CA2375407A1 - Rna metabolism proteins - Google Patents

Abstract

The invention provides human RNA metabolism proteins (RMEP) and polynucleotides which identify and encode RMEP. 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 expresssion of RMEP.

Description

RNA METABOLISM PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of RNA
metabolism proteins and to the use of these sequences in the diagnosis, treatment, and prevention of nervous system, autoimmune/inflammatory, and cell proliferative disorders, including cancer.
BACKGROUND OF THE INVENTION
Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, CTP, 1Q UTP, and GTP. In most organisms, RNA is transcribed as a copy of deoxyribonucleic acid (DNA), the genetic material of the organism. In retroviruses RNA rather than DNA
serves as the genetic material. RNA copies of the genetic material encode proteins or serve various structural, catalytic, or regulatory roles in organisms. RNA is classified according to its cellular localization and function.
Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate mRNA into polypeptides. Transfer RNAs (tRNAs) are cytosolic adaptor molecules that function in mRNA
translation by recognizing both an mRNA codon and the amino acid that matches that codon.
Heterogeneous nuclear RNAs (hnRNAs) include mRNA precursors and other nuclear RNAs of various sizes. Small nuclear RNAs (snRNAs) are a part of the nuclear spliceosome complex that removes intervening, non-coding sequences (introns) and rejoins exons in pre-mRNAs.
Proteins are associated with RNA during its transcription from DNA, RNA
processing, and translation of mRNA into protein. Proteins are also associated with RNA as it is used for structural, catalytic, and regulatory purposes.
Various proteins are necessary for processing of transcribed RNAs in the nucleus. Pre-mRNA processing steps include capping at the 5' end with methylguanosine, polyadenylating the 3' end, and splicing to remove introns. The spliceosomal complex is comprised of five small nuclear ribonucleoprotein particles (snRNPs) designated U1, U2, U4, U5, and U6. Each snRNP contains a single species of snRNA and about ten proteins. The RNA components of some snRNPs recognize and base-pair with intron consensus sequences. The protein components mediate spliceosome assembly and the splicing reaction.
An early step in pre-mRNA cleavage involves the cleavage factor Im (CF Im).
The human CF Im protein aids in the recruitment and assembly of processing factors that make up the 3' end processing complex (Ruegsegger, U. et al (1998) Mol. Cell. 1:243-253). The marine formin binding proteins (FBP's) FBP11 and FBP12 are components of pre-mRNA splicing complexes that facilitate the bridging of 5' and 3' ends of the intron. These proteins function through bridging interactions invloving U1 and U2 snRNPs. Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry W.H. Freeman and Company, New York NY, p. 863).
Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that have roles in splicing, exporting of the mature RNAs to the cytoplasm, and mRNA translation (Biamonti, G. et al.
(1998) Clin. Exp. Rheumatol. 16:317-326). Some examples of hnRNPs include the yeast proteins Hrplp, involved in cleavage and polyadenylation at the 3' end of the RNA;
Cbp80p, involved in capping the 5' end of the RNA; and Npl3p, a homolog of mammalian hnRNP A1, involved in export of mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691).
HnRNPs have been shown to be important targets of the autoimmune response in rheumatic diseases (Biamonti, su ra .
Many snRNP and hnRNP proteins are characterized by an RNA recognition motif (RRM).
(Reviewed in Birney, E. et al. (1993) Nucleic Acids Res. 21:5803-5816.) The RRM is about 80 amino acids in length and forms four ~i-strands and two a-helices arranged in an a/(3 sandwich. The RRM
