit of RUBISCO or wo ooiosiss Pcrn.rs99n~~~~
heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. (1984) Science 224:838-843; and 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. (See, e.g., The McGraw Hill Yearbook of Science and Technolo~v (1992) McGi~aw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding HRAP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E 1 or E3 region of the viral genome may be used to obtain infective virus which expresses HRAP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat Genet.
15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of HRAP in cell lines is preferred. For example, sequences encoding HRAP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk or apr~ cells, respectively.
(See, e.g., Wigler, M. et al. ( 1977) Cell I 1:223-232; 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 G-418;
and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
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. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,.anthocyanins, green fluorescent proteins (GFP; Clontech),13 glucuronidase and its substrate 13-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C.A.
(1995) Methods Mol.
Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding HRAP is inserted within a marker gene sequence, transformed cells containing sequences encoding HRAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HRAP
under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding HRAP
and that express HRAP may be identified by a variety of procedures known to those of skill in the art.
These procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for detecting and measuring the expression of HRAP 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 HRAP is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art.
(See, e.g., Hampton, R. et al. ( 1990) Serological Methods. a Laboratory Manual, APS Press, St Paul MN, Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in ImmunoloQV, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols. Humans Press, Totowa NJ).
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 HRAP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding HRAP, 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 polymerise 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 Phanmacia Biotech, Promega (Madison WI), 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 nucleotide sequences encoding HRAP 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 HRAP may be designed to contain signal sequences which direct secretion of HRAP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences 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"
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, Hel:,a, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Manassas, VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding HRAP 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 HRAP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HRAP
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 immunoafl~;nity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the HRAP
encoding sequence and the heterologous protein sequence, so that HRAP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel ( 1995, sutira, ch 10). A variety of commercially available kits may also be IO used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled HRAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (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, preferably "S-methionine.
Fragments of HRAP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, sera. pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Various fragments of HRAP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of HRAP and human receptor-associated proteins. In addition, the expression of HRAP is closely associated with cell proliferative disorders, such as cancer, and with inflammation and the immune response. HRAP are expressed in libraries from cancerous tissues, hematopoietic tissues, reproductive tissues, cardiovascular tissues, and gastrointestinal tissues (Table 3). Therefore, HRAP appears to play a role in cell proliferative, autoimmune/inflammatory, reproductive, cardiovascular, and gastrointestinal disorders.
Therefore, in the treatment of disorders associated with increased expression or activity of HRAP, it is desirable to decrease the expression or activity of HRAP. In the treatment of disorders associated with decreased expression or activity of HRAP, it is desirable to provide the protein or to increase the expression of HRAP.
Therefore, in one embodiment, HRAP or a fragment or derivative thereof may be wo ooiosiss rcrius99n~~~~
administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HRAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, 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, a cancer 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;
and an autoimmune/inflammatory disorder such as actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic denmatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjdgren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.
In another embodiment, a vector capable of expressing HRAP 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 HRAP including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified HRAP 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 HRAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of HRAP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HRAP including, but not limited to, those listed above.
_2~_ wo ooroaiss rcims~il~~~~
In a further embodiment, an antagonist of HRAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HRAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, 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, a cancer 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; an autoimmune/inflammatory disorder such as actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic purpura, ulcerative colitis, uveitis, Wemer syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and heiminthic infections, and trauma; a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, an autoimmune disorder, an ectopic pregnancy, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male breast, and gynecomastia; a cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, and vascular tumors, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, and congenital heart disease; and a gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, an infection of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, and acquired immunodeficiency syndrome (AIDS) enteropathy. In one aspect, an antibody which specifically binds HRAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HRAP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HRAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HRAP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary 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 HRAP may be produced using methods which are generally known in the art. In particular, purified HRAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HRAP.
Antibodies to HRAP 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 wo uoiosi<ss hcr~rs99ii~~~~
inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HRAP or with any fragment or oiigopeptide 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 lysoiecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to HRAP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, 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 and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HRAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to HRAP 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. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;
Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci.
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Mornson, S.L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HRAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad.
Sci. 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. ( 1989) Proc.
Natl. Acad. Sci. 86:

wo oorosiss Pc~./usml~~~~
3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for HRAP may also be generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. 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 HRAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HRAP epitopes is preferred, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for HItAP.
Affinity is expressed as an association constant, K" which is defined as the molar concentration of HRAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K, determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple HRAP epitopes, represents the average affinity, or avidity, of the antibodies for HRAP. The K, determined for a preparation of monoclonal antibodies, which are monospecific for a particular HRAP epitope, represents a true measure of affinity. High-affinity antibody preparations with K, ranging from about 109 to 10'Z L/mole are preferred for use in immunoassays in which the HItAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K, ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of HRAP, preferably in active form, from the antibody (Catty, D.
( 1988) Antibodies.
Volume I: A Practical Approach, IItL Press, Washington, DC; Liddell, J. E. and Cryer, A. ( 1991 ) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
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 S-10 mg specific antibody/ml, is preferred for use in procedures requiring precipitation of HRAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.) In another embodiment of the invention, the polynucleotides encoding HRAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding HRAP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HRAP. Thus, complementary molecules or fragments may be used to modulate HRAP activity, or to achieve regulation of gene function.
Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or contml regions of sequences encoding HRAP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HRAP. (See, e.g., Sambrook, supra; Ausubel, 1995, suvra.) Genes encoding HRAP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding HRAP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding HRAP. 0ligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, are preferred.
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. (See, e.g., Gee, J.E. et al.
(1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

wo ooiosiss pcr,~s99u~~~~
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 sequences encoding HRAP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These IS 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 sequences encoding HRAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
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. (See, e.g., Goldman, C.K. et al.
( 199Z) Nature Biotechnology 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HRAP, antibodies to HRAP, and mimetics, agonists, antagonists, or inhibitors of I-IRAP. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra-arterial, intramedullary,~ intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, IS enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurnes, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.

Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identiftcation or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable iipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, tactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1 % to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of HRAP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical 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.
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, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example HRAP or fragments thereof, antibodies of HRAP, and agonists, antagonists or inhibitors of HRAP, 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 EDT (the dose therapeutically effective in 50% of the population) or LDP (the dose lethal to 50% of the population) statistics. The dose ratio of therapeutic to toxic effects is the therapeutic index, and it can be expressed as the ED~/I,Ds° ratio. Pharmaceutical 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 EDs° with tittle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0. I ~g to 100,000 /cg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.

Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HRAP may be used for the diagnosis of disorders characterized by expression of HRAP, or in assays to monitor patients being treated with HRAP or agonists, antagonists, or inhibitors of HRAP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for HRAP include methods which utilize the antibody and a label to detect HRAP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring HRAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of HRAP
expression. Normal or standard values for HRAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HRAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of HRAP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding HRAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HRAP
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of HRAP, and to monitor regulation of HRAP
levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HRAP or closely related molecules may be used to identify nucleic acid sequences which encode HRAP.
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 (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurnng sequences encoding HRAP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the HRAP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:17-32 or from genomic sequences including promoters, enhancers, and introns of the HRAP gene.
Means for producing specific hybridization probes for DNAs encoding HRAP
include the cloning of polynucleotide sequences encoding HRAP or HRAP 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
polymerises and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as'ZP or'sS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding HRAP may be used for the diagnosis of disorders associated with expression of HRAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, 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, a cancer 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;
an autoimmune/inflammatory disorder such as actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjiigren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, 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 reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, an autoimmune disorder, an ectopic pregnancy, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male breast, and gynecomastia; a cardiovascular disorder such as arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, and vascular tumors, congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease, and congenital heart disease;
and a gastrointestinal disorder such as dysphagia, peptic esophagitis, esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral or pyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, an infection of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis, passive congestion of the liver, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, and acquired immunodeficiency syndrome (AIDS) enteropathy. The polynucleotide sequences encoding HRAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR
technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patients to detect altered lvIRAP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding I-IRAP may be useful in assays wo oorosiss ~.~us~n~»~
that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding I-IRAP may be labeied 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 quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HRAP in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with expression of HRAP, 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 I-IRAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding I-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 HRAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding HRAP, 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 quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of HRAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
(1993) J. Immunol.
Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microamay. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and 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, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et al. ( 1996) Proc. Natl.
Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc.
Natl. Acad. Sci.
94:2150-2155; and Heller, M.J. et al. ( 1997) U.S. Patent No. 5,605,662.) In another embodiment of the invention, nucleic acid sequences encoding HRAP
may be used to' generate hybridization probes useful in mapping the naturally occurring genomic sequence. 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. (See, e.g., Harnngton, J.J. et al. (1997) Nat Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7: i49-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., 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) site.
Correlation between the location of the gene encoding HRAP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA
associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
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 number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, HRAP, 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 HRAP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al.
(1984) PCT application W084/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with HRAP, or fragments thereof, and washed. Bound HRAP is then detected by methods well known in the art.
Purified HRAP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HRAP specifically compete with a test compound for binding HRAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HRAP.
In additional embodiments, the nucleotide sequences which encode HRAP 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 wo ooiosiss rcrius~n~~~~
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.
The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in anyway whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. [Atty. Docket No. PF-0571 P] filed August 7, 1998, and U.S. Ser. No.
60/098,703, filed September 1, 1998, are hereby expressly incorporated by reference.
EXAMPLES
I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life IS Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI 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, Valencia CA), 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 TX).
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 (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription was initiated using 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 Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the wo ooioaiss rcrnrs99n~~~~
polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTI plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH 1 OB, or ElectroMAX DH 1 OB from Life Technologies.
II. . Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector system (Stratagene) or cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep pwification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, Pius Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep 96 plasmid kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4 °C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCB in a high-throughput format (Rao, V.B. ( 1994) Anal. Biochem. 216:1-14). Host cell lysis and IS 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 OR) and a Fluoroskan II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Bobbins Scientific) or MICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI
protocols, base calling software, and kits. In one alternative, cDNAs were sequenced using the MEGABACE
1000 DNA sequencing system (Molecular Dynamics). In another alternative, the cDNAs were amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced using solutions and dyes from Arnersham Pharmacia Biotech. Reading frames for the ESTs were determined using standard methods (reviewed in Ausubel, 1997, ssupra, unit 7.7). Some of the cDNA
sequences were selected for extension using the techniques disclosed in Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table 5 summarizes the software programs, descriptions, references, and threshold parameters used. The first column of Table 5 shows the tools, programs, wo ooiosiss Pcr~s99n~~~~
and algorithms used, the second column provides a brief description thereof, the third column presents the references which are incorporated by reference herein, 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 probability the greater the homology). Sequences were analyzed using. MACDNASIS PRO software (Hitachi Software Engineering, S. San Francisco CA) and LASERGENE software (DNASTAR).
The polynucleotide sequences were validated by removing vector, linker, and polyA
sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation, using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and 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 amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, PFAM, and Prosite.
IV. Northern Analysis 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. (See, e.g., Sambrook, supra, ch. 7;
Ausubel, 1995, s_ upra. ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). 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:
%~s uence identity x % maximum BLAST score The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar wo oorosiss molecules are usually identified by selecting those which show product scores between IS and 40, although lower scores may identify related molecules.
The results of northern analyses are reported a percentage distribution of libraries in which the transcript encoding HRAP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The orgat~/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease categories included cancer, inflammation/trauma, fetal, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease expression are reported in Table 3.
V. Extension of HRAP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:17-22 were produced by extension of the component fragments described in Table I, Column 5, using oligonucleotide primers based on those fragments. One primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide.
Primers were used to facilitate the extension of the known sequence "outward"
generating amplicons containing new unknown nucleotide sequence for the region of interest. The initial primers were designed from the cDNA using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (Life Technologies) were used to extend the sequence. If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region.
High fidelity amplification was obtained by following the instructions for the XI,-PCR kit (Perkin-Elmer Corp.) and thoroughly mixing the enzyme and reaction mix. PCR
was performed using the PTC-200 thermal cycler (MJ Research), beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:

Step 1 94 C for 1 min (initial denaturation) Step 2 65 C for 1 min Step 3 68 C for 6 min SAP 4 94 C for 15 sec Step 5 65 C for 1 min Step 6 68 C for 7 min Step 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94 C for 15 sec Step 9 65 ° C for 1 min Step 10 68° C for 7:15 min Step I I Repeat steps 8 through 10 for an additional 12 cycles Step 12 72 ° C for 8 min Step I 3 4 ° C (and holding) A 5 /cl to 10 ~l aliquot of the 'reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6% to 0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using QIAQUICK purification kit (QIAGEN Inc.), and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
Ai3er ethanol precipitation, the products were redissolved in 13 ~cl of ligation buffer, l~l T4-DNA ligase (IS units) and 1~1 T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16° C.
Competent E. coli cells (in 40 /cl of appropriate media) were transformed with 3 ul of ligation mixture and cultured in 80 ~cl of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C, the E. coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sarnbrook, supra, Appendix A, p. 1 ) containing carbenicillin (2x carb). The following day, several colonies were randomly picked from each plate and cultured in I50 /cl of liquid LB/2x carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ,ul of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 ~1 from each sample was transferred into a PCR array.
For PCR amplification, 18 ul of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerise, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
Step I 94 C for 60 sec Step 2 94 C for 20 sec Step 3 55 C for 30 sec Step 4 72 C for 90 sec Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6 72 C for 180 sec Step 7 4 C (and holding) Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and appropriate clones were selected, ligated into plasmid, and sequenced.
In like manner, the nucleotide sequences of SEQ ID N0:17-22 is used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and _97_ WU 0008155 PCT/US99~t~~~~
an appropriate genomic library.
The full length nucleic acid sequences of SEQ ID N0:23-32 were 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; 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 SO% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)ZSO4, and ~-mercaptoethanol, Taq DNA polymerise (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (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, I 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, I min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ul PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1 XTE and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), 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 S ~cl to 10 ~cl aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). 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 _98_ clones were relegated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerise (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerise (Amersham Pharmacia Biotech) and Pfu DNA polymerise (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequence of SEQ iD N0:23-32 is used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:17-32 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 oiigomer, 250 uCi of [Y aZp] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 supe~ne size exclusion dextrin bead column (Amersham Pharmacia Biotech).
An aliquot containing 10' 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, Xbal, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to the blots to film for several hours, hybridization patterns are compared visually.
VII. Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array 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 by hand or using available methods and machines and contain any appropriate number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarily and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynncleotides Sequences complementary~to the HRAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HRAP.
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 HRAP. 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 HRAP-encoding transcript.
IX. Expression of HRAP
Expression and purification of HRAP is achieved using bacterial or virus-based expression systems. For expression of HRAP 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 TS 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 HRAP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of HRAP 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 HRAP 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 fru ~~iperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl.
Acad. Sci. USA
IS 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.) In most expression systems, HRAP 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 ia~onicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be proteolytically cleaved from HRAP 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 ( 1995, supra, ch 10 and 16).
Purified HRAP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of HRAP Activity Receptor activity of HRAP is determined in a ligand-binding assay using candidate ligand molecules in the presence of '~I-labeled HRAP. HRAP is labeled with'ZSI
Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate ligand molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HRAP, washed, and any wells with labeled HRAP complex are assayed. Data obtained using different concentrations of HRAP are used to calculate values for the number, affinity, and association of wo oarosiss »,NS~iI~~~~
HRAP with the ligand molecules.
Alternatively, HRAP activity is determined by measuring the stimulation of DNA
synthesis in Swiss mouse 3T3 cells. Plasmids containing polynucleotides encoding HRAP are added to quiescent 3T3 cultured cells using transfection methods well known in the art and the transfected cells are then incubated in the presence of [3H]thymidine, a radioactive DNA
precursor. Varying amounts of HRAP ligand are then added to the cultured cells. Incorporation of ['H]thymidine into acid-precipitable DNA is measured over an appropriate time interval using a radioisotope counter, and the amount incorporated is directly proportional to the amount of newly synthesized DNA. A linear dose-response curve over at least a hundred-fold HRAP ligand concentration range is indicative of receptor activity. One unit of activity per milliliter is defined as the concentration of HRAP producing a 50% response level, where 100%
represents maximal incorporation of ['H]thymidine into acid-precipitable DNA (McICay, I. and Leigh, L, eds. (1993) Growth Factors: A Practical Approach, Oxford University Press, New York, NY, page 73).
XI. Functional Assays HRAP function is assessed by expressing the sequences encoding HRAP 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 (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.
5-10 ~cg of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 ug 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 properties, for example, their apoptotic state. 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 wo oorosiss pornrs~n~~~~
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. ( 1994) Flow Cvtometry, Oxford, New York NY.
The influence of HRAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HRAP 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 NY). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding HRAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of HRAP Specific Antibodies HRAP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HRAP 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. (See, e.g., Ausubel, 1995, su~,ra, ch. 11.) Typically, oligopeptides 15 residues in length are synthesized using an ABI

Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH
(Sigma-Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-ICI,H complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring )(TRAP Using Specific Antibodies Naturally occurring or recombinant HRAP is substantially purified by immunoaffinity chromatography using antibodies specific for HRAP. An immunoaffinity column is constructed by covalently coupling anti-HRAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing HRAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HRAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/HRAP 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 HR.AP is collected.
Various modifications and variations of the described 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. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

~o . .
.

w ~ ~ ' N M I~ eh ~

w N E H . N 0 10 .H

~

M V~ O M ei In w ' M
O

~ ~ ~ O
x . c p~ ~ ~ I~ N 00 O V7 z 01 ~ ~ M ~ 0 a n e-I U N ~ i x w _ N ,1 ~ A' sr . ,- o '' -ii o o C-0 ~

c OD N M w ri N o1 1 --N e-i sr O 10 . ~ -I 10 vD

x ~ . .1 ~O
~ fY. _ '-i N w _ ' ~.O v-i ' O .
CO D O~ If1 H O O N .~ N
O O

H 01 E .~ w o H ~n .~ o a H
o ao ~ ~ N a ~
~ n o ~ O l0 O In r-I O r-1 O E~ O
N ~ ~ 1f1 ~ GO

N ~ O
Ul (~ O ~ z N ~
N

M ~ ~ ~ N

' " a a o v o r~ a ~ w -I _ v ~~ ~ x~ _ ~ v ~

e ~ d M rl ~ N l~ W N , -I 00 , O ,-~

e O n-1 ~--f ~ O 1p r O .~ O ,1 O O ri N r.i O p lf1 I~ Lf1 M z p r-I 10 ~ O O
rf 10 N , ~ H x w M M Pa M 00 f~
l~ 1 01 00 ~ I~ E
U1 l~

~

w e P v-1 M 01 l~ 01 I~ l0 a U1 I~ ~ (Z,' ~ UI
i M w o ai er w ~

. m o a o N n H ~
e1 . , u m E
.- -~

' N -1 N ~ M 00 N N '-' ri . W ~ M ~ ~ '-' v-I
M cr ~ cn ~ w ' 'w o r ~ H W ~ _ . . ,.. wo ~ N .
E ... ~ _ ,~ ~ w O

e-i !11 U1 N .-' '-I ... . .~ CwD
OO lf1 v-i e1 ri tx N '- 1xp OD CO i l~

O t~ O O !n r-i O If1 r-I e-1 N
M O O O (~ In l17 l~ N r-1 0 U ~ E N O Ea O O O t~
~ ~' ~ O 10 V~ O M M O 10 E
V7 fll O tIf 1 ~ O W ~ '"'~ O [-~ O
w '.~ N O
E ~ N
a O1 W If1 U~ M N VI ~, 'i N
O rl H N ~ .~.~
~ W
-~ ~ O e-1 PG ~ N N
M x e-I fn H CJ ~
o ~
w o ~ . ~n x . ~ > _ a ' E ' -i -a y ~ ... ~ O ~ .-' U ~ ~ ~ ~ ~
..

. . c l v v t0 e-1 10 r r O W n-1 O v-1 O rl -1 4; x w O v-1 -m N OW O
~ E w x . ,~ ~ , x ~-, ,-, ~ E x ~w ~o w E x o E
N xo~ ~o x~

a~o x xo~ Nw w ~ Z H

H 10 ~ IJ1 er f~ l~ l~ 10 N
O t~ t0 ~r M (l1 CJ
10 ~ rn oo ~ o0 O
M ~n n O O pf, O O O M p; ~ 1p .~ H ~
~r O o~ CD O , ~
v ~o M a ~ oa '~
w o o, H ~ ~r oo ~ ~ w M
H 01 N e-1 ~ M a w 'd~ oa a N M N ~ ~ tf1 e-1 '~ I~ ~
~ Vl N

M ll1 tl1 r1 OD l~ 111 e-i f'~ N

~'' E' ~ H H E H

rt C'J f/1 ~

H
a ~ o o ~

a E > o a x ~Z r a o c a a. ~
w 1 ' ~ N 1D ~ " ~-i tn N

l ~ I~ 00 O
~O ~

Q) 01 O~ C' l0 ~ ~ M t~ N

Q '~

l O O OD O t~ ~ N if1 t0 00 01 01 O ~ ~ O N N M

V H N N M M M ~ ,1 -~

z O t~ 00 01 O r! N M 'Ch lf1 A

H '"~ ~"~ f"'~N N N

r~
N N N N

U
d W

zw ~

z ~
A

H ~ N M d~ In 10 h OD 01 O

H

W
W

W

PG'IYtJS99/17777 ' _ _ _~ _~ E
M rl M d~ N '-I N
e-1 10 O e1 O .-I '-1 r~ O N N E .-i M ~ O ~C u~ M ~
N M ~ (~ [-~ O ~ O H N ~ 4l1 '~-1 r' M 01 O 01 ~l,' O x e~ d~ ~ M '(f O 00 O al OD al 01 [~ I~ al 1p a N t.,"
r-1 N M '-' !n ~ ri ~~ tn V N ~ N IO

'x-, .W _.x .~.~ .vc x ~x~w ~ V~ N ~. wl M N
r-i V~ O 01 01 M N 01 O OD ~ OND rl M O N
E oo E o N E o 00 (-~ o M .-I
~0~0 EN O~ oN ~~ ono H N ',~ HO N ~ C~.h N ~ N "~., N ~ a-I ,'~ '~-1 vl H . W W
°° .'~ a owc ~ .°u '. c~ ~. o, m o, ,v a ~: ~. ~ .., ~ ,. ' w H x H .-i x '",i N vo o ~ o w o .--i ~
~, ~o,Zo~o w~aN xo~ ~o ~o N~N~
M H ~-~I tEll O O~ ~ tn ~ 'E-I aO ~ O Vz1 l~ ~7 O ~ 01 O r-1 M ~ Ov 01 L~ lp O
U~~ ~w~ ~a o~oEx-~ ME SOD
e-I N N ~ d~ N ~ ,-~ ~. .~ .~ M .". N "r O~
W . ~O '-1 ~O . e-I . v-1 . 10 ~ W .... [~, . ~, .-. _ _ _ 01 01 M l0 .-. M ch .-. ri tf1 N ~ ~ ,-I N M a-i N
O vD O ~O v-1 O O sr L~ ~ Op M M o O I~ O I~
E N H .-~ o ~n E, o M E o o u~ E E ~c ~o O o O tn Ew ao W E an O wo O p m o0 H ~"~ ~ M z rl ~ O N O N
U f'-1 ~ a-i M W fl.' ,p ~ ,~ ~ .A ~ .w O "~"~ ~N-- aN ~, ~~-- ,-~....:...
U w ~ ~ o o ~ N vo o wo .~ o ~ 0 0 Mo~~ ww~ ao ~ '°~w xHx~E
~z~ ~ ~ ~,z o ~z~ ~~ o z N H 00 ~ CO er ~O II1 01 tn N 10 N '-1 () W
00 ~ N C~ l0 ~ 00 M M ep O 01 O
LY N ~ pC, Npp.M-iA oUa~o ~~ ~U ~WoMO ~ ~~U
N O N N ~ .-I ~ 01 ~ N M ~ M
M rl N cf~ N N
H z a H ~O dW -1 N N
O ~ N M O ,-.pp O M M 00 U1 l0 r-I '"~ u1 In 10 U N N N N N M
'b O
~.~ z A t'~ CO 01 O ,~ N
N N N M M M
r~
ua ~w z ~n .~ z°
A H N M cr V1 H W -1 .-1 rl .-i O
~' a ~w '., x '~~x ~ ~'~d~ '~~av~Za ~
~

, p ' '."1 N ' p,P. P~ ~7 04 ~ ~ a1 Lx GO G4 a' ~

44 W ~

LL

N

O 1 ~ 0 . a ~ o o _ N U _ _ O
~ N U ,-i ro U ~ O U OD I o ~," yp d~

N o ~ N o -~ HM u~lt~oN~

-girl ,-I p ~ " o O

. r-1 .1 .1 (, ~ r J.
~ -I J

G1 ~G! H N ~ ~, H N
ro ro ' ~ ' ~

H .~ w H t7 ~ ~ ~?' ~ ~ ~ t~

.

ar a~
U

b1 N
b N

U O -rl N
~

O 1 ~ ..
,j~ ~ 01 Q O
~i O

0 O ~ H ~ ~i ~''' G O U

!

~
' ~ ~

fV ' ~ a z w ""I "
~
v G7 E ,.1 E

~, O

d~
O

ro z z N
~o ro w ao ,-1 .~
ro z zzz M
1J M O ~
'~I

W C7 z N N O ~ N
Cl~ M l0 e-I

z z zzz zz O O M M r-I N
ri tf1 ' ~ OD lt7 lI1N If1 N I

O E~ N ~ ~ ~ N E'' ~ E
~ E

O M U1. . '-1 r-1 [~r M
-1 -I !l1 v-1 ~ OD
I'' r1 M e)t UI M VI E Ll1 10 In e!
O

~ ~ E ~ N N
E H
E

t . ~ ,. ~ t /1 -i ..I l1 ~

ro r E E ~ ~ E W n ~-I o N a~

s ,. .
rl a M .~ -1 d~ M v-1 O t0 (W CO N 1D
N -I
M

v-I W -I N M
a~ .-i 0oM v~ H E E M
~ t4 t4 ~ t~ v1 tn ~n M 01 '-1 J~ N O ~ 10 01 C1 v-i tJ1 M M 00 01 O ~'.,M N M N e1 (W -1 c!t 1p rl N N ~

-/ N M

w fi, W c~ m try c~ t~ E r cn E E , v1 ~ E E c~
E

N

N

.~ u1 L~N 01 I~ AD
r~

~ M ~
!

f d M M M M

N

P,' .~
z a~
A

N M ~N l11 ~O

WO 00/08155 PG"f/US99/17777 H ~ H ~ H ~ ~ H ~ ~
x ~ ~

H H
~ ~ ~ ~ ~ ~
~ ~

O O G~ W O O O
'~'~' ~ ~ ~' ~ ~i W' c~, ~
~

~, ~ QI ~r a p y o v ~ o a v ~ ~ ~'' ~ ' ' a ' ro ~ '' ~ ~
O ~ ~
, ;
~

tC ~ U a U
O

~ .~u ~ m ~ Ei N ov4 p, a~

U

O

O QJ O

N ..~ 't7 .rl ri O
N M M ~ N ~, a H H

sr H a N ~
u1 N

e ,~; ~ 7 ~ ~ d, E .-y x 01 ri C ~ '-W -i ~ ao ~

~ ., M In ri p ~ ~ ~"~ c? a N
~ A ~i ~ ~ ' N ~ ~ ~ ~
O N ~ ~

V Y~ - ~
C cn H -1 .7 (~ ' cx N

o .a cd ~ ~ N z ~ o N

N N M
o z zz z u~
y ll1 N ~D
~

O r ch rl M d~ '-i e-1 l --rlz z z N

zzz z 00 OD ~ N 00 N
N l~

o H N can can ~
H ~ E ~

p .-i ~ H N E
~

O u1 E w o m twn t~ .-I E v1 c .-~
l~ M r M IW-1 cr U1 (y l'~ M I~
JJ W -1 dW -1 10 l N ~D d~
N d~
~

N tn y-~1 N M
F f.~., ~ ~ E N ~ E N
~ u1 ~ ~ t4 ~ E
~ E c4 m nN u~o~o~~a~o~ E~ E N
~ ~N

O 00 00 In N N d ,-~ a O
~ N N d~ M 01 rl o a O v1 W -1 N c-i e-1 . ~ E E uI ~ E ~ E u) E E
~ a u1 ~ u1 E
~n N G) ~ ~N 1D 01 ~ M M 01 ~ M ~O 1D ~

O 1~ L 01 ~ M M 01 If1 M l~ 01 O ,T; l11 N M d~ OD I~ N rl ,~ 01 u-1 N ~"'~ O
r~l l s~ M i -1 v-i ~
rl r r e-1 ,...pl w c!~ W t~ E c~ E I E v~ r tn E ~ r-I E t~
V1 ~ En v~
a~ H

O

.-i o o, 0 0 d' ca tW o o~ u~
M N 'd~

N ,-I er ~

z .,, v A t~ 00 01 O ~ N

O ~ ~-1 .-a '' a ~ w a b E W
E E

p, H H
~
~

b m ~~ ~o~'~ oa ~o a~
~

~ ov w w ~

O

ro ,Q

~ a i a ~ .R r ~ j U

v r E

H H N S-i G; O

N ~D
U

M ri O! 'i U7 t rl N ~ 1p ~ O sr d .Na v ~ ~ a ~~' ~.I oo . a M
01 N ~ r-1 ' ~
p ' , sr ~ o ; a ~ a ~ , ; ~ o N
,, ~ N
~ ~ .~ '~
1 C~7 o r"~ y n ~, . .- ,, , . r n ~ a a >

~ E E ro ~ ~ o dmo ~ ~

V ~ ~ '" tr~ M c. ao '~ -E H ~J E H H

N

z z 'fit 1p M
1) O ri I~ N
rl ~cn~n z z lf1 E ~

~ M E N ~ C
-~

O N E vo M
rl 01 tJ1 sr 01 ~ tf1 sr M N ~ ~ N M

fO E H E ~ E E
. E
En E ~

lf1 GWD ~ ~ U7 l17 p -i N lf1 JJ ~ ~ v E ~ N M
~ ~ ~ ~ N

~ E W E

N 4) tn E ~ ~
1f1 'd~ ri E U1 tf1 M
O 1! M p r-I ep ~ 1f1 O
~ H N v-i w-I

~v cn~ ~alt~n Hc~r EE

~

1 c n a~

d b o vo N d~

U -r1 d' ~ cW o N

M rl M

~

z .,., y Ca M d~ 1f1 1p H

O r-I e-1 v-I rI

~' a ' w w p ~ ~ ~ E M ~., ~.~., E-~

" z z z o ~ z o 0 U

~

a a , ~ ~, w o0 N

_ _ O O

01 CO _ _ 01 M M O .~ e-1 ,O v (d ~ N N M ~ ~ ~'a ,-1 ri ,~-"

yJ ' -,~
O O O O

_ O O JJ O 1.!
V O

M O r-I ~ ~ ~ " .-. _ .., ~ ...

N w 4; OD Ci M f"" l~ O tll v Gi ( M ~.,' ll1 e1 f"'., ~' ' N
M In N In O O M d~ O M M O cn . w O r ~ O .-i ~ O O N O W
.-i O O

r1 ~ . .,.p-I
O O N . ~ ~

~ O 1J O iJ O 1J O JJ rl J-1 O 1J rl O

O O ~ O O ~ O O
_ ~

10 N v 10 N v r1 N rl rl a a U ri O

v (fS r-i U r-1 r-1 U r-WOU ri t0 ~ -1 U rt U 1~ U r-I 1 ,-1 .-I

t~. ~ rt ~ ~ ~ ~ ~ '~-' ~ ~ ~ w , H v .u w r w ~

A "' H U U H H U ~ ~ ~ F-~W[~, V H H
Ea fs, E U H ~ V

., U

M
m ~, ~:, ,. ..

M
M d~

_ M H d~
O C1 O ~ -. O O N
N N . .-.
O ~ .. N

O ~ ~ ~-1 O fV IV tIf O O N
O O ~

~ O O N ~ O N O
N

4~, M N ~ M N '-1 td -1 e d~ ~ O M
~ ~ ~ O

-rl I~ O la b O O b O ~. -1 O O .-.

r O .. H b tn w ~ ~ O~ w ~ w ... ... O H O
Ei ~ ao ~0 -~
H N ~
u~

U! U -rl rl U r1 U rl u1 IJ t . 01 U -ri N w rl N JJ -rl ri O ~-1 (a U ~
J..1 v-1 1~ 01 ri N v N N J.J i N

r N -rl rl J..
N O JJ "J Ll iJ 'J i "~' ' N
W N Ul 5 Ul :~ O 7 ' 'J ',~ J

, r , 1.~
v v r~ v v v ri l O - i W
~ v .-I v - J
O

r N U -ri v N
('., rl JJ -ri -ri U ~ O -rl J-~ JJ 1J 1 J ~ v IJ ~-- JJ 1 LJ

. J aG J-~ -ri O C; U G O ~ . !II N ~ J-~
U U O U -rl 1J
G

U td uI U O O ~
a ~ ~ 4f U
~ ~ U

- a -,~ a ~ ~ v~ o ~ o ~ a -.~
v ~ o ~n a -,~ > -~, a a o ro ro o o o ro ro .-~ ro 0 o U o ro o ~ o ~ ro ~ o v, 0 ~ s~ o s~ ~ s~ ~ o o o a ~ 0 0 y s o o o s~ -.~ 0 ~ o ~n ~ s~ ~ a +r s~ s~ U ~
ro ~, ~ >
ro ~ ro s~ ~ N
~w ro~ ~~ ~ ~ > ~
a ~

- v ro~ r a v~~ ~~N ~v rtaar a E x c~ x c~ x c~ x x a z z U x z x c~
x z x c~ U x x x ~ro a a v o, U

W ~

v a-i M

ro M

U z v ,~, v v.

roo -~
z O Ca I~ 00 01 O ~-I N M 'd~

H H ~"~ ~ N N

N N N

ua z n p ~ ~ H ~ ~ E

z z ~

v o z w a a p j r p, , n w w N e-I O If1 OD -i 01 ~-1 O \O M 10 N v L~

sr d~ 10 O Ln . .
O O O O O O

O .-. .-. ~ _ _ ri M I~ M v O

O O M O M
M M

-~ ~ ~ O M O N

E ~ tJ .IJ JJ JJ JJ 1) JJ O
O O O O O O

m N ~ ~ ~ '.' rt qy "
'~

~ a~ y, v ~ ~
~

o ~ a v ~ o a ~ a o o w o w w o w o w o w o .~ o . ..~ .~' -~ -, .'' ..' -~ ~ ~

a~
0 o m ' a a ~ ro ~ .~ ~, m ~ ~ w r-i ~ w ~ w ~ w ~ w w w A " U U H H H

H U U U U !-1 U H U H

U

~o ~ io 0 O N O
M N

~-I M M

o o o ~ o u, b' U v '-' .~ ~ N ~ ..
N

1 ~ N L'~ .
t11 N Gl IW --I ~ ~ ~ W -I W -1 y-I t~ Ci O t ... C.." O
~ I~
O
d~

OD 10 M ~
~ N O y-1 e-1 N e-1 ~-i .. O
~ O ~ O -1 J ri r y ~ H ~ O O H ~ H Rf ~ ~
~ b .~. O O v O H

m E ~ .-.
m ~ a M ~ ~ v V O
v 0 of U rt o 1 U -~ it o - D U -r ~
U ~

1-1 N N -rl M N G1 ri rl M O N rl O rl J-1 ri r-1 l~ JJ ri O) ~ ~ N ' n-1 N N
' ' !