contains a core RNP-1 octapeptide motif along with surrounding conserved sequences. In addition to snRNP proteins, examples of RNA-binding proteins which contain the above motifs include heteronuclear ribonucleoproteins which stabilize nascent RNA and factors which regulate alternative splicing. Alternative splicing factors include developmentally regulated proteins, specific examples of which have been identified in lower eukaryotes such as Drosophila melano~aster and Caenorhabditis elegans. These proteins play key roles in developmental processes such as pattern formation and sex determination, respectively. (See, for example, Hodgkin, J. et al. (1994) Development 120:3681-3689.) Ribonucleases (RNases) catalyze the hydrolysis of phosphodiester bonds in RNA
chains, thus cleaving the RNA. For example, RNase P is a ribonucleoprotein enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation process. RNase H digests the RNA strand of an RNA/DNA
hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle. RNase H domains are often found as a domain associated with reverse transcriptases. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15:529-536).
Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections.
Degradation of mRNAs having premature termination or nonsense codons is accomplished through a surveillance mechanism that has been termed nonsense-mediated mRNA
decay (NMD).
This mechanism helps eliminate flawed mRNAs that might code for nonfunctional or deleterious polypeptides. Various NMD components are linked to both yeast and human RNA
metabolism disorders (Hentze, M. and Kulozik, A. (1999) Cell 96:307-310).
The conversion of information in the form of mRNA to protein involves the many ribosomal proteins of the translation machinery of the cell. The eukaryotic ribosome is composed of a 60S
(large) subunit and a 40S (small) subunit, which together form the 80S
ribosome. In addition to the 18S, 28S, SS, and 5.8S rRNAs, the ribosome also contains more than fifty proteins. The ribosomal proteins have a prefix which denotes the subunit to which they belong, either L (large) or S (small).
Initiation of translation requires the participation of several initiation factors, many of which contain multiple subunits. One eukaryotic initiation factor (EIF~ EIFSA is an 18-kD protein containing the unique amino acid residue, hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) (Rinaudo, M. et al. (1993) Gene 137:303-307).
The release factor eRF carries out termination of translation. eRF recognizes stop codons in the mRNA, leading to the release of the polypeptide chain from the ribosome.
The discovery of new RNA metabolism 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 nervous system, autoimmune/inflammatory, and cell proliferative disorders, including cancer.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, RNA metabolism proteins, referred to collectively as "RMEP" and individually as "RMEP-1 ", "RMEP-2", "RMEP-3", "RMEP-4", "RMEP-5", "RMEP-6", "RMEP-7", "RMEP-8", "RMEP-9", "RMEP-10", "RMEP-11", "RMEP-12" and "RMEP-13". In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:l-13.
The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-13. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-13. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID N0:14-26.
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-13. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.
The invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID
NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13.
The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleodde sequence selected from the group consisting of SEQ
ID N0:14-26, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.

Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.
The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID N0:14-26, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
The invention further provides a pharmaceutical composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-13. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional RMEP, comprising administering to a patient in need of such treatment the pharmaceutical composition.
The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a pharmaceutical composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional RMEP, comprising administering to a patient in need of such treatment the pharmaceutical composition.
Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a pharmaceutical composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional RMEP, comprising administering to a patient in need of such treatment the pharmaceutical composition.
The invention further provides a method of screening for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypepdde.

The invention further provides a method of screening for a compound that modulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:1-13, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ
ID NO:1-13. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypepdde in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID N0:14-26, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full-length sequences encoding RMEP.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of RMEP.
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis;
diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA
was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding RMEP were isolated.
Table 5 shows the tools, programs> and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
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 fo 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
"RMEP" refers to the amino acid sequences of substantially purified RMEP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, marine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the biological activity of RMEP. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of RMEP either by directly interacting with RMEP or by acting on components of the biological pathway in which RMEP
participates.
An "allelic variant" is an alternative form of the gene encoding RMEP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding RMEP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as RMEP or a polypeptide with at least one functional characteristic of RMEP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding RMEP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding RMEP. 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 RMEP. 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 RMEP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine;
and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the biological activity of RMEP. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of RMEP either by directly interacting with RMEP or by acting on components of the biological pathway in which RMEP
participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant.
Antibodies that bind RMEP polypepddes can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA; RNA;
peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative" or "minus" can refer to the antisense strand, and the designation "positive" or "plus" can refer to the sense strand of a reference DNA molecule.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" or "immunogenic"
refers to the capability of the natural, recombinant, or synthetic RMEP, or,of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution Compositions comprising polynucleotide sequences encoding RMEP or fragments of RMEP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;
SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
S "Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala ~s Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val - Ile, Leu, Thr Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
A "fragment" is a unique portion of RMEP or the polynucleotide encoding RMEP
which is identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25 % or 50% of a polypeptide) as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
A fragment of SEQ ID N0:14-26 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:14-26, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID N0:14-26 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:14-26 from related polynucleotide sequences. The precise length of a fragment of SEQ
ID N0:14-26 and the region of SEQ ID N0:14-26 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO:1-13 is encoded by a fragment of SEQ ID N0:14-26. A
fragment of SEQ ID NO:1-13 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-13. For example, a fragment of SEQ ID NO:l-13 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-13.
The precise length of a fragment of SEQ ID NO:1-13 and the region of SEQ ID NO:1-13 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A "full-length" polynucleodde sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A "full-length" polynucleodde sequence encodes a "full-length" polypeptide sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wn. CLUSTAL V is described in Higgins, D.G.
and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992) CABIOS 8:189-191.
For pairwise alignments of polynucleotide sequences, the default parameters are set as follows:
Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted"
residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBn Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.govBLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST
programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2Ø12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2 Open Gap: 5 and Extension Gap: 2 penalties Gap x drop-off.' S0 Expect: 10 Word Size: 1l Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity"
between aligned polypeptide sequence pairs.
Aiternadvely the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version 2Ø12 (Apr-21-2000) with blastp set at default parameters. Such default parameters may be, for example:
Matrix: BLOSUM62 Open Gap: 1l and Extension Gap: 1 penalties Gap x drop-off.' S0 Expect: l0 Word Size: 3 Filter: on Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured "Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity.
Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing steps) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 ~g~ml sheared, denatured salmon sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T"~ for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J.
et al., 1989, Molecular Clonine: A Laboratory Manual, 2°d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour.
Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ~g/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizadons. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two nucleic 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" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of RMEP
which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term "immunogenic fragment" also includes any polypeptide or oligopeptide fragment of RMEP which is useful in any of the antibody production methods disclosed herein or known in the art.
The term "microarray" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of RMEP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of RMEP.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an RMEP may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of RMEP.
"Probe" refers to nucleic acid sequences encoding RMEP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. "Primers" are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al., 1989, Molecular Cloning: A Laborato~ Manual, 2"d ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; Ausubel, F.M. et a1.,1987, Current Protocols in Molecular Biolo~v, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M. et al., 1990, PCR
Protocols. A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU
primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence.
This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, su ra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs).
Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA

stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding RMEP, or fragments thereof, or RMEP 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" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
A "transcript image" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism," as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants, and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. ( 1989), su ra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length A variant may be described as, for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides 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 nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.