r N ,~ ~ J 1~ JJ . ...
O - ,~ m m l U 1 1J 'J 'J
l r G O ri r1 N 0) Ol O U O U
~ N U O -ri Gl ra -- rl O ri m ~ ,.- ~ .~, .~ -- -- m O +. a.~ ~ m .- a.r a o +~
.u -,~ .u m m U of O U b U O O O ~ O c0 rt U
m ~ ~ m ~ O U U m -~ m > U D ~ a ~ a -~ 9 a m ~ ~
~ a ~

~zs 0 ors ~ 0 oro o o2t ~ s~ 0 ob o ors o ~ 0 U O O ~ O O O .IJ t~ 1 r1 H ri O S-1 f1 O U

. O ri m R! a '~ ~ is 'd N b a~ ~ ~ O b O
H D H N ~ 23 f.~

-~ a~ a~ a ~ a ~ ~ ~ ~ ~ ro a w ro ro a ~ ~ v m i a~ a a ro ~ ro H zxU xc~x r r ~ a a r r zxU xzx xux xc~x zc zUx U

m ~ro U
a a~
o, to M
N U
~

.-I u1 .-1 ~ ~ rn O

G4 ~ rn ~D ~r .1 N

u~ .-~ u1 am "i U

vw '~
o x ~o -.~
z N N N r N
N

N M M M

U o~

ow z ~n wo ooiosiss 0 0 ' 1 N GJ O ,i," G) W W

ar .~.~ ro " ~

~ ro N NN ~
~ ~
~

) ~ blro ' 1) ' ! -r l~Ur "
"
"

. r~ H .C
. ir L, G; i1 dl U

m N l~ l~ _ N ~ U m ~ ro O

~
w - ro ~ ~ ~ U

U ro n ro (n W Gl L1 C; v rl r l O PD

iL W ro ro W

~ N
'fit M U G N ro ~ _ ~

a.~ ~ o ~' ro ro ar a .,~
ro U ro ~ rl ~ ~ ro O 1~
..i o0 a..~ .~ .~ ~ U~ ro ro ro H U W
~

of 3 rn r.' 'u .
~
~
~

~ .
?' ov ~
~ ~

, ~
, ~
.

~ s ~~' ' s ~' ~
~3U
~

.~ s ~
a a ~~
.

W W W ro ~n ro ro N a ~ ~ M ~ ~
~
ro ~n ro ro ro x~ o as m v > ~
o a v rn ro a~

a~ ar u, ~ ro ..~ 0 0 a~ 0~

H w a.yr u~ .,~
U ro ro ro ~

r- r r O N
i .~ -I -I ~~
~
~~

.
E O .IJ O .U .

..
fi ,?, rl U N V7 .~ ~ W ~ o > ro ro w ~ z . ~, ro ~ i ~ ~ r' ~
~

a ~ ar ~ ~ ro a r N "...~ ~1 W N U
1~.1 ti ~" ri ~.

Ci '~ b .
rl fi JJ IW i N

~i ~i ,~
y CL ~ .~ t~ r ~ O v 4 O ~ O ~ O
l '~'f' .' f r- -I ' ~ ~ '1 p ' .' r r ' -rl E Cl ..

~ . ro~, , ~ av ur ro ro ro ~
~

v .Q d ar m -,~
. ~ o a ~ ~

.~

a ~ ~ ~ ~ 3 > gar m ~
~ ~ ~n ~
~

. ~ .
_ o .1 ~, a~
G

3v ~ N ~
~ W ro ro Ul NU Ul ./",N
N 7 ~ ~
~ r iO.4"

N ~ v! ~ .~ H ro .~ r-a ro m f.~ i~ U

o ~ o x o x .n ,~ ,~ ~ ~ ro o o o W N

s ~ o ~~
~ 'c ro o ro ro ro ~

N -1 n ? Q1 ro a r -~

, . f0 H O
ro i0 r-1 ro ri N aj W O Gl ro W N O p O 1~ ro N ~
O H y .u ~ N

3 ~ g ~ 3 ~ , N N , .
N w a~ o N ro w ro !-i LI ~
10 ~d 10 a N ro 1-I ro l ~

~ a~ ro ro r O d ! 1 .~ ar ro ro a ~
~ ro a "

Q N W r-1 f Ul ro ., Gl ro ri O U
ro ' ro .~., ?
~

r r ~ rl y .-1 -ri O
r1 0 ~ M .~ ~ J.roJ ~ ,/~~
~ M H r-1 w r-i f.~1 U

a~rU aroro aro'7 J.'L,"Ur-zOH1 d M tf1 O O

O

a E E o N

w o, (~ l0 M .

O C~ O O CO

,~ OD 01 p~ O

U ~ N N M

ro o -~
z y A t~ o0 0, o r . r-i a -i ri N

ua ~w z ~n ~.
N

a : b ,~ '-, r >
~

O .u ~ ~ ~ N I H
~ O

v U ~
W
rt ~, ~ ~ ~ r- ~-~ I I o ~

~ ro v G7 w N
1 v I

l v N i -a ~
O H ~ -r H " UW1 N U' N

.Ca N a ro ~ a~ a . "
H ro ~ .Li U W O
H U

~ v 1.) N O v U G
, rt 1~ Ov > U ~ ~ w ~
3 o ~
h ~ ~ ~ y O v v ,~ o .~
~ v N -~I ro b~
a~ ~ 7 w rti o -~ w bi 10 - i 3 c .-~ -.~ c o ~a ~ -~ 3 r i y ~ ~ o o o ro a a ro ~ ~ ro v . a ~ W H N m o r ~i ~ 1~ rt ~
U

vo ro ~ b ~p .-i - - w o o a ~ H ~
.~ -~ ~
H

_ . .,~
b ~ H H N N .
rtf H ~
f n U O ~ o O
~ U ~ a a y i rt ro ~ "
"~

H v ~ '~ o ro ro G
a a a ~ r tn a v N N ~'v m'~ .
o a v ~ ~
.-I v ~

. v ~a ro a v o ~ .,~~, N
rt ~ r~
~

, N a v -~I ,~
ri (~.,L; fir" di ro v v 1J v U ro ~'., ~

, . larl N O .N a~ O b~ N ro ~-1 ro a N
N -r1 vI H ro ~ ~

o N ~ N ~I ~ -v v a tn r1 v ~ N ro ~.~roy H ~ H
O H ~ ~ O -.~v H ~ H
a W .N v H v ~
. H

N v w a ro ~ ~ o ro ~ rort v v ~
a N N N v a .~I
~ -~I a~ .u J. ~OC,'i01'.7.~r"Q~S.~. .~.f~.'~NOU f~.IF~'~..iHl~'~' ~ror-IN 1 a J- O O r1 ro rt O 'i.a' U H .L! O O U
ri H H H ro U H v i.~"H U b H ~ W W a " U
",~ N ro ~ ~
J-~

" ~" w U rl v tW O U .~ , ro (; ~ v H .-1 N O ~ v ro H O
-a r ~I H b N -n U -ri v ro ro-1 H IO ro .N
~ .u ro r.~ ~ ~ v w v ~ .Q x v ~
N w ' b >

, v v v . t~ v ~ 0 0 0 ~ ~ ~ ~ ~ ~ ,~ ro w 3 to v o ~ v ~ >
.

. c + ~ r, -~I o .u v .u U U N O to o ~ ~
O

~$ Rf id.C',N H 1~ r~
U ~ v ' ~ w r-1 ,L~
~ ID ro N N
-i N

. . ~ .-1 rl, rtS O ri ro H
N N H ro ., O -.iO U r-I
O ro N N
'~ l ' ' O ' , r O b1 ., U O Rf O
rtf of ~ rt N N ~ -ri p .~., w Id O O -~I.t. -a r-1 H N ~ N H
v O N
+~

3 3 v ,,~ ro -.I ..~~d ro ro w ~a ~ r., H

_ _ .a cn ~ ~ o H U

.4 ro N ~ M v ~ ~ ~ ~ w ~ -ri J ~
U v a G v . + ~ xd ~ N O ro ~ ?~
>~ o, 3 ~ rt ~ ~
ro u,~' ro o o .; w ~
~ Mro~ . ' ~ H
o o N ~~ '~
~

.I t U~ .UO N
.a ~ I~ v v ~ ~ ~s n p o, v ~ .~ v ro ro a~ ~ ro ~' ro o .~ a ~o .a ro H ~ O U H .a v ~
, a ~.r ~ . H U ~ o a ~ ro .~ ~d ~ to v ., .~.~ a , .~, ro ,-~ ~ ro v .~ ro -, v o ro H O O H $ N N ro ro '~ N N Id H Id N ~.,''r1 ri L ~' H ~ U
o ~

r H ~ o ~ ~ ~-,ro .-~ ~ >, .~
U o -.~ ~ N N ~ --~~ o -~I
o ,~ w ~ as o ro . v o v v ~ ~
~I -.~
~, , ,-., a -.~ a a ro roI N ~ ro v ro H N a ~

o N ro o o v v H it N v ro ~ H br a ro w ~ -~I~ H
w v N .~ ~ o v ~' ~

ro v .r.l~ v b O sr > IO .N U U x N w v H H a.i ~ U v N ~ U
O E O $
N

, w v O O v rl '~' ,~!'~ rl ~ b1 i." rl r-WO '~' 1.1 U

.~ w ,~ r-i ro H H I ro 1.~ H v ~a ~ a~ v dl ~ ~ O t!! N ro y ~ N a ~
.~ u, ~
-~, -. ~ h .~ a ~ ,-al ., ~ H r~ o N N , v -,~ r, ;~
..~ .a o. a w v v a N ~ ~ N
~ -~ ~
.o N v 3 rtf v ~ ~ a . -~I , N
3 H .-IN .~ -~ t~ o O rt v ~ a~
!

. a O O rt ~ 1.i O U a!
rt ~ ~1 O Pr H U v N O O ~ O U U ~ 3 a ~ N x U

. ~ v ~ N N ~ E W N v .4 O rl rl '~ .Q ~ " ~
N -rl fr" O O
.~ w O

~ N H N v v ~ '~ -I f H lr ~ N

r o N H rC 10 11 H
rt v v - r-~ E
d ~ rt ~

. 3 3 w M 3 ~, o a N v H ~ 0 w ,~ o ro ~ v . N ~ ro ~
a~ o, o v + a . w a ~ y v v v ~ ~' ~ ~ H ro v M o v v Rf 10 IWO U ~ Wd ~ >~ o ~ ~
ro f ro -rl ., . v O N r-1 ,Ja H -.~ ~ o , r-I td a ~ v .r .

~. ~ ro ro> v rt .N ~ N ~ H v ,~ o H H N N -.~
~ H
o H H O 1.~ ro H ~ H
.Q O O v ,l~?~ ,~ O rt b v ,~ O v ,fl ~ .!~ ro .4 ~
t0 tll 1 U H
t~

~~x wa rt x~'~a~ a ~ v ~~ a ~ x ~ a~' ~w ~~

u x , h '~ ~' o H

w z A

v ~ d~ M h h 0 h 00 p rl ~ V~ ~O
N

U ~"~ M OD N

h -z A '-'~ N M

r~
N N N N

U
d W

z ~

ro'd -a ~ ro ~
ar ro ro ~

~
~

m pl d - o .4" . ~ J m N ya ,d O

' Ud C~l~~~' N
!

am C a~roa~
H ~ ro 3 ~ a~

a~ -~ro s.l ro H a ~
W .Ci 'i~ " m b M
C; ~ ai l , C GJ b1 1J m ro a O ~ d H m 5,ro m ,5 ~
O m ~
m ro ,-I m a~m a~
m H-a N m a b ~
'b -I
m . p -a ~ a~ N O O~ a~U a d rt3 a m m f a ~ a! ro !~

~ . ro a O a ~ ~ a ~~ ~ -a O a.l H H H
U a H a U

N H U m~ ~ U ~
N ~
~ U
C ~
'~

. H -1 ror O
. -ad lN a .l ~
. O .rCQ~
~ a m a H ro a ~
W ~

G ~ a ~ m a~ ~ ~ w Tf ro ~ O
~ m O a ro ~r" 'Cf U b -a v W tnH ~ro yJ JJ 1.1 a G! t~ r-i r . ~ H H N a s~O ro ro O O r-1 ~ ~m ~ m .-i U

ro .~ U ro.4 G!
H tn a~ ~ bl -aa arb a a~ ro a~ ro 2f m >
u ar ~ . a -a a~
.O _>~ ~ ~ Gf ryi ,~ m 3 m a ro H
, ~ ~

W N H O a .~'. f1 E'ir-I W~f.~" U ~, .~ a~
~ .r. ~ O O v H ~
O ~

ro O a O ~ -~ .,.Ii U O
H r 4 .
1W -1 't7 rl H r-~ N H ~ Ct' 1~ U U ~0 -~

~ ~ ~ ~ b U W
~ W a~ ~ d '~ror o '~ ' H ro ~ U

~ ~' '~ r u ,' iI
~ ~ m g v ~ ~a ar ~'a v ro o ~ ~ ro ro w rt .~ f ~ . far ~s v ~ ~ ~

a-a o ~ m .
v ~ ~ m m w tn ~
u ro v w c~ rov a~ m ro H ~ airo.
w r ci, -a -a ro >~ ro .i ~-IN Ho ro~ _ ~ U .~ ~
CJ
O
~

r O . O ml.lroHN~ N Ob1'C3 ~
J --i H m ~ ~ ~ m UN m m ~ N N R m > a a~ ~d O

. ~
ro -a ro ro -aro Orl a -a a ~ .~
G U H -a ~ ro ~

_ m a V O ~'U 'OH ro d - t) ~

.G R', f~ a a r-I
a m - ~ ~W roo ~ ~~ix ~ N ~' ro a x ~
roes H

~ , ~' ~i ro~ ~ ~ ~ ro~~
' w -N

, ro a a bl ro N b~~ ~ r O ~I

O MN ~ bl r-I N m ~
J"r C: U .~'. 'i..''CS m 11m ro 1~-a>~
tJ r-1 ,-i Gi ro ~
1J H " O m .Wd -a ~ m a O G!N roa a H b ., O ., a a ro ~

m m Zf O a m N TJro m d C~ ro .
~ ~ .~ ro~ ~ m a a~
-i a~

O O O ' U ~ ~ w .~ H ~ m ro ~ b- O ro W U ~
~

?i b ~Gl rob f~ . H -II~ U W
~ 1 0 O H 1~ N Gl m ~".,G! ro ror O O
rl O N U ~ O
ro U ro O a~ a O
ro la H
~ b H
-a ~ U t~ ro G U ro ~0! H U m w O m bl bl 7 r~ .~ ro U U ~
f ~ ~

,~..~.~ ~ a rom O h ro U > roh O U ro I '1 'f.,' . -r1 W ~ ~ p~ U y ..
ro a - ..

n ~ . ~ ~ ~ U O ~
~

m M >~ N m U ~m U m ro O 1-Im O 0!
U '~ N O O

!~ O ~ ~ ~ ~-IH -~I~ U .-I U ~ I
ro -a Q1 a ~ .Q ~ w ~ 0i a o ro ~ro .uo o ro a ro.
U m a ~ o H .u ro m 1~ m U U p,rl O U 't7 s~i-IU ro N >~ -a G! a .i: .IJ
O N

H ~ H ~ ~ m O N N W
m O ~ O m G1.~ ~ s~
~ 'C1 .-i m O ro U ?
N
U

, p, H U m ?, ro H C1~ ro 'Jm rlro I ~ O
-a ri r1 U
C! ~' ., N ro I
3 w .-r 3 0 ro a 3 H -I O
o 0 ~cs ~
-a -a . ~ 3 ~ ~
ro ~-1 f~ I m~ m ro m a O p, H

U s~~ O H
~''od~?~ >.HS~ roo o dm~v m o -aro a s~ H ro ~' ~' ~' a~ a~ a~

. ~H a ~, ro o a ro y ro > ~ o ~t m ~ .~ ~ ar roar' ~ H ro I .a H w ro a H O .c .4 ~ H o o a N O U H ~, ~ Q7H M H N U H ~ N
~ ~; ?a 4 a O J a O ' N
I ~
~ H
i m ~ . 1 ~ .O W d ..4ro.Ct O
O U m m a.t ..7 H .~ v-7 ~U ~ O ~ '~'~H w H
" U c~ ~ ~
~".. U rl ~' . .. r7 ro 1 ~.7 . ' 0 O

M
M O O

o E

p a z A

H

G) N

dp N

pp ~ N 01 M

U
N N

roo -~, z a A y v ~ o N N

N N

U
d O
W

z~

W~ ~/~I55 ' ~ o ro a o r N H
~
r~l O H
~

- ro ~, ~ H V
.i O ~
t ~ ~ ~ ~

ro ~ 1.1 ' ~ ~ ~
~

~ d' ~ ~ a ~ o ~

o a a~ -o ~ , ~'~ a o v a N N H o V

C V
l ~ N

H a ~I U ~ v ~ v~ ~ N
a ar ~ -~ ro m ~ ro ~ '~ '., ~ ~ ' - , ~ ~
, ~

UO ~ H E
V U
U w v - .~ ~
I H
U
I
H

~ O V b U ~ 0 O

(Il Rf 3 f. C r-. H bf 1 C

H b .
~ W .
~

u~ ~ ro ,~ ~, H o H ~
o C' 1 l ro C7 ~., O ~ J.1 . y G"' Jx H

, , ., , O ~

R1 N JJ ~ O
~

~, al O .-I ~ W
-.~ ~ ~ "

, O N r1 O 10 H oror,ro x O .i H , o ~ o ~

,~-~ H a v W w a b ~ -ro ~ ~ ro "
w .~ ~ ~
ro N ~ W ro ~ ro a a m w ~ ~' O ~1 N ~ Q ' .
" " "
~
J

., 1.1 (", JJ '~
U , (,., ~-i (; Ul .J-1 J
rtf O
~ v! H

. r0 O O t0 ~ b .-I
O H >, I

r- r-1 ri r-1 U r-I
(/1 u1 1~ W O U O (''., rl O

Ul ri 47 O .-I U '~' O 1 11 -ri O -rl ul ~ t~ ~ m r-I t0 m m H
,~ U ro H ~C

o .
ro ~ ro 0 H a~ ~~
~ a ~ a ~

a - o ov H H u-.~ .1 d o ro ~
eh bl 3 H ~ bn ce ~

O ~ bl r Cil ~ ai ~ ro 1 10 ~n ro ~' i .' a ~ a ~ to a o ~ ~
U H

N td ~ 4-I ?i ~ ~

~-- ro roiN ro x ro o~o ~~ a~'~rtro ' a mro ~
' ' ~

a ro o m a ~ ~
o ~ -~I I .u a ro H of , a H
s ~

o ~, .u ~o .
r-, H f~ ~
H N U O ~ ~ O ~

I N
a 10 b ro N 1~ y.~ .N J~ H 1~
.N V H ~
W U G.~" ~p U!
fi "~ f W
' rl "
!' . 1! Ul r N ro -ri ~

O ~ O O U ~ O
10 ~ U

~ O W ro ~
U H
w H O R) ,f,~

ulroa~w~H~ m~ vl~OwUN tn0 10 r"I a1 ~ ~ O O ro rt V
m C1 W O .~ -.~1 3 0 3 ~
-I a ~ 3 g .~ 3 3 ~ t ~ r~
~I

H O x ~ ro ~ ~r V H O .Orr ~' ~ ~ ~ ~ W ro ~
' 1 >, a ar H as ar , rt a~ ro -.~ m o am o ~ ~' . la I~ H ~ ~ ro H ' ~ ~

fir H ,Li H H t~ 00 H F., rl Gl N I r1 m 1.1 td .4 I ,ra .Q .R .G m ,R O
it ~ a~ M U v~ H N

ao .I ~ o -~I ~I ~ r~ -~I
H x -.~ rt1 H H

a u, ,-I a a N .u u~ a w a, w .a ~ ~I a ,~
b N ~ N N

N p ,..I p 'O

U
a U

o~

H

r N N

o p O

N M p r-I 1p O ~ ~n ~o U N N N M

-~

z ~ o, o ,-I N
A

H N M M M

as ~

z 0 o a ' C W
II ~ g w v "~ ~ OG ~ ~ ~ ,u a II ~ ~ ~ ~ ~ v ~ ~ II
a ~' ~- ~ o .. a '= ~o > ".. H~ v ~o gz a, ~'w (il ~ ~ W '° ' p4 ~ o O
. .err. ~ . .r-c Wo Zs c~ ~o'o o c :o ~ ~5.
V ~ ~ ~ j ! ~_ 'O ~ ~ ~ I ii ~ II 7 _ '' ~ " ~ ~ at ". ~ ° °: '>
>~ ~ °u ~~ > ~<a=W~ ~ Ji '~~ ~ v~.g a . at ~ of 8 3 ~ a :fl ~ iii ~'o$~, ~ ~ oo ~~~ E t~vo°~o a . ~ e~a ~ o~
_. .. ~ o_ .:.
C !~ ~ ao a, ~, ~ ~ ~ ~ ~ oo ~ ~ ~ ~ ~ o °. cNn ..w~ ~'~_ ~ ,.°r uct~~' ~.ad o'a 'a~a a in ~ ~'. ~~ "~ ' 3 ~ ~C " " N -: ..y ..~e~ G~,~viq -~ '~yC~ ..
'U 'U 't~ ric:~
rr ~ . ~ ~ ~r N n N
czlU G~U ts7U wcn c t~~~ L"~~~., '~
c ~ a .. a ~~~ 4~.~~ ~~~~ s_..'_u ~ C~ ~ ~ "" ~° ~ w ~ a o ~
..~ aNz a.z~~~ ac~'x~ ~ ~~z :E o '~ 3 .. r?
.=
_ ~ ~~ a x = . .~ , .v ~ ~ _ 8 a ._ ~ ~ y~E
> ~ ~ .S~ ~ ~ ~ a y.. ~ ~' ~ ~ '$
..
wv ~ ~v~~ 0. ~ ~ ~'~~ ~ _ °' was..
~c ~ ~. ~. a ~.
'u E
w ~ d ..
a ~a a as u, m a~'""

V
R
r ~ ' a s~' ' ~a O
vo II
N
a - > N eo .z ~a~a ~
v ~.. oo~~ So C ~p4 ....
W v ~ ~ ''' ~ ~ ~ ~e ;~
u~~ ~~ ~~ oa ~ -N =
o°°o ~ '~ (~ ~~ °° E op ° '~ > U
ai ~ 3 v~ o ,~.. c~
..,~ ~N ~,3 ~ v~~~ a ~ ~;~~ 3 acd ~.:
v w ~ ~, N ~ ~~L v rr ~ ~ '~' (~ 3 in1 V at ~ N . ad ~ ~ 00 '' ivy' Gt;N'~~a ~
V O O. ~ ~ ~ ~ ~~,. . Er _ _ °°
G7 ~ wxCWn~3~~ a~
Z-c~ m~°.t7 H
Vl ~ ~G ~ C r.
.~. H
.. -., = a a ~ .~ $ "~ :g a o ~s ~ ~~ .~ H~
.>
~'o ~5 ,e ._ a ~» a ~
v ~ ~ H
V
a ~~ a ~ auk ~ a a ~ a~

H
4_~

SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
YUE, Henry LAL, Preeti .
TANG, Y. Tom GORGONE, Gina A.
GUEGLER, Karl J.
CORLEY, Neil C.
BAUGHN, Mariah R.
<120> HUMAN RECEPTOR-ASSOCIATED PROTEINS
<130> PF-0571 PCT
<140> To Be Assigned <141> Herewith <150> 09/130,884; Unassigned; 60/098,703 <151> 1998-08-07; 1998-08-07; 1998-09-O1 <160> 32 <170> PERL Program <2I0> 1 <211> 345 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> incyte Clone No: 1877651 <400> 1 Met His Leu Val Gly Gly Ser Cys Glu Val Trp Phe Pro Asp Val Leu Gln Gln Val Pro Leu Pro Cys Leu Trp Ala Pro Ser Met Ala Asn Ser Ala Met Asp Thr Arg Val Leu Cys Cys Ala Val Ile Cys Leu Leu Gly Ala Gly Leu Ser Asn Ala Gly Val Met Gln Asn Pro Arg His Leu Val Arg Arg Arg Gly Gln Glu Ala Arg Leu Arg Cys Ser Pro Met Lys Gly His Ser His Val Tyr Trp Tyr Arg Gln Leu Pro Glu Glu Gly Leu Lys Phe Met Val Tyr Leu Gln Lys Glu Asn Ile Ile Asp Glu Ser Gly Met Pro Lys Glu Arg Phe Ser Ala Glu Phe Pro Lys Glu Gly Pro Ser Ile Leu Arg Ile Gln Gln Val Val Arg Gly Asp Ser Ala Ala Tyr Phe Cys Ala Ser Ser Pro His Ser 1~g wo oorosiss pcrius~n~~~7 Lys Gln Ala Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly <210> 2 <211> 487 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2906971 <400> 2 Met Ala Ser Ser Ala Glu Gly Asp Glu Gly Thr Val Val Ala Leu Ala Gly Val Leu Gln Ser Gly Phe Gln Glu Leu Ser Leu Asn Lys Leu Ala Thr Ser Leu Gly Ala Ser Glu GIn Ala Leu Arg Leu Ile Ile Ser Ile Phe Leu Gly Tyr Pro Phe Ala Leu Phe Tyr Arg His Tyr Leu Phe Tyr Lys Glu Thr Tyr Leu Ile His Leu Phe His Thr Phe Thr Gly Leu Ser Ile Ala Tyr Phe Asn Phe Gly Asn Gln Leu Tyr His Ser Leu Leu Cys Ile Val Leu Gln Phe Leu Ile Leu Arg Leu Met Gly Arg Thr Ile Thr Ala Val Leu Thr Thr Phe Cys Phe Gln Met Ala Tyr Leu Leu Ala Gly Tyr Tyr Tyr Thr Ala Thr Gly wo oorosiss poi,~usml~~~~

Asn Tyr Asp Ile Lys Trp Thr Met Pro His Cys Val Leu Thr Leu Lys Leu Ile Gly Leu Ala Val Asp Tyr Phe Asp Gly Gly Lys Asp Gln Asn Ser Leu Ser Ser Glu Gln Gln Lys Tyr Ala Ile Arg Gly 170 . 175 180 Val Pro Ser Leu Leu Glu Val Ala Gly Phe Ser Tyr Phe Tyr Gly Ala Phe Leu Val Gly Pro Gln Phe Ser Met Asn His Tyr Met Lys Leu Val Gln Gly Glu Leu Ile Asp Ile Pro Gly Lys Ile Pro Asn Ser Ile Ile Pro Ala Leu Lys Arg Leu Ser Leu Gly Leu Phe Tyr Leu Val Gly Tyr Thr Leu Leu Ser Pro His Ile Thr Glu Asp Tyr Leu Leu Thr Glu~Asp Tyr Asp Asn His Pro Phe Trp Phe Arg Cys Met Tyr Met Leu Ile Trp Gly Lys Phe Val Leu Tyr Lys Tyr Val Thr Cys Trp Leu Val Thr Glu Gly Val Cys Ile Leu Thr Gly Leu Gly Phe Asn Gly Phe Glu Glu Lys Gly Lys Ala Lys Trp Asp Ala Cys Ala Asn Met Lys Val Trp Leu Phe Glu Thr Asn Pro Arg Phe Thr Gly Thr Ile Ala Ser Phe Asn Ile Aen Thr Asn Ala Trp Val Ala Arg Tyr Ile Phe Lys Arg Leu Lys Phe Leu Gly Asn Lys Glu Leu Ser Gln Gly Leu Ser Leu Leu Phe Leu Ala Leu Trp His Gly Leu His Ser Gly Tyr Leu Val Cys Phe Gln Met Glu Phe Leu Ile Val Ile Val Glu Arg Gln Ala Ala Arg Leu Ile Gln Glu Ser Pro Thr Leu Ser Lys Leu Ala Ala Ile Thr Val Leu Gln Pro Phe Tyr Tyr Leu Val Gln Gln Thr Ile His Trp Leu Phe Met Gly Tyr Ser Met Thr Ala Phe Cys Leu Phe Thr Trp Asp Lys Trp Leu Lys Val Tyr Lys Ser Ile Tyr Phe Leu Gly His Ile Phe Phe Leu Ser Leu Leu Phe Ile Leu Pro Tyr Ile His Lys Ala Met Val Pro Arg Lys Glu Lys Leu Lys Lys Met Glu <210> 3 <211> 312 <212> PRT
<213> Homo sapiens wo ooiosiss ~,ius99n7~77 <220>
<221> misc_feature <223> Incyte Clone No: 2907954 <400> 3 Met Gly Thr Arg Leu Leu Cys Leu Leu Trp Ala Ala Leu Cys Gly 1 5 - l0 15 Ala Glu Leu Thr Glu Ala Gly Arg Tyr Val Ala Gln Ser Pro Lys Ile Ile Glu Lys Arg Gln Ser Phe Trp Asn Pro Val Ala Cys Ile Ser Gly His Ala Thr Leu Tyr Gln Gln Leu Gly Trp Tyr Ile Gln Gly Pro Lys Leu Leu Ile Gln Asn Asn Val Val Phe Gln Gly Asp Aep Ser Gln Leu Pro Lys Asp Ser Ala Arg Leu Arg phe Glu Lys Gly Val Asp Ser Thr Leu Lys Pro Ala Leu Glu Ile Gln Lys Asp Ser Ala Val Tyr Leu Cys Ala Phe Leu Arg Asn Ser Ser Asp Asn Glu Gln Phe Phe Gly Pro Gly Leu Thr Leu Glu Thr Arg Val Asp Leu Lys Asn Val Phe Pro Pro Ala Val Glu Pro Glu Val Phe Ser Glu Ala Glu Ile Ser His Thr Ala Thr Val Cys Gln Lys Leu Leu Ala Thr Gly Phe Tyr Pro Asp Glu Leu Trp Trp His Val Ser Val Asn Gly Lys Glu Val His Ser Ser Thr Pro Gln Gly Val Asp Pro Leu Lys Glu Gln Pro Ala Leu Ser Arg Cys Leu Asn Asp Tyr Ser Ser Arg Leu Arg Val Ser Ala Trp Gln Pro Arg Thr Phe Asn Asn His Phe Arg Cys Gln Val Gln Gly Leu Glu Asn Phe Tyr Ser Asp Glu Trp Thr Gln Asp Arg Ala Val Thr Ile Val Lys Pro Gln Ser Ala Glu Ala Trp Gly Arg Ala Gly Phe Ser Glu Asp Cys Thr Ser Tyr Gln Gln Gly Val Leu Ser Ile Leu Glu Ile Ala Thr Tyr Leu Leu Gly Lys Ala Thr Leu Tyr Leu Val Ala Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Ser Arg Lys Asp Gly <210> 4 <211> 309 <212> PRT
<213> Homo sapiens <220>