A "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2Ø9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%
or greater sequence identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human RNA metabolism proteins (RMEP), the polynucleotides encoding RMEP, and the use of these compositions for the diagnosis, treatment, or prevention of nervous system, autoimmune/inflammatory, and cell proliferative disorders, including cancer.
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding RMEP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each RMEP were identified, and column 4 shows the cDNA
libraries from which these clones were isolated Column 5 shows Incyte clones and their corresponding cDNA libraries.
Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each RMEP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each 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 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding RMEP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID N0:14-26 and to distinguish between SEQ ID N0:14-26 and related polynucleotide sequences.
The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
Column 3 lists tissue categories which express RMEP as a fraction of total tissues expressing RMEP.
Column 4 lists diseases, disorders, or conditions associated with those tissues expressing RMEP as a fraction of total tissues expressing RMEP. Column 5 lists the vectors used to subclone each cDNA
library. Of particular note is the expression of SEQ ID N0:24 in muscle tumor and fetal brain.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding RMEP were isolated. Column 1 references the nucleotide SEQ ID
NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA
libraries in column 2.
SEQ ID N0:14 maps to chromosome 3 within the interval from 176.40 to 179.80 centiMorgans. SEQ ID NO:15 maps to chromosome 22 within the interval from 24.30 to 36.60 centiMorgans, to chromosome 16 within the interval from 19.70 to 33.30 centiMorgans, and to chromosome 5 within the interval from 174.30 centiMorgans to q-terminus. SEQ
ID N0:17 maps to chromosome 11 within the interval from 70.90 to 72.10 centiMorgans. SEQ ID
N0:26 maps to chromosome 8 within the interval from 64.60 to 78.80 centiMorgans.
The invention also encompasses RMEP variants. A preferred RMEP variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the RMEP amino acid sequence, and which contains at least one functional or structural characteristic of RMEP.
The invention also encompasses polynucleotides which encode RMEP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:14-26, which encodes RMEP. The polynucleotide sequences of SEQ ID N0:14-26, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding RMEP. In particular, such a variant polynucleotide sequence will have at least about 80%, or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding RMEP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID
N0:14-26 which has at least about 80%, or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:14-26.
Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of RMEP.
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 RMEP, some bearing minimal similarity to the polynucleodde sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring RMEP, and all such variations are to be considered as being specifically disclosed Although nucleotide sequences which encode RMEP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring RMEP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding RMEP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryodc host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding RMEP 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 RMEP
and RMEP 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 RMEP 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
N0:14-26 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.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE
Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases 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 MICROLAB 2200 liquid transfer system (Hamilton, Reno N~, PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (PE
Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biolo~v, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
(1995) Molecular Bioloey and Biotechnology, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding RMEP 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 unlmown 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 unlmown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth 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 S' 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, PE Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode RMEP may be cloned in recombinant DNA molecules that direct expression of RMEP, 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 RMEP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter RMEP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of RMEP, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
In another embodiment, sequences encoding RMEP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, RMEP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY, pp.
55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (PE Biosystems). Additionally, the amino acid sequence of RMEP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing.
(See, e.g., Creighton, supra, pp. 28-53.) In order to express a biologically active RMEP, the nucleotide sequences encoding RMEP 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 RMEP. Such elements may vary in their strength and specificity.
Specific initiation signals may also be used to achieve more efficient translation of sequences encoding RMEP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding RMEP and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (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 RMEP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, 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 RMEP. 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. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544;
Scorer, C.A. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al.
(1994) Proc. Natl.
Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;
Brogue, R. et al.
(1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105; The McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York NY, pp.
191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993) Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleodde sequences encoding RMEP. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding RMEP 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 RMEP into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem 264:5503-5509.) When large quantities of RMEP are needed, e.g. for the production of antibodies, vectors which direct high level expression of RMEP may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of RMEP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra;

Bitter, supra; and Scorer, supra.) Plant systems may also be used for expression of RMEP. Transcription of sequences encoding RMEP 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 heat shock promoters may be used. (See, e.g., Coruzzi, supra; Brogue, supra; and Winter, supra.) 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 y (1992) McGraw 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 RMEP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses RMEP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (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 RMEP in cell lines is preferred. For example, sequences encoding RMEP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk- and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;
Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J.
Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), B
glucuronidase and its substrate B-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 RMEP is inserted within a marker gene sequence, transformed cells containing sequences encoding RMEP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding RMEP 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 RMEP
and that express RMEP 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 RMEP 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 RMEP 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 Immunoloay, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana 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 RMEP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.