wo oo/u8iss <221> misc_feature <223> Incyte Clone No: 3083742 <400> 4 Met Asn Gly Thr Tyr Asn Thr Ser Ser Asp Leu Trp Cys Gly Thr Pro Pro Ala Ile Lys Leu Gly Ala Tyr Leu Gly Leu Phe Tyr Val Leu Val Leu Gly Leu Leu Leu Leu Ala Leu Trp Phe Asn Ser Val Cys Cys Arg Met Gln Gln Trp Thr Arg Ile Tyr Thr Thr Glu Met Asn Leu Ala Val Ala Asp Leu Leu Cys Thr Leu Phe Cys Leu Pro Val Leu His Ser Leu Arg Asp Asp Thr Pro Leu Gln Thr Ser Cys Leu Ser Gln Gly Ile Tyr Leu Arg Tyr Met Ser Ser Thr Asn Ile Leu Val Thr Ala Ile Ala Val Tyr Val Ala Val His Asp Arg Arg Pro Leu Arg Ala Arg Gly Leu Pro Arg Gln Ala Ala Arg Ser Ala Val Cys Ala Val Leu Trp Val Ile Gly Ser Leu Ala Leu Val Val Arg Trp Leu Leu Gly Ile Gln Gly Phe Cys Phe Ser Glu Gly Arg Thr Arg His Asn Phe Asn Ser Phe Pro Leu Leu Phe Met Ala Gly Tyr Leu Pro Leu Ala Val Val Cys Ser Leu Lys Val Val Phe Val Thr Ala Leu Ala Gln Arg Pro Asp Val Gly Gln Glu Pro Thr Ala Ala Thr Arg Lys Ala Ala Arg Trp Ala Asn Leu Val Met Val Leu Phe Val Val Cys Phe Leu Pro Val Gly Leu Thr Arg Leu His Val Leu Ala Val Gly Trp Asn Ala Leu Leu Glu Thr Arg Cys Ala Ile Arg Ala Leu Tyr Ile Thr Ser Ser Asp Ala Asn Cys Lys Leu Cys Leu Asp Ala Ile Cys Tyr Tyr Ala Lys Glu Phe Glu Tyr Met Gln Ala Ser Ala Leu Ala Val Ala Ser Pro Arg Ala Lys Ala His Lys Gln Asp Ser Leu Cys Val Thr Leu Ala <210> 5 <211> 367 <212> PRT
<213> Homo sapiens <220>
<221> misc feature size wo oorosiss po,I,NS99n~77~
<223> Incyte Clone No: 3407686 <400> 5 Met Ile Arg Asn Trp Leu Thr Ile Phe Ile Leu Phe Pro Leu Lys 1 . 5 10 15 Leu Val Glu Lys Cys Glu Ser Ser Val Ser Leu Thr Val Pro Pro 20 . 25 30 Val Val Lys Leu Glu Asn Gly Ser Ser Thr Asn Val Ser Leu Thr Leu Arg Pro Pro Leu Asn Ala Thr Leu Val Ile Thr Phe Glu Ile Thr Phe Arg Ser Lys Asn Ile Thr Ile Leu Glu Leu Pro Asp Glu Val Val Val Pro Pro Gly Val Thr Asn Ser Ser Phe Gln Val Thr Ser Gln Asn Val Gly Gln Leu Thr Val Tyr Leu His Gly Asn His Ser Asn Gln Thr Gly Pro Arg Ile Arg Phe Leu Val Ile Arg Ser Ser Ala Ile Ser Ile Ile Asn Gln Val Ile Gly Trp Ile Tyr Phe Val Ala Trp Ser Ile Ser Phe Tyr Pro Gln Val Ile Met Asn Trp Arg Arg Lys Ser Val Ile Gly Leu Ser Phe Asp Phe Val Ala Leu Asn Leu Thr Gly Phe Val Ala Tyr Ser Val Phe Asn Ile Gly Leu Leu Trp Val Pro Tyr Ile Lys Glu Gln Phe Leu Leu Lys Tyr Pro Asn Gly Val Asn Pro Val Asn Ser Asn Asp Val Phe Phe Ser Leu His Ala Val Val Leu Thr Leu Ile Ile Ile Val Gln Cys Cys Leu Tyr Glu Arg Gly Gly Gln Arg Val Ser Trp Pro Ala Ile Gly Phe Leu Val Leu Ala Trp Leu Phe Ala Phe Val Thr Met Ile Val Ala Ala Val Gly Val Ile Thr Trp Leu Gln Phe Leu Phe Cys Phe Ser Tyr Ile Lys Leu Ala Val Thr Leu Val Lys Tyr Phe Pro Gln Ala Tyr Met Asn Phe Tyr Tyr Lys Ser Thr Glu Gly Trp Ser Ile Gly Asn Val Leu Leu Asp Phe Thr Gly Gly Ser Phe Ser Leu Leu Gln Met Phe Leu Gln Ser Tyr Asn Asn Asp Gln Trp Thr Leu Ile Phe Gly Asp Pro Thr Lys Phe Gly Leu Gly Val Phe Ser Ile Val Phe Asp Val Val Phe Phe Ile Gln His Phe Cys Leu Tyr Arg Lys Arg Pro Gly Tyr Asp Gln Leu Asn <210> 6 <211> 386 <212> PRT
<213> Homo sapiena <220>
<221> misc_feature <223> Incyte Clone No: 3472455 <400> 6 Met Gly Leu Trp Gly Gln Ser Val Pro Thr Ala Ser Ser Ala Arg Ala Gly Arg Tyr Pro Gly Ala Arg Thr Ala Ser Gly Thr Arg Pro Trp Leu Leu Asp Pro Lys Ile Leu Lys Phe Val Val Phe Iie Val Ala Val Leu Leu Pro Val Arg Val Asp Ser Ala Thr Ile Pro Arg Gln Asp Glu Val Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg Arg Ser Leu Lys Glu Glu Glu Cys Pro Ala Gly Ser His Arg Ser Glu Tyr Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly Val Asp Tyr Thr Ile Ala Ser Asn Asn Leu Pro Ser Cys Leu Leu Cys Thr Val Cys Lys Ser Gly Gln Thr Asn Lys Ser Ser Cys Thr Thr Thr Arg Asp Thr Val Cys Gln Cys Glu Lys Gly Ser Phe Gln Asp Lys Asn Ser Pro Glu Met Cys Arg Thr Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser Asp Ile Lys Cys Lys Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr Pro Ala Ala Glu Glu Thr Val Thr Thr Ile Leu Gly Met Leu Ala Ser Pro Tyr His Tyr Leu Ile Ile Ile Val Val Leu Val Ile Ile Leu Ala Val Val Val Val Gly Phe Ser Cys Arg Lys Lys Phe Ile Ser Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Gly Pro Glu Arg Val His Arg Val Leu Phe Arg Arg Arg Ser Cys Pro Ser Arg Val Pro Gly Ala Glu Asp Asn Ala Arg Asn Glu Thr Leu Ser Asn Arg Tyr Leu Gln Pro Thr Gln Val Ser Glu Gln Glu Ile Gln Gly Gln Glu Leu Ala Glu Leu Thr Gly Val Thr Val Glu Ser Pro Glu Glu Pro Gln Arg Leu Leu Glu Gln Ala Glu Ala Glu Gly Cys Gln Arg Arg Arg Leu Leu Val Pro Val Asn Asp Ala Asp Ser Ala Asp Ile Ser Thr Leu Leu Asp Ala Ser Ala Thr Leu Glu Glu Gly His Ala Lys Glu Thr Ile Gln Asp Gln Leu Val Gly Ser Glu Lys Leu Phe Tyr Glu Glu Asp Glu Ala Gly Ser Ala Thr Ser Cys Leu <210> 7 <211> 346 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 786873 <400> 7 Met Glu Ser Asn Leu Ser Gly Leu Val Pro Ala Ala Gly Leu Val Pro Ala Leu Pro Pro Ala Val Thr Leu Gly Leu Thr Ala Ala Tyr Thr Thr Leu Tyr Ala Leu Leu Phe Phe Ser Val Tyr Ala Gln Leu Trp Leu Val Leu Leu Tyr Gly His Lys Arg Leu Ser Tyr Gln Thr Val Phe Leu Ala Leu Cys Leu Leu Trp Ala Ala Leu Arg Thr Thr Leu Phe Ser Phe Tyr Phe Arg Asp Thr Pro Arg Ala Asn Arg Leu Gly Pro Leu Pro Phe Trp Leu Leu Tyr Cys Cys Pro Val Cys Leu Gln Phe Phe Thr Leu Thr Leu Met Asn Leu Tyr Phe Ala Gln Val Val Phe Lys Ala Lys Val Lys Arg Arg Pro Glu Met Ser Arg Gly Leu Leu Ala Val Arg Gly Ala Phe Val Gly Ala Ser Leu Leu Phe Leu Leu Val Asn Val Leu Cys Ala Val Leu Ser His Arg Arg Arg Ala Gln Pro Trp Ala Leu Leu Leu Val Arg Val Leu Val Ser Asp Ser Leu Phe Val Ile Cys Ala Leu Ser Leu Ala Ala Cys Leu Cys Leu Va1 Ala Arg Arg Ala Pro Ser Thr Ser Ile Tyr Leu Glu Ala Lys Ala Asp Leu Val Asn Asp Leu Gly Asn Lys Gly Tyr Leu Val Phe Gly Leu Ile Leu Phe Val Trp Glu Leu Leu Pro Thr Thr Leu Leu Val Gly Phe Phe Arg Val His Arg Pro Pro Gln Asp Leu Ser Thr Ser His Ile Leu Asn Gly Gln Val Phe Ala Ser Arg Ser Tyr Phe Phe Asp Arg Ala'Gly His Cys Glu Asp Glu Gly Cys Ser Trp Glu His Ser Arg Gly Glu Ser Thr Ser Met Ser Gly Ser Leu Gly Ser Gly Ser Trp Tyr Gly Ala Ile Gly Arg Glu Pro Gly Trp Tyr Gly Gly Ser Gln Thr Lys Thr Thr Pro Leu Leu Phe Ser Gln Val Pro,Gly Pro Gly Gly His His His Ser Leu Tyr Ser Thr Pro Gln Thr <210> 8 <211> 241 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1220371 <400> 8 Met Gly Thr Ala Ser Arg Ser Asn Ile Ala Arg His Leu Gln Thr Asn Leu Ile Leu Phe Cys Val Gly Ala Val Gly Ala Cys Thr Leu Ser Val Thr Gln Pro Trp Tyr Leu Glu Val Asp Tyr Thr His Glu Ala Val Thr Ile Lys Cys Thr Phe Ser Ala Thr Gly Cys Pro Ser Glu Gln Pro Thr Cys Leu Trp Phe Arg Tyr Gly Ala His Gln Pro Glu Asn Leu Cys Leu Asp Gly Cys Lys Ser Glu Ala Asp Lys Phe Thr Val Arg Glu Ala Leu Lys Glu Asn Gln Val Ser Leu Thr Val Asn Arg Val Thr Ser Asn Asp Ser Ala Ile Tyr Ile Cys Gly Ile Ala Phe Pro Ser Val Pro Glu Ala Arg Ala Lys Gln Thr Gly Gly Gly Thr Thr Leu Val Val Arg Glu Ile Lys Leu Leu Ser Lys Glu Leu Arg Ser Phe Leu Thr Ala Leu Vai Ser Leu Leu Ser Val Tyr Val Thr Gly Val Cys Val Ala Phe Ile Leu Leu Ser Lys Ser Lys Ser Asn Pro Leu Arg Asn Lys Glu Ile Lys Glu Asp Ser Gln Lys Lys Lys Ser Ala Arg Arg Ile Phe Gln Glu Ile Ala Gln Glu Leu Tyr His Lys Arg His Val Glu Thr Asn Gln Gln Ser Glu Lys Asp Asn Asn Thr Tyr Glu Asn Arg Arg Val Leu Ser Asn Tyr Glu Arg Pro <210> 9 <211> 450 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1258785 <400> 9 Met Gly Glu Thr Met Ser Lys Arg Leu Lys Leu His Leu Gly Gly Glu Ala Glu Met Glu Glu Arg Ala Phe Val Asn Pro Phe Pro Asp Tyr Glu Ala Ala Ala Gly Ala Leu Leu Ala Ser Gly Ala Ala Glu Glu Thr Gly Cys Val Arg Pro Pro Ala Thr Thr Asp Glu Pro Gly Leu Pro Phe His Gln Asp Gly Lys Ile Ile His Asn Phe Ile Arg Arg Ile Gln Thr Lys Ile Lys Asp Leu Leu Gln Gln Met Glu Glu Gly Leu Lys Thr Ala Asp Pro His Asp Cys Ser Ala Tyr Thr Gly Trp Thr Gly Ile Ala Leu Leu Tyr Leu Gln Leu Tyr Arg Val Thr Cys Asp Gln Thr Tyr Leu Leu Arg Ser Leu Asp Tyr Val Lys Arg 125 , 130 135 Thr Leu Arg Asn Leu Asn Gly Arg Arg Val Thr Phe Leu Cys Gly Asp Ala Gly Pro Leu Ala Val Gly Ala Val Ile Tyr His Lys Leu Arg Ser Asp Cys Glu Ser Gln Glu Cys Val Thr Lys Leu Leu Gln Leu Gln Arg Ser Val Val Cys Gln Glu Ser Asp Leu Pro Asp Glu Leu Leu Tyr Gly Arg Ala Gly Tyr Leu Tyr Ala Leu Leu Tyr Leu Asn Thr Glu Ile Gly Pro Gly Thr Val Cys Glu Ser Ala Ile Lys Glu Val Val Asn Ala Ile Ile Glu Ser Gly Lys Thr Leu Ser Arg Glu Glu Arg Lys Thr Glu Arg Cys Pro Leu Leu Tyr Gln Trp His Arg Lys Gln Tyr Val Gly Ala Ala His Gly Met Ala Gly Ile Tyr Tyr Met Leu Met Gln Pro Ala Ala Lys Val Asp Gln Glu Thr Leu Thr Glu Met Val Lys Pro Ser Ile Asp Tyr Val Arg His Lys Lys Phe Arg Ser Gly Asn Tyr Pro Ser Ser Leu Ser Asn Glu Thr Asp Arg Leu Val His Trp Cys His Gly Ala Pro Gly Val Ile His Met Leu Met Gln Ala Tyr Lys Val Phe Lys Glu Glu Lys Tyr Leu Lys Glu Ala Met Glu Cys Ser Asp Val Ile Trp Gln Arg Gly Leu Leu Arg Lys Gly Tyr Gly Ile Cys His Gly Thr Ala Gly Asn Gly Tyr Ser Phe Leu Ser Leu Tyr Arg Leu Thr Gln Asp Lys Lys Tyr Leu 380 ~ 385 390 Tyr Arg Ala Cys Lys Phe Ala Glu Trp Cys Leu Asp Tyr Gly Ala His Gly Cys Arg Ile Pro Asp Arg Pro Tyr Ser Leu Phe Glu Gly Met Ala Gly Ala Ile His Phe Leu Ser Asp Val Leu Gly Pro Glu Thr Ser Arg Phe Pro Ala Phe Glu Leu Asp Ser Ser Lys Arg Asp <210> 10 <211> 269 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1361202 <400> 10 Met Glu Thr Leu Leu Gly Leu Leu ile Leu Trp Leu Gln Leu Gln Trp Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Asp Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser <210> 11 <211> 190 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2132846 <400> 11 Met Gly Lys Ser Asn Ser Lys Leu Thr Pro Glu Val Val Glu Glu Leu Thr Arg Lys Thr Tyr Phe Thr Glu Lys Glu VaI Gln Gln Trp Tyr Lys Gly Phe Ile Lys Asp Cys Pro Ser Gly Gln Leu Asp Ala Ala Gly Phe Gln Lys Ile Tyr Lys Gln Phe Phe Pro Phe Gly Asp Pro Thr Lys Phe Ala Thr Phe Val Phe Asn Val Phe Asp Glu Asn Lys Asp Gly Arg Ile Glu Phe Ser Glu Phe Ile Gln Ala Leu Ser Val Thr Ser Arg Gly Thr Leu Asp Glu Lys Leu Arg Trp Ala Phe Lys Leu Tyr Asp Leu Asp Asn Asp Gly Tyr Ile Thr Arg Asn Glu Met Leu Asp Ile Val Asp Ala Ile Tyr Gln Met Val Gly Asn Thr Val Glu Leu Pro Glu Glu Glu Asn Thr Pro Glu Lys Arg Val Asp Arg Ile Phe Ala Met Met Asp Lys Asn Ala Asp Gly Lys Leu Thr Leu Gln Glu Phe Gln Glu Gly Ser Lys Ala Asp Pro Ser Ile Val Gln Ala Leu Ser Leu Tyr Asp Gly Leu Val <210> 12 <211> 450 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2539294 <400> 12 Met Ser Asp Met Glu Asp Asp Phe Met Cys Asp Asp Glu Glu Asp 1 . 5 ~ 10 15 Tyr Asp Leu Glu Tyr Ser Glu Asp Ser Asn Ser Glu Pro Asn Val Asp Leu Glu Asn Gln Tyr Tyr Asn Ser Lys Ala Leu Lys Glu Asp Asp Pro Lys Ala Ala Leu Ser Ser Phe Gln Lys Val Leu Glu Leu Glu Gly Glu Lys Gly Glu Txp Gly Phe Lys Ala Leu Lys Gln Met Ile Lys Ile Asn Phe Lys Leu Thr Asn Phe Pro Glu Met Met Asn Arg Tyr Lys Gln Leu Leu Thr Tyr Ile Arg Ser Ala Val Thr Arg Asn Tyr Ser Glu Lys Ser Ile Asn Ser Ile Leu Asp Tyr Ile Ser Thr Ser Lys Gln Asn Ser Asp Phe Leu Cys Gln Met Asp Leu Leu Gln Glu Phe Tyr Glu Thr Thr Leu Glu Ala Leu Lys Asp Ala Lys Asn Asp Arg Leu Trp Phe Lys Thr Asn Thr Lys Leu Gly Lys Leu Tyr Leu Glu Arg Glu Glu Tyr Gly Lys Leu Gln Lys Ile Leu Arg Gln Leu His Gln Ser Cys Gln Thr Asp Asp Gly Glu Asp Asp Leu i85 190 195 Lys Lys Gly Thr Gln Leu Leu Glu Ile Tyr Ala Leu'Glu Ile Gln Met Tyr Thr Ala Gln Lys Asn Asn Lys Lys Leu Lys Ala Leu Tyr Glu Gln Ser Leu His Ile Lys Ser Ala Ile Pro His Pro Leu Ile Met Gly Val Ile Arg Glu Cys Gly Gly Lys Met His Leu Arg Glu Gly Glu Phe Glu Lys Ala His Thr Asp Phe Phe Glu Ala Phe Lys Asn Tyr Asp Glu Ser Gly Ser Pro Arg Arg Thr Thr Cys Leu Lys Tyr Leu Val Leu Ala Asn Met Leu Met Lys Sex Gly Ile Asn Pro Phe Asp Ser Gln Glu Ala Lys Pro Tyr Lys Asn Asp Pro Glu Ile Leu Ala Met Thr Asn Leu Val Ser Ala Tyr Gln Asn Asn Asp Ile Thr Glu Phe Glu Lys Ile Leu Lys Thr Asn His Ser Asn Ile Met Asp Asp Pro Phe Ile Arg Glu His Ile Glu Glu Leu Leu Arg Asn Ile Arg Thr Gln Val Leu Ile Lys Leu Ile Lys Pro Tyr Thr Arg Ile His Ile Pro Phe Ile Ser Lys Glu Leu Asn Ile Asp Val Ala wo oorosiss rcrius~n~~~~
Asp Val Glu Ser Leu Leu Val Gln Cys Ile Leu Asp Asn Thr Ile His Gly Arg Ile Asp Gln Val Asn Gln Leu Leu Glu Leu Asp His Gln Lys Arg Gly Gly Ala Arg Tyr Thr Ala Leu Asp Lys Trp Thr Asn Gln Leu Asn Ser Leu Asn Gin Ala Val Val Ser Lys Leu Ala <210> 13 <211> 240 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2589371 <400> 13 Met Leu Leu Gln Ser Gln Thr Met Gly Val Ser His Ser Phe Thr Pro Lys Gly Ile Thr Ile Pro Gln Arg Glu Lys Pro Gly His Met Tyr Gln Asn Glu Asp Tyr Leu Gln Asn Gly Leu Pro Thr Glu Thr Thr Val Leu Gly Thr Val Gln Ile Leu Cys Cys Leu Leu Ile Ser Ser Leu Gly Ala Ile Leu Val Phe Ala Pro Tyr Pro Ser His Phe Asn Pro Ala Ile Ser Thr Thr Leu Met Ser Gly Tyr Pro Phe Leu 80 ~ 85 90 Gly Ala Leu Cys Phe Gly Ile Thr Gly Ser Leu Ser Ile Ile Ser Gly Lys Gln Ser Thr Lys Pro Phe Asp Leu Ser Ser Leu Thr Ser Asn Ala Val Ser Ser Val Thr Ala Gly Ala Gly Leu Phe Leu Leu Ala Asp Ser Met Val Ala Leu Arg Thr Ala Ser Gln His Cys Gly Ser Glu Met Asp Tyr Leu Ser Ser Leu Pro Tyr Ser Glu Tyr Tyr Tyr Pro Ile Tyr Glu Ile Lys Asp Cys Leu Leu Thr Ser Val Ser Leu Thr Gly Val Leu Val Val Met Leu Ile Phe Thr Val Leu Glu Leu Leu Leu Ala Ala Tyr Ser Ser Val Phe Trp Trp Lys Gln Leu Tyr Ser Asn Asn Pro Gly Ser Ser Phe Ser Ser Thr Gln Ser Gln Asp His Ile Gln Gln Val Lys Lys Ser Ser Ser Arg Ser Trp Ile <210> 14 <211> 316 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2656082 <400> 14 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His Leu Val Met Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Leu Gly His Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn Ser Val Pro Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe Leu His Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Gln Val His Pro Gly Leu Ala Gly Gly Leu Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu ~Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly <210> 15 <211> 172 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature .
<223> Incyte Clone No: 2762182 <400> 15 Met Ala Thr Ile Thr Glu Lys Glu Val Gln Gln Trp Tyr Lys Gly Phe Ile Lys Asp Cys pro Ser Gly Gln Leu Asp Ala Ala Gly Phe Gln Lys Ile Tyr Lys Gln Phe Phe Pro Phe Gly Asp Pro Thr Lys Phe Ala Thr Phe Val Phe Asn Val Phe Asp Glu Asn Lys Asp Gly Arg Ile Glu Phe Ser Glu Phe Ile Gln Ala Leu Ser Val Thr Ser Arg Gly Thr Leu Asp Glu Lys Leu Arg Trp Ala Phe Lys Leu Tyr Asp Leu Asp Asn Asp Gly Tyr Ile Thr Arg Asn Glu Met Leu Asp Ile Val Asp Ala Ile Tyr Gln Met Val Gly Asn Thr Val Glu Leu Pro Glu Glu Glu Asn Thr Pro Glu Lys Arg Val Asp Arg Ile Phe Ala Met Met Asp Lys Asn Ala Asp Gly Lys Leu Thr Leu Gln Glu Phe Gln Glu Gly Ser Lys Ala Asp Pro Ser Ile Val Gln Ala Leu Ser Leu Tyr Asp Gly Leu Val <210> 16 <211> 364 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3140659 <400> 16 Met Ser Val Met Val Val Arg Lys Lys Val Thr Arg Lys Trp Glu Lys Leu Pro Gly Arg Asn Thr Phe Cys Cys Asp Gly Arg Val Met Met Ala Arg Gln Lys Gly Ile Phe Tyr Leu Thr Leu Phe Leu Ile Leu Gly Thr Cys Thr Leu Phe Phe Ala Phe Glu Cys Arg Tyr Leu Ala Val Gln Leu Ser Pro Ala Ile Pro Val Phe Ala Ala Met Leu Phe Leu Phe Ser Met Ala Thr Leu Leu Arg Thr Ser Phe Ser Asp Pro Gly Val Ile Pro Arg Ala Leu Pro Asp Glu Ala Ala Phe Ile Glu Met Glu Ile Glu Ala Thr Asn Gly Ala Val Pro Gln Gly Gln 110 ~ 115 120 Arg Pro Pro Pro Arg Ile Lys Asn Phe Gln Ile Asn Asn Gln Ile Val Lys Leu Lys Tyr Cys Tyr Thr Cys Lys Ile Phe Arg Pro Pro Arg Ala Ser His Cys Ser Ile Cys Asp Asn Cys Val Glu Arg Phe Asp His His Cys Pro Trp Val Gly Asn Cys Val Gly Lys Arg Asn Tyr Arg Tyr Phe Tyr Leu Phe Ile Leu Ser Leu Ser Leu Leu Thr Ile Tyr Val Phe Ala Phe Asn Ile Val Tyr Val Ala Leu Lys Ser Leu Lys Ile Gly Phe Leu Glu Thr Leu Lys Glu Thr Pro Gly Thr Val Leu Glu Val Leu Ile Cys Phe Phe Thr Leu Trp Ser Val Val Gly Leu Thr Gly Phe His Thr Phe Leu Val Ala Leu Asn Gln Thr Thr Asn Glu Asp Ile Lys Gly Ser Trp Thr Gly Lys Asn Arg Val Gln Asn Pro Tyr Ser His Gly Asn Ile Val Lys Asn Cys Cys Glu Val Leu Cys Gly Pro Leu Pro Pro Ser Val Leu Asp Arg Arg Gly Ile Leu Pro Leu Glu Glu Ser Gly Ser Arg Pro Pro Ser Thr Gln Glu Thr Ser Ser Ser Leu Leu Pro Gln Ser Pro Ala Pro Thr Glu His Leu Asn Ser Asn Glu Met Pro Glu Asp Ser Ser Thr Pro Glu Glu Met Pro Pro Pro Glu Pro Pro Glu Pro Pro Gln Glu Ala Ala Glu Ala Glu Lys <210> 17 <211> 1270 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1877651 <400> 17 gacctcacag gaagatgcat cttgtaggag gcagctgtga ggtctggttc cccgacgtgc 60 tgcagcaagt gcctttgccc tgcctgtggg ctccctccat ggccaactct gctatggaca 120 ccagagtact ctgctgtgcg gtcatctgtc ttctgggggc aggtctctca aatgccggcg 180 tcatgcagaa cccaagacac ctggtcagga ggaggggaca ggaggcaaga ctgagatgca 240 gcccaatgaa aggacacagt catgtttact ggtatcggca gctcccagag gaaggtctga 300 aattcatggt ttatctccag aaagaaaata tcatagatga gtcaggaatg ccaaaggaac 360 gattttctgc tgaatttccc aaagagggcc ccagcatcct gaggatccag caggtagtgc 420 gaggagattc ggcagcttat ttctgtgcca gctcaccaca ttcgaagcag gctgagcagt 480 tcttcgggcc agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg ttcccacccg 540 aggtcgctgt gtttgagcca tcagaagcag.agatctccca cacccaaaag gccacactgg 600 tgtgcctggc cacaggcttc taccccgacc acgtggagct gagctggtgg gtgaatggga 660 aggaggtgca cagtggggtc agcacagacc cgcagcccct caaggagcag cccgccctca 720 atgactccag atactgcctg agcagccgcc tgagggtctc ggccaccttc tggcagaacc 780 cccgcaacca cttccgctgt caagtccagt tctacgggct ctcggagaat gacgagtgga 840 cccaggatag ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg ggtagagcag 900 actgtggctt cacctccgag tcttaccagc aaggggtcct gtctgccacc atcctctatg 960 agatcttgct agggaaggcc accttgtatg ccgtgctggt cagtgccctc gtgctgatgg 1020 ccatggtcaa gagaaaggat tccagaggct agctccaaaa ccatcccagg tcattcttca 1080 tcctcaccca ggattctcct gtacctgctc ccaatctgtg ttcctaaaag tgattctcac 1140 tctgcttctc atctcctact tacatgaata cttctctctt ttttctgttt ccctgaagat 1200 tgagctccca acccccaagt acgaaatagg ctaaaccaat aaaaaattgt gtgttgggcc 1260 tggttgcatt <210> 18 <211> 2234 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2906971 <400> 18 aggttttgcc cgcattcggg gcgcgcggac tggggggtcc ctgtggggct cccggagtta 60 agatggcgtc ctcagcggag ggggacgagg ggactgtggt ggcgctggcg ggggttctgc 120 agtcgggttt ccaggagctg agccttaaca agttggegac gtccctgggc gcgtcagaac 180 aggcgctgcg gctgatcatc tccatcttcc tgggttaccc ctttgctttg ttttatcggc 240 attacctttt ctacaaggag acctacctca tccacctctt ccataccttt acaggcctct 300 caattgctta ttttaacttt ggaaaccagc tctaccactc cctgctgtgt attgtgcttc 360 agttcctcat ccttcgacta atgggccgca ccatcactgc cgtcctcact accttttgct 420 tccagatggc ctaccttctg gctggatact attacactgc caccggcaac tacgatatca 480 agtggacaat gccacattgt gttctgactt tgaagctgat tggtttggct gttgactact 540 ttgacggagg gaaagatcag aattccttgt cctctgagca acagaaatat gccatacgtg 600 gtgttccttc cctgctggaa gttgctggtt tctcctactt ctatggggcc ttcttggtag 660 ggccccagtt ctcaatgaat cactacatga agctggtgca gggagagctg attgacatac 720 caggaaagat accaaacagc atcattcctg ctctcaagcg cctgagtctg ggccttttct 780 acctagtggg ctacacactg ctcagccccc acatcacaga agactatctc ctcactgaag 840 actatgacaa ccaccccttc tggttccgct gcatgtacat gctgatctgg ggcaagtttg 900 tgctgtacaa atatgtcacc tgttggctgg tcacagaagg agtatgcatt ttgacgggcc 960 tgggcttcaa tggctttgaa gaaaagggca aggcaaagtg ggatgcctgt gccaacatga 1020 aggtgtggct ctttgaaaca aacccccgct tcactggcac cattgcctca ttcaacatca 1080 acaccaacgc ctgggtggcc cgctacatct tcaaacgact caagttcctt ggaaataaag 1140 aactctctca gggtctctcg ttgctattcc tggccctctg gcacggcctg cactcaggat 1200 acctggtctg cttccagatg gaattcctca ttgttattgt ggaaagacag gctgccaggc 1260 tcattcaaga gagccccacc ctgagcaagc tggccgccat tactgtcctc cagcccttct 1320 actatttggt gcaacagacc atccactggc tcttcatggg ttactccatg actgccttct 1380 gcctcttcac gtgggacaaa tggcttaagg tgtataaatc catctatttc cttggccaca 1440 tcttcttcct gagcctacta ttcatattgc cttatattca caaagcaatg gtgccaagga 1500 aagagaagtt aaagaagatg gaataatcca tttccctggt ggcctgtgcg ggactggtgc 1560 1$/28 agaaactact cgtctccctt ttcacagcac tcctttgccc cagagcagag aatggaaaag 1620 ccagggaggt ggaagatcga tgcttccagc tgtgcctctg ctgccagcca agtcttcatt 1680 tggggccaaa ggggaaactt ttttttggag aaggcgtctt gctttgtcac ccacgctgga 1740 atgcagtggc gggatctcag ctcaccgcaa cctccacctc ctgggttcaa gtgattttcc 1800 tgcctcagcc tcccaagtag ctgggaatac aggcacgcca ccatgcccag ctaatttttg 1860 tattttcagt agaaacggga tttcaccacg ttggccaggc tggtctcgaa ctcctgaccg 1920 caagtgatcc acccgcctcc gcctcccaaa gtgctgggat tacaggcgtg agccaccgtg 1980 cccggcccaa aggggaaact cttgtgggag gagcagaggg gctcacatct cccctctgat 2040 tcccccatgc acattgcctt atctctcccc atctagccag gaatctattg tgtttttctt 2100 ctgccaattt actatgattg tgtatgtgcc gctaccacca ccccccccat gggggggtgg 2160 agaggggtgc aaggccctgc ctgctccact ttttctacct tggaactgta ttagatatgg 2220 tcacttctgt gtgt 2234 <210> 19 <211> 1552 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2907954 <400> 19 tgaccctgat tgggcaaagc tcccatcctt ccctgaccct gccatgggca ccaggctcct 60 ctgctgggcg gccctctgtc tcctgggagc agaactcaca gaagctggag ttgcccagtc 120 tcccagatat aagattatag agaaaaggca gagtgtggct ttttggtgca atcctatatc 180 tggccatgct accctttact ggtaccagca gatcctggga cagggcccaa agcttctgat 240 tcagtttcag aataacggtg tagtggatga ttcacagttg cctaaggatc gattttctgc 300 agagaggctc aaaggagtag actccactct caagatccaa cctgcaaagc ttgaggactc 360 ggccgtgtat ctctgtgcca gcagcttctt agaccgaaac aatgagcagt tcttcgggcc 420 agggacacgg ctcaccgtgc tagaggacct gaaaaacgtg ttcccacccg aggtcgctgt 480 gtttgagcca tcagaagcag agatctccca cacccaaaag gccacactgg tgtgcctggc 540 cacaggcttc taccccgacc acgtggagct gagctggtgg gtgaatggga aggaggtgca 600 cagtggggtc agcacagacc cgcagcccct caaggagcag cccgccctca atgactccag 660 atactgcctg agcagccgcc tgagggtctc ggccaccttc tggcagaacc cccgcaacca 720 cttccgctgt caagtccagt tctacgggct ctcggagaat gacgagtgga cccaggatag 780 ggccaaacct gtcacccaga tcgtcagcgc cgaggcctgg ggtagagcag actgtggctt 840 cacctccgag tcttaccagc aaggggtcct gtctgccacc atcctctatg agatcttgct 900 agggaaggcc accttgtatg ccgtgctggt cagtgccctc gtgctgatgg ccatggtcaa 960 gagaaaggat tccagaggct agctccaaaa ccatcccagg tcattcttca tcctcaccca 1020 ggattctcct gtacctgctc ccaatctgtg ttcctaaaag tgattctcac tctgcttctc 1080 atctcctact tacatgaata cttctctctt ttttctgttt ccctgaagat tgagctccca 1140 acccccaagt acgaaatagg ctaaaccaat aaaaaatggt gtgttgggcc tggttgcatt 1200 tcaggagtgt ctgtggagtt ctgctcatca ctgacttatc ttctgattta gggaaagcag 1260 cattcccttg gacatctgaa gtgacagccc tctttctctc cacccaatgc tgctttctcc 1320 tgttcatcct gatggaagtc ctcaaacacc atttccatac ccaggcattc tgggtcccca 1380 ctggagggtt agtctgaagg gcaatggctg ggctttggaa aaccagcaag atgaggacag 1440 agaggaaggc acacagcaaa ccataagccc ttacccagtg caggacagag gatgcgggca 1500 gacctatggg ttacaatgtc tggtcatttc ccaattccag attaaatatg to 1552 <210> 20 <211> 1369 <212> DNA
<213> Homo sapiens WO 00/0$155 PGT/US99/17777 <220>
<221> misc_feature <223> Incyte Clone No: 3083742 <400>. 20 gggtcacaca ctccacccgg gaggccaaag ctgcctgcag gaccatgaat ggcacctaca 60 acacctgtgg ctccagcgac ctcacctggc ccccagcgat caagctgggc ttctacgcct 120 acttgggcgt cctgctggtg ctaggcctgc tgctcaacag cctggcgctc tgggtgttct 180 gctgccgcat gcagcagtgg acggagaccc gcatctacat gaccaacctg gcggtggccg 240 acctctgcct gctgtgcacc ttgcccttcg tgctgcactc cctgcgagac acctcagaca 300 cgccgctgtg ccagctctcc cagggcatct acctgaccaa caggtacatg agcatcagcc 360 tggtcacggc catcgccgtg gaccgctatg tggccgtgcg gcacccgctg cgtgcccgcg 420 ggctgcggtc ccccaggcag gctgcggccg tgtgcgcggt cctctgggtg ctggtcatcg 480 gctccctggt ggctcgctgg ctcctgggga ttcaggaggg cggcttctgc ttcaggagca 540 cccggcacaa tttcaactcc atggcgttcc cgctgctggg attctacctg cccctggccg 600 tggtggtctt ctgctccctg aaggtggtga ctgccctggc ccagaggcca cccaccgacg 660 tggggcaggc agaggccacc cgcaaggctg cccgcatggt ctgggccaac ctcctggtgt 720 tcgtggtctg cttcctgccc ctgcacgtgg ggctga.cagt gcgcctcgca gtgggctgga 780 acgcctgtgc cctcctggag acgatccgtc gcgccctgta cataaccagc aagctctcag 840 atgccaactg ctgcctggac gccatctgct actactacat ggccaaggag ttccaggagg 900 cgtctgcact ggccgtggct ccccgtgcta aggcccacaa aagccaggac tctctgtgcg 960 tgaccctcgc ctaagaggcg tgctgtgggc gctgtgggcc aggtctcggg ggctccggga 1020 ggtgctgcct gccaggggaa gctggaacca gtagcaagga gcccgggatc agccctgaac 1080 tcactgtgta ttctcttgga gccttgggtg ggcagggacg gcccaggtac ctgctctctt 1140 gggaagagag agggacaggg acaagggcaa gaggactgag gccagagcaa ggccaatgtc 1200 agagaccccc gggatggggc ctcacacttg ccacccccag aaccagctca cctggccaga 1260 gtgggttcct gctggccagg gtgcagcctt gatgacacct gccgctgccc ctcggggctg 1320 gaataaaact ccccacccag agtcaaaaaa aaaaaaaaga aaaaaaaaa 1369 <210> 21 <211> 2096 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3407686 <400> 21 cggacccgcc tctcccaaag tctagccggg caagggaacg cggtgcattc ctgaccggca 60 cctggcgagg ctcatgcgtc ccgtgagggc ggttcctcga gcctgggggc gctcagattg 120 ctttggagac gctgagagag cctttgcgag agcgccggtt gacgtgcgga gtgcggggct 180 ccgggggact gagcagcacg agaccccatc ctcccctccg ggttttcaca ctgggcgaag 240 ggaggactcc tgagctctgc ctcttccagt aacattgagg attactgtgt tttgtgagag 300 ctcgctaggc gccctaagca acagagttct gagaaatcga gaaacatgat aaggaattgg 360 ctgactattt ttatcctttt tcccctgaag ctcgtagaga aatgtgagtc aagcgtcagc 420 ctcactgttc ctcctgtcgt aaagctggag aacggcagct cgaccaacgt cagcctcacc 480 ctgcggccac cattaaatgc aaccctggtg atcacttttg aaatcacatt tcgttccaaa 540 aatattacta tccttgagct ccccgatgaa gttgtggtgc ctcctggagt gacaaactcc 600 tcttttcaag tgacatctca aaatgttgga caacttactg tttatctaca tggaaatcac 660 tccaatcaga ccggcccgag gatacgcttt cttgtgatcc gcagcagcgc cattagcatc 720 ataaaccagg tgattggctg gatctacttt gtggcctggt ccatctcctt ctaccctcag 780 gtgatcatga attggaggcg gaaaagtgtc attggtctga gcttcgactt cgtggctctg 840 aacctgacgg gcttcgtggc ctacagtgta ttcaacatcg gcctcctctg ggtgccctac 900 atcaaggagc agtttctcct caaatacccc aacggagtga accccgtgaa cagcaacgac 960 gtcttcttca gcctgcacgc ggttgtcctc acgctgatca tcatcgtgca gtgctgcctg 1020 20/2$

tatgagcgcg gtggccagcg cgtgtcctgg cctgccatcg gcttcctggt gctcgcgtgg 1080 ctcttcgcat ttgtcaccat gatcgtggct gcagtgggag tgatcacgtg gctgcagttt 1140 ctcttctgct tctcctacat caagctcgca gtcacgctgg tcaagtattt tccacaggcc 1200 tacatgaact tttactacaa aagcactgag ggctggagca ttggcaacgt gctcctggac 1260 ttcaccgggg gcagcttcag cctcctgcag atgttcctcc agtcctacaa caacgaccag 1320 tggacgctga tcttcggaga cccaaccaag tttggactcg gggtcttctc catcgtcttc 1380 gacgtcgtct tcttcatcca gcacttctgt ttgtacagaa agagaccggg gtatgaccag 1440 ctgaactagc acccagggac ccagtgtacc cagcctctgg cctcgtgccc tgctggggaa 1500 ggcctcaccc agcgaaggcc ggagaagcgg ttgggccctg gcacacaggg ctggctcagt 1560 gtgcggacag aggagaccac tctgctcctg gggccagagg ccattcaata gcctgccttc 1620 gtccgggccc ctcctgggcc tccceggcca ggcacgtggc accgtcgcct tgacaccgcc 1680 atctcttttc tttaaggctt caggcagcgc gcacaggctc tggcagccgt ctcaggcagg 1740 actgggcagc aagcttgcag ccgaagcgtt gccccaaact accagcgttt ctgcaagcag 1800 cttgaagggc tgaccttgca gccgggtgag ccaagggcac tttgctgcca ccgctgcatt 1860 cccagagatc aagcagcccg gtgccgtggc cagtgaactc agaggtgctg gtgggacggg 1920 ctaggacttt ggggttaggc catggggcgt ctttctctgg aggccacttt cctgacgtac 1980 tctctgtaca taattcagcg tccgtgactg cagtaacgag cagccctagc cgaggatttc 2040 tgagccatga ggggcccacg gaattggttc taaattgatc atgcccagcc attaga 2096 <210> 22 <211> 1431 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3472455 <400> 22 gaacctttgc acgcgcacaa actacgggga cgatttctga ttgcaatcag gcgcattcga 60 tccaccctcc tcccttctca tgggactttg gggacaaagc gtcccgaccg cctcgagcgc 120 tcgagcaggg cgctatccag gagccaggac agcgtcggga accagaccat ggctcctgga 180 ccccaagatc cttaagttcg tcgtcttcat cgtcgcggtt ctgctgccgg tccgggttga 240 ctctgccacc atcccccggc aggacgaagt tccccagcag acagtggccc cacagcaaca 300 gaggcgcagc ctcaaggagg aggagtgtcc agcaggatct catagatcag aatatactgg 360 agcctgtaac ccgtgcacag agggtgtgga ttacaccatt gcttccaaca atttgccttc 420 ttgcctgcta tgtacagttt gtaaatcagg tcaaacaaat aaaagttcct gtaccacgac 480 cagagacacc gtgtgtcagt gtgaaaaagg aagcttccag gataaaaact cccctgagat 540 gtgccggacg tgtagaacag ggtgtcccag agggatggtc aaggtcagta attgtacgcc 600 ccggagtgac atcaagtgca aaaatgaatc agctgccagt tccactggga aaaccccagc 660 agcggaggag acagtgacca ccatcctggg gatgcttgcc tctccctatc actaccttat 720 catcatagtg gttttagtca tcattttagc tgtggttgtg gttggctttt catgtcggaa 780 gaaattcatt tcttacctca aaggcatctg ctcaggtggt ggaggaggtc ccgaacgtgt 840 gcacagagtc cttttccggc ggcgttcatg tccttcacga gttcctgggg cggaggacaa 900 tgcccgcaac gagaccctga gtaacagata cttgcagccc acccaggtct ctgagcagga 960 aatccaaggt caggagctgg cagagctaac aggtgtgact gtagagtcgc cagaggagcc 1020 acagcgtctg ctggaacagg cagaagctga agggtgtcag aggaggaggc tgctggttcc 1080 agtgaatgac gctgactccg ctgacatcag caccttgctg gatgcctcgg caacactgga 1140 agaaggacat gcaaaggaaa caattcagga ccaactggtg ggctccgaaa agctctttta 1200 tgaagaagat gaggcaggct ctgctacgtc ctgcctgtga aagaatctct tcaggaaacc 1260 agagcttccc tcatttacct tttctcctac aaagggaagc agcctggaag aaacagtcca 1320 gtacttgacc catgccccaa caaactctac tatccaatat ggggcagctt accaatggtc 1380 ctagaacttt gttaacgcac ttggagtaat ttttatgaaa tactgcgtgt g 1431 <210> 23 wo oorosiss <211> 1788 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 786873 <400> 23 cctacgacga ctatagggaa tttggccctc gagcacggac attccggcac gatggcgagc 60 ctccccctcc tccccaggac gacatgaacg accgaggcca gggagtcctc tccttgggcc 120 tctgcatccc cccatccttg gctctggggt aggcccaggg aggagacacc cccaacccct 180 atccggtctg tcctggagaa aagagactgc ccttccatgc ccctgagtga ggggcctggg 240 gcccaggctg cctgtgttcc ccaagggcaa gggtctctct gttgaggagg aggggcctgt 300 cagccacaac ttctttcctc ctgagcgccc catctccctc tctgcaccct gcaattccca 360 cccctccgta tttatttccc tggtcccgcc gacagtccct ccttgtctgt ctccgggatt 420 caggcctccc tccctgacat ggagagtaac ctgtctggcc tggtgcctgc tgccgggctg 480 gtgcctgcgc tgccacctgc tgtgaccctg gggctgacag ctgcctacac caccctgtat 540 gccctgctct tcttctccgt ctatgcccag ctctggctgg tgcttctgta tgggcacaag 600 cgtctcagct atcagacggt gttcctggcc ctctgtctgc tctgggccgc cttgcgtacc 660 accctcttct ccttctactt ccgagatact ccccgcgcca accgcctggg gcccttgccc 720 ttctggcttc tctactgctg ccccgtctgc ctgcagttct tcaccttgac gcttatgaac 780 ctctactttg cccaggtggt gttcaaggcc aaggtgaagc gtcggccgga gatgagccga 840 ggcttgctcg ctgtccgagg ggcctttgtg ggggcctcgc tgctctttct gctggtgaac 900 gtgctgtgtg ctgtgctctc ccatcggcgc cgggcacagc cctgggccct gctgcttgtc 960 cgcgtcctgg tgagcgactc cctgttcgtc atctgcgcgc tgtctcttgc tgcctgcctc 1020 tgcctcgtcg ccaggcgggc gccctccact agcatctacc tggaggccaa ggcggacctg 1080 gtgaatgacc tggggaacaa aggctacctg gtatttggcc tcatcctctt cgtgtgggag 1140 ctactgccca ccaccctgct ggtgggcttc ttccgggtgc accggccccc acaggacctg 1200 agcaccagcc acatcctcaa tgggcaggtc tttgcctctc ggtcctactt ctttgaccgg 1260 gctgggcact gtgaagatga gggctgctcc tgggagcaca gccggggtga gagcaccagt 1320 atgtcgggca gtctaggctc tgggagctgg tatggtgcca tcgggcgtga gccgggctgg 1380 tatgggggca gccagacgaa gaccactcct ctgctcttct cccaggtgcc aggaccaggc 1440 ggccaccacc acagtctcta ctccacccca cagacgtgat ccccctccct cccccacaga 1500 atacccaggc cccagtcccc ctcaccctag gcccctgtgc caagtttgtc tgccgcttct 1560 tgcccaggat cctgggggtc gtggctaccc cctcctctgg ccggctcctt gctgctcctg 1620 tcatagtgag cttgtgccgt ccccctagga tggggggcat ggccctggct gccagatgcc 1680 cacagcaccc tggcatgacc tgccacctct gcttccacac cggagccagc tacctctcct 1740 gtgcctgcca ctcaataaac agtgtctgcg ccccacaaaa aaaaaaaa 1788 <210> 24 <211> 1044 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1220371 <400> 24 gaaagccaac tttcctttca aatacacacc ccaacccgcc ccggcataca cagaaatggg 60 gactgcgagc agaagcaaca tcgctcgcca tctgcaaacc aatctcattc tattttgtgt 120 cggtgctgtg ggcgcctgta ctctctctgt cacacaaccg tggtacctag aagtggacta 180 cactcatgag gccgtcacca taaagtgtac cttctccgca accggatgcc cttctgagca 240 accaacatgc ctgtggtttc gctacggtgc tcaccagcct gagaacctgt gcttggacgg 300 gtgcaaaagt gaggcagaca agttcacagt gagggaggcc ctcaaagaaa accaagtttc 360 wo ooiosiss ~,NS~m,~',~
cctcactgta aacagagtga cttcaaatga cagtgcaatt tacatctgtg gaatagcatt 420 ccccagtgtg ccggaagcga gagctaaaca gacaggagga gggaccacac tggtggtaag 480 agaaattaag ctgctcagca aggaactgcg gagcttcctg acagctcttg tatcactgct 540 ctctgtctat gtgaccggtg tgtgcgtggc cttcatactc ctctccaaat caaaatccaa 600 ccctctaaga aacaaagaaa taaaagaaga ctcacaaaag aagaagagtg ctcggcgtat 660 ttttcaggaa attgctcaag aactatacca taagagacat gtggaaacaa atcagcaatc 720 tgagaaagat aacaacactt atgaaaacag aagagtactt tccaactatg aaaggccata 780 gaaacgtttt aattttcaat gaagtcactg aaaatccaac tccaggagct atggcagtgt 840 taatgaacat atatcatcag gtcttaaaaa aaaataaagg taaactgaaa agacaactgg 900 ctacaaagaa ggatgccaga atgtaaggaa actataacta atagtcatta ccaaaatact 960 aaaacccaac aaaatgcaac tgaaaaatac cttccaaatt tgccaagaaa aaaaattcta 1020 ttttaaactt gtaaaaaaaa aaaa 1044 <210> 25 <211> 1605 <212> DNA
<213> Homo sapiens <220>
<221> mi.sc_feature <223> Incyte Clone No: 1258785 <400> 25 gtccccgccc gcgcgccgta ccgcggcgga gatgggcgag accatgtcca agaggctgaa 60 gctccacctg ggaggggagg cagaaatgga ggaacgggcg ttcgtcaacc ccttcccgga 120 ctacgaggcc gccgccgggg cgctgctcgc ctccggagcg gccgaagaga caggctgtgt 180 tcgtcccccg gcgaccacgg atgagcccgg cctccctttt catcaggacg ggaagatcat 240 tcataatttc ataagacgga tccagaccaa aattaaagat cttctgcagc aaatggaaga 300 agggctgaag acggctgatc cccatgactg ctctgcttat actggctgga caggcatagc 360 ccttttgtac ctgcagttgt accgggtcac atgtgaccaa acctacctgc tccgatccct 420 ggattacgta aaaagaacac ttcggaatct gaatggccgc agggtcacct tcctctgtgg 480 ggatgctggc cccctggctg ttggagctgt gatttatcac aaactcagaa gtgactgtga 540 gtcccaggaa tgtgtcacaa aacttttgca gctccagaga tcggttgtct gccaagaatc 600 agaccttcct gatgagctgc tttatggacg ggcaggttat ctgtatgcct tactgtacct 660 gaacacagag ataggtccag gcaccgtgtg tgagtcagct attaaagagg tagtcaatgc 720 tattattgaa tcgggtaaga ctttgtcaag ggaagaaaga aaaacggagc gctgcccgct 780 gttgtaccag tggcaccgga agcagtacgt tggagcagcc catggcatgg ctggaattta 840 ctatatgtta atgcagccgg cagcaaaagt ggaccaagaa accttgacag aaatggtgaa 900 acccagtatt gattatgtgc gccacaaaaa attccgatct gggaattacc catcatcatt 960 aagcaatgaa acagaccggc tggtgcactg gtgccacggc gccccggggg tcatccacat 1020 gctcatgcag gcgtacaagg tctttaagga ggagaagtac ttgaaagagg ccatggagtg 1080 tagcgatgtg atttggcagc gaggtttgct gcggaagggc tacgggatat gccatgggac 1140 tgctggcaac ggctattcct tcctgtccct ttaccgtctc acgcaggata agaagtacct 1200 ctaccgagct tgcaagtttg cagagtggtg tctagattac ggagcacacg ggtgccgcat 1260 tcctgacaga ccctattcgc tctttgaagg catggctggc gctattcact ttctctctga 1320 tgtcctggga ccagagacat cacggtttcc agcatttgaa cttgactctt cgaagaggga 1380 ttaaaaggtg caaaaagaca actaaaatac ccatttggac caaaagccgc cagattgctt 1440 agtgcctgac acagaaacaa ctgggaatcc tgaaagagaa gcagacaccg tcacaggccc 1500 ctctggttag actagcatga gtgaccgaag ccatccatca acattttcta acagcaccct 1560 catcaatata aaatatgact tcttcacata cagaaaaaaa aaaaa 1605 <210> 26 <211> 1464 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1361202 <400> 26 gagatagcaa agcagagtgt ttatcttgtg agccattctc catatttcag atataagatt 60 tcagttctca gtgagtctaa gtgacagaag gaatggagac cctcttgggc ctgcttatcc 120 tttggctgca gctgcaatgg gtgagcagca aacaggaggt gacacagatt cctgcagctc 180 tgagtgtccc agaaggagaa aacttggttc tcaactgcag tttcactgat agcgctattt 240 acaacctcca gtggtttagg caggaccctg ggaaaggtct cacatctctg ttgcttattc 300 agtcaagtca gagagagcaa acaagtggaa gacttaatgc ctcgctggat aaatcatcag 360 gacgtagtac tttatacatt gcagcttctc agcctggtga ctcagccacc tacctctgtg 420 ctgtgaggga caatgacatg cgctttggag cagggaccag actgacagta aaacccaata 480 tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 540 tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 600 tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 660 ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 720 ttccagaaga caccttcttc cccagcccag aaagttcctg tgatgtcaag ctggtcgaga 780 aaagctttga aacagatacg aacctaaact ttcaaaacct gtcagtgatt gggttccgaa 840 tcctcctcct gaaagtggcc gggtttaatc tgctcatgac gctgcggctg tggtccagct 900 gagatctgca agattgtaag acagcctgtg ctccctcgct ccttcctctg cattgcccct 960 cttctccctc tccaaacaga gggaactctc ccacccccaa ggaggtgaaa gctgctacca 1020 cctctgtgcc cccccggcaa tgccaccaac tggatcctac ccgaatttat gattaagatt 1080 gctgaagagc tgccaaacac tgctgccacc ccctctgttc ccttattgct gcttgtcact 1140 gcctgacatt cacggcagag gcaaggctgc tgcagcctcc cctggctgtg cacattccct 1200 cctgctcccc agagactgcc tccgccatcc cacagatgat ggatcttcag tgggttctct 1260 tgggctctag gtcctgcaga atgttgtgag gggtttattt ttttttaata gtgttcataa 1320 agaaatacat agtattcttc ttctcaagac gtggggggaa attatctcat tatcgaggcc 1380 ctgctatgct gtgtatctgg gcgtgttgta tgtcctgctg ccgatgcctt cattaaaatg 1440 attgggagag cagaaaaaaa aaaa <210> 27 <211> 960 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2132846 <400> 27 acggccagtg caagctaaaa ttaaccctca ctaaagggaa taagcttgcg ccgccagagg 60 atggggaaat ccaacagcaa gttgacgccc gaagttgtgg aggagctgac caggaagacc 120 tactttaccg agaaggaggt ccagcagtgg tacaaaggct tcatcaagga ctgccccagt 180 gggcagctgg atgcggcagg cttccagaag atctacaagc aattcttccc gttcggagac 240 cccaccaagt ttgccacatt tgttttcaac gtctttgatg aaaacaagga cgggcgaatt 300 gagttctccg agttcatcca ggcgctgtcg gtgacctcac ggggaaccct ggatgagaag 360 ctacggtggg ccttcaagct ctacgacttg gacaatgatg gctacatcac caggaatgag 420 atgctggaca ttgtggatgc catttaccag atggtgggga ataccgtgga gctcccagag 480 gaggagaaca ctcctgagaa gagggtggac cggatctttg ccatgatgga taagaatgcc 540 gacgggaagc tgaccctgca ggagttccag gaggggtcca aggcagaccc gtccattgtg 600 caggcgctgt ccctctacga cgggctggta tagtcccagg ctggagctgg atgcctggga 660 accactcacc tccttctgtg ccatgaggcc acctcagccc tgacaccaac cccgtgcgtc 720 cacccagcct tcttccgcat ccacacacag ccggctgccc ttgacccggg aggccccggc 780 tctcctctcc cctgtcctgc acccatcccc cgcctgaagc caccggctcc aattgccagc 840 aacctctgct tgtccggaaa acgacaacac gaaatggaaa aggctacagc cctctgcata 900 z4ns wo oo~osiss Pcrius99n~7~~
aaccaaggac ttggctgcct cgcaggcagc gtccgttcct cccgctctct tgcgcgtgtg 960 <210> 28 <211> 1990 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2539294 <400> 28 tctcctcccc ctcccggcca agatgtctga catggaggat gatttcatgt gcgatgatga 60 ggaggactac gacctggaat actctgaaga tagtaactcc gagccaaatg tggatttgga 120 aaatcagtac tataattcca aagcattaaa agaagatgac ccaaaagcgg cattaagcag 180 tttccaaaag gttttggaac ttgaaggtga aaaaggagaa tggggattta aagcactgaa 240 acaaatgatt aagattaact tcaagttgac aaactttcca gaaatgatga atagatataa 300 gcagctattg acctatattc ggagtgcagt cacaagaaat tattctgaaa aatccattaa 360 ttctattctt gattatatct ctacttctaa acagaattct gattttttat gtcagatgga 420 tttactgcag gaattctatg aaacaacact ggaagctttg aaagatgcta agaatgatag 480 actgtggttt aagacaaaca caaagcttgg aaaattatat ttagaacgag aggaatatgg 540 aaagcttcaa aaaattttac gccagttaca tcagtcgtgc cagactgatg atggagaaga 600 tgatctgaaa aaaggtacac agttattaga aatatatgct ttggaaattc aaatgtacac 660 agcacagaaa aataacaaaa aacttaaagc actctatgaa cagtcacttc acatcaagtc 720 tgccatccct catccactga ttatgggagt tatcagagaa tgtggtggta aaatgcactt 780 gagggaaggt gaatttgaaa aggcacacac tgattttttt gaagccttca agaattatga 840 tgaatctgga agtccaagac gaaccacttg cttaaaatat ttggtcttag caaatatgct 900 tatgaaatcg ggaataaatc catttgactc acaggaggcc aagccgtaca aaaatgatcc 960 agaaatttta gcaatgacga atttagtaag tgcctatcag aataatgaca tcactgaatt 1020 tgaaaagatt ctaaaaacaa atcacagcaa catcatggat gatcctttca taagagaaca 1080 cattgaagag cttttgcgaa acatcagaac acaagtgctt ataaaattaa ttaagcctta 1140 cacaagaata catattcctt ttatttctaa ggagttaaac atagatgtag ctgatgtgga 1200 gagcttgctg gtgcagtgca tattggataa cactattcat ggccgaattg atcaagtcaa 1260 ccaactcctt gaactggatc atcagaagag gggtggtgca cgatatactg cactagataa 1320 atggaccaac caactaaatt ctctcaacca ggctgtagtc agtaaactgg cttaacagag 1380 aacaagcttt tacagacgtc cttaaggcaa cagtgcagag atgtaatcct taaaagaact 1440 gggaatggca aaactactgt cggttgatgt gtcctgaaaa ttattggagt tatggcagaa 1500 gtgctttttt gatcaactgg tttgtgtttt gctgctgcat ttatcccaag aaaaacagct 1560 ttaatctcca gaagaaaacc saaataccat gggatttatg ctgtattgac atcttgccct 1620 aaacgtacaa catcatagta atttgtcatg ggcaacatga ccagagagaa gatttttgtc 1680 atgattttaa atacactgac acgctactgt tggttaaatt taaacatgtt ttacctgcag 1740 aaattctctc acaaataacc tgcaataact tgaaatgcat acccttttga acacttcctt 1800 ttctcatgta taaattaaaa tgtttgctgc attttgcaaa atgtcaattc tctaaaaatg 1860 tgtccgtata tttctgtacc tgcagtgtag taaaggttta gacgaaaccc cataattata 1920 gtggcatact gtcacttagg tttcaagcag caaaataaac agtgcagctc agaaattgta 1980 aaaaaaaaaa 1990 <210> 29 <211> 1038 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2589371 <400> 29 cataaatact gctctaagaa agggacagga agtctcagag gctggagagc agagcaccaa 60 gatcgttctg gcaggaacag ccagtgggag gttccagctg agcgctcccc agaggtgagc 120 tgatccccag ccacagcaca caggaccagg ctgcgagaac agcatcatca gcatcatgct 1B0 attacaatcc caaaccatgg gggtttctca cagctttaca ccaaagggca tcactatccc 240 tcaaagagag aaacctggac acatgtacca aaacgaagat tacctgcaga acgggctgcc 300 aacagaaacc accgttcttg ggactgtcca gatcctgtgt tgcctgttga tttcaagtct 360 gggggccatc ttggtttttg ctccctaccc ctcccacttc aatccagcaa tttccaccac 420 tttgatgtct gggtacccat ttttaggagc tctgtgtttt ggcattactg gatccctctc 480 aattatctct ggaaaacaat caactaagcc ctttgacctg agcagcttga cctcaaatgc 540 agtgagttct gttactgcag gagcaggcct cttcctcctt gctgacagca tggtagccct 600 gaggactgcc tctcaacatt gtggctcaga aatggattat ctatcctcat tgccttattc 660 ggagtactat tatccaatat atgaaatcaa agattgtctc ctgaccagtg tcagtttaac 720 aggtgtccta gtggtgatgc tcatcttcac tgtgctggag ctcttattag ctgcatacag 780 ttctgtcttt tggtggaaac agctctactc caacaaccct gggagttcat tttcctcgac 840 ccagtcacaa gatcatatcc aacaggtcaa aaagagttct tcacggtctt ggatataagt 900 aactcttggc ctcagaggaa ggaaaagcaa ctcaacactc atggtcaagt gtgattagac 960 tttcctgaaa tctctgccat tttagatact gtgaaacaaa aaaaaaaaaa aaaagctttt 1020 gttttgtaaa aaaaaaaa 1038 <210> 30 <211> 1260 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2656082 <400> 30 gaaaaaccac caaccaaggc caaggagacc agagcccagc acctcaccca gaggacccca 60 gtcagaggcc ccatctcaga cccgaggcta gcatgggctg caggctgctc tgctgtgcgg 120 ttctctgtct cctgggagcg gtccccatgg aaacgggagt tacgcagaca ccaagacacc 180 tggtcatggg aatgacaaat aagaagtctt tgaaatgtga acaacatctg ggtcataacg 240 ctatgtattg gtacaagcaa agtgctaaga agccactgga gctcatgttt gtctacagtc 300 ttgaagaacg ggttgaaaac aacagtgtgc caagtcgctt ctcacctgaa tgccccaaca 360 gctctcactt attccttcac ctacacaccc tgcagccaga agactcggcc ctgtatctct 420 gcgccagcag ccaagtacat ccgggactag cgggaggatt aaatgagcag ttcttcgggc 480 cagggacacg gctcaccgtg ctagaggacc tgaaaaacgt gttcccaccc gaggtcgctg 540 tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg gtgtgcctgg 600 ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg aaggaggtgc 660 acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc aatgactcca 720 gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac ccccgcaacc 780 acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg acccaggata 840 gggccaaacc cgtcacccag atcgtcagcg ccgaggcctg gggtagagca gactgtggct 900 tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat gagatcttgc 960 tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg gccatggtca 1020 agagaaagga ttccagaggc tagctccaaa accatcccag gtcattcttc atcctcaccc 1080 aggattctcc tgtacctgct cccaatctgt gttcctaaaa gtgattctca ctctgcttct 1140 catctcctac ttacatgaat acttctctct tttttctgtt tccctgaaga ttgagctccc 1200 aacccccaag tacgaaatag gctaaaccaa taaaaaattg tgtgttggga aaaaaaaaaa 1260 <210> 31 <211> 1551 <212> DNA

WU 00~08I55 <213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2762182 <400> 31 , agagcccggg ctgaccacag agaggggctg cagggacagg ctgacaggga gggtggtcca 60 cccttctgct gagagctgcc ccttttcagc ctagctcccc ccaccccccc acataggtgg 120 ggcagccgag tgcctgggga agccagggcc ttccctgaca tcccgtcccc agggattgag 180 agatggcaac gattaccgag aaggaggtcc agcagtggta caaaggcttc atcaaggact 240 gccccagtgg gcagctggat gcggcaggct tccagaagat ctacaagcaa ttcttcccgt 300 tcggagaccc caccaagttt gccacatttg ttttcaacgt ctttgatgaa aacaaggacg 360 ggcgaattga gttctccgag ttcatccagg cgctgtcggt gacctcacgg ggaaccctgg 420 atgagaagct acggtgggcc ttcaagctct acgacttgga caatgatggc tacatcacca 480 ggaatgagat gctggacatt gtggatgcca tttaccagat ggtggggaat accgtggagc 540 tcccagagga ggagaacact cctgagaaga gggtggaccg gatctttgcc atgatggata 600 agaatgccga cgggaagctg accctgcagg agttccagga ggggtccaag gcagacccgt 660 ccattgtgca ggcgctgtcc ctctacgacg ggctggtata gtcccaggct ggagctggat 720 gcctgggaac cactcacctc cttctgtgcc atgaggccac ctcagccctg acaccaaccc 780 cgtgcgtcca cccagccttc ttccgcatcc acacacagcc ggctgccctt gacccgggag 840 gccccggctc tcctctcccc tgtcctgcac ccatcccccg cctgaagcca ccggctccaa 900 ttgccagcaa cctctgcttg tccggaaaac gacaacacga aatggaaaag gctacagccc 960 tctgcataaa ccaaggactt ggctgcctcg caggcagcct ccgttcctcc cgctctcttg 1020 cgcgtgtgct tttgtttttt attttgaaca gacgttttaa aagaaaaaaa aacaactacc 1080 ttctgtccta gaagacacag actgacagat ggggtgaagg cctggggacc tcagagaact 1140 ctgccttgcc ctcgtccctc gtccttcggc agccggagag gctgtgggtg ggccgagggt 1200 gtctaggggt tctgcctggt caacgttatt tgtcgtccca tcttttggca gcaaaaccac 1260 ctgcgtggct aggatgatta attatgagga tgatgatttt ttttgtgata acagtattgt 1320 gctttttgtg gggaaagtga ggtttttttt tatatacata tataattgat atctttaatt 1380 tattggttgt taactgttgc tgctgcctgg. tgtgtcctca gctcccaggg ctgcgggccc 1440 accgtttaca tgtgcacgcc ctgacccacc tgcccacgcc gacttgggag gatggtggcc 1500 tgcagcggcc aagaagccaa aaaaaatttt ttttttttca aaaaaaaaaa a 1551 <210> 32 <211> 2889 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No:3140659 <400> 32 gccacagctg gtttagggcc ccgaccactg gggccccttg tcaggaggag acagcctccc 60 ggcccgggga ggacaagtcg ctgccacctt tggctgccga cgtgattccc tgggacggtc 120 cgtttcctgc cgtcagctgc cggccgagtt gggtctccgt ggttcaggcc ggctccccct 180 tcctggtctc ccttctcccg ctgggccggt ttatcgggag gagattgtct tccagggcta 240 gcaattggac ttttgatgat gtttgaccca gcggcaggaa tagcaggcaa cgtgatttca 300 aagctgggct cagcctctgt ttcttctctc gtgtaatcgc aaaacccatt ttggagcagg 360 aattccaatc atgtctgtga tggtggtgag aaagaaggtg acacggaaat gggagaaact 420 cccaggcagg aacacctttt gctgtgatgg ccgcgtcatg atggcccggc aaaagggcat 480 tttctacctg acccttttcc tcatcctggg gacatgtaca ctcttcttcg cctttgagtg 540 ccgctacctg gctgttcagc tgtctcctgc catccctgta tttgctgcca tgctcttcct 600 tttctccatg gctacactgt tgaggaccag cttcagtgac cctggagtga ttcctcgggc 660 gctaccagat gaagcagctt tcatagaaat ggagatagaa gctaccaatg gtgcggtgcc 720 wo oo~osiss ccagggccag cgaccaccgc ctcgtatcaa gastttccag ataaacaacc agattgtgaa 780 actgaaatac tgttacacat gcaagatctt ccggcctccc cgggcctccc attgcagcat 840 ctgtgacaac tgtgtggagc gcttcgacca tcactgcccc tgggtgggga attgtgttgg 900 aaagaggaac taccgctact tctacetctt catectttct ctctccctcc tcacaatcta 960 tgtcttcgcc ttcaacatcg tctatgtggc cctcaaatct ttgaaaattg gcttcttgga 1020 gacattgaaa gaaactcctg gaactgttct agaagtcctc atttgcttct ttacactctg 1080 gtccgtcgtg ggactgactg gatttcatac tttcctcgtg gctctcaacc agacaaccaa 1140 tgaagacatc aaaggatcat ggacagggaa gaatcgcgtc cagaatccct acagccatgg 1200 caatattgtg aagaactgct gtgaagtgct gtgtggcccc ttgcccccca gtgtgctgga 1260 tcgaaggggt attttgccac tggaggaaag tggaagtcga cctcccagta ctcaagagac 1320 cagtagcagc ctcttgccac agagcccagc ccccacagaa cacctgaact caaatgagat 1380 gccggaggac agcagcactc ccgaagagat gccacctcca gagcccccag agccaccaca 1440 ggaggcagct gaagctgaga agtagcctat ctatggaaga gacttttgtt tgtgtttaat 1500 tagggctatg agagatttca ggtgagaagt taaacctgag acagagagca agtaagctgt 1560 .
cccttttaac tgtttttctt tggtctttag tcacccagtt gcacactggc attttcttgc 1620 tgcaagcttt tttaaatttc tgaactcaag gcagtggcag aagatgtcag tcacctctga 1680 taactggaaa aatgggtctc ttgggccctg gcactggttc tccatggcct cagccacagg 1740 gtccccttgg accccctctc ttccctccag atcccagccc tcctgcttgg ggtcactggt 1800 ctcattctgg ggctaaaagt ttttgagact ggctcaaatc ctcccaagct gctgcacgtg 1860 ctgagtccag aggcagtcac agagacctct ggccagggga tcctaactgg gttcttgggg 1920 tcttcaggac tgaagaggag ggagagtggg gtcagaagat tctcctggcc accaagtgcc 1980 agcattgccc acaaatcctt ttaggaatgg gacaggtacc ttccacttgt tgtatttatt 2040 agtgtagctt ctcctttgtc tcccatccac tctgacacct aagccccact cttttcccat 2100 tagatatatg taagtagttg tagtagagat aataattgac atttctcgta gactacccag 2160 aaactttttt aatacctgtg ccattctcaa taagaattta tgagatgcca gcggcatagc 2220 ccttcacact ctctgtctca tctctcctcc tttctcatta gcccctttta atttgttttt 2280 ccttttgact cctgctccca ttaggagcag gaatggcagt aataaaagtc tgcactttgg 2340 tcatttcttt tcctcagagg aagcctgagt gctcacttaa acactatccc ctcagactcc 2400 ctgtgtgagg cctgcagagg ccctgaatgc acaaatggga aaccaaggca cagagaggct 2460 ctcctctcct ctcctctccc cegatgtacc ctcaaaaaaa aaaaaaatgc taaccagttc 2520 ttccattaag cctcggctga gtgagggaaa gcccagcact gctgccctct cgggtaactc 2580 accctaaggc ctcggcccac ctctggctat ggtaaccaca ctgggggctt cctccaagcc 2640 ccgctcttcc agcacttcca ccggcagagt cccagagcca cttcaccctg ggggtgggct 2700 gtggccccca gtcagctctg ctcaggacct gctctatttc agggaagaag atttatgtat 2760 tatatgtggc tatatttcct agagcacctg tgttttcctc tttctaagcc agggtcctgt 2820 ctggatgact tatgcggtgg gggagtgtaa accggaactt ttcatetatt tgaaggcgat 2880 taaactgtg

Claims (20)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ
ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and fragments thereof.

2. A substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of claim 1.

3. An isolated and purified polynucleotide encoding the polypeptide of claim 1.

4. An isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity to the polynucleotide of claim 3.

5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.

6. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 3.

7. A method for detecting a polynucleotide, the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one nucleic acid in a sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide in the sample.

8. The method of claim 7 further comprising amplifying the polynucleotide prior to hybridization.

9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:17-32 and fragments thereof.

10. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 9.

11. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 9.

12. An expression vector comprising at least a fragment of the polynucleotide of claim 3.

13. A host cell comprising the expression vector of claim 12.

14. A method for producing a polypeptide, the method comprising the steps of:
a) culturing the host cell of claim 13 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

15. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.

16. A purified antibody which specifically binds to the polypeptide of claim 1.

17. A purified agonist of the polypeptide of claim 1.

18. A purified antagonist of the polypeptide of claim 1.

19. A method for treating or preventing a disorder associated with decreased expression or activity of HRAP, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 15.

20. A method for treating or preventing a disorder associated with increased expression or activity of HRAP, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 18.