Alternatively, the sequences encoding RMEP, 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 Pharmacia 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 RMEP 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 polynucleoddes which encode RMEP may be designed to contain signal sequences which direct secretion of RMEP 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" or "pro" form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding RMEP 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 RMEP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of RMEP activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the RMEP encoding sequence and the heterologous protein sequence, so that RMEP
may be cleaved away from the heterologous moiety following purification.
Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled RMEP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35S-methionine.
RMEP of the present invention or fragments thereof may be used to screen for compounds that specifically bind to RMEP. At least one and up to a plurality of test compounds may be screened for specific binding to RMEP. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the natural ligand of RMEP, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, Coligan, J.E. et al. (1991) Current Protocols in Immunoloev 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which RMEP
binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express RMEP, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coll. Cells expressing RMEP or cell membrane fractions which contain RMEP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either RMEP or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with RMEP, either in solution or affixed to a solid support, and detecting the binding of RMEP to the compound.
Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compounds) may be free in solution or affixed to a solid support.
RMEP of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of RMEP. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for RMEP
activity, wherein RMEP is combined with at least one test compound, and the activity of RMEP in the presence of a test compound is compared with the activity of RMEP in the absence of the test compound. A change in the activity of RMEP in the presence of the test compound is indicative of a compound that modulates the activity of RMEP. Alternatively, a test compound is combined with an in vitro or cell-free system comprising RMEP under conditions suitable for RMEP activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of RMEP
may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
In another embodiment, polynucleotides encoding RMEP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R.
(1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding RMEP may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding RMEP can also be used to create "knockin" humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding RMEP is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress RMEP, e.g., by secreting RMEP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of RMEP and RNA metabolism proteins. In addition, the expression of RMEP is closely associated with cell proliferation, cancer, and inflammation.
Therefore, RMEP appears to play a role in nervous system, autoimmune/inflammatory, and cell proliferative disorders, including cancer. In the treatment of disorders associated with increased RMEP
expression or activity, it is desirable to decrease the expression or activity of RMEP. In the treatment of disorders associated with decreased RMEP expression or activity, it is desirable to increase the expression or activity of RMEP.
Therefore, in one embodiment, RMEP 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 RMEP. Examples of such disorders include, but are not limited to, a nervous system disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome;
fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorder of the central nervous system, cerebral palsy, a neuroskeletal disorder, an autonomic nervous system disorder, a cranial nerve disorder, a spinal cord disease, muscular dystrophy and other neuromuscular disorder, a peripheral nervous system disorder, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathy; myasthenia gravis, periodic paralysis; a mental disorder including mood, anxiety, and schizophrenic disorders;
seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
In another embodiment, a vector capable of expressing RMEP 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 RMEP including, but not limited to, those described above.
In a firrttrer embodiment, a pharmaceutical composition comprising a substantially purified RMEP 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 RMEP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of RMEP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of RMEP including, but not limited to, those listed above.
In a further embodiment, an antagonist of RMEP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of RMEP.
Examples of such disorders include, but are not limited to, those nervous system, autoimmune/inflammatory, and cell proliferative disorders, including cancer described above. In one aspect, an antibody which specifically binds RMEP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express RMEP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding RMEP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of RMEP 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 RMEP may be produced using methods which are generally known in the art.
In particular, purified RMEP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind RMEP.
Antibodies to RMEP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with RMEP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG
(bacilli Calmette-Guerin) and Corvnebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to RMEP
have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein.
Short stretches of RMEP 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 RMEP 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. USA
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., Morrison, S.L. et al. (1984) Proc.
Natl. Acad. Sci. USA 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 RMEP-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. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for RMEP 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 RMEP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering RMEP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for RMEP. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of RMEP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple RMEP epitopes, represents the average affinity, or avidity, of the antibodies for RMEP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular RMEP
epitope, represents a true measure of amity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 L/mole are preferred for use in immunoassays in which the RMEP-antibody complex must withstand rigorous manipulations. Low-amity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of RMEP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer ( 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 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of RMEP-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, swpra, and Coligan et al., su ra.) In another embodiment of the invention, the polynucleotides encoding RMEP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding RMEP.
Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding RMEP.
(See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. ( 1990) Blood 76:271; Ausubel, su ra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Moms, M.C. et al. (1997) Nucleic Acids Res.
25(14):2730-2736.) In another embodiment of the invention, polynucleotides encoding RMEP may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX
deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and Somia, N. (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and TrYpanosoma cruzi). In the case where a genetic deficiency in RMEP expression or regulation causes disease, the expression of RMEP from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in RMEP
are treated by constructing mammalian expression vectors encoding RMEP and introducing these vectors by mechanical means into RMEP-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev.
Biochem. 62:191-217;
Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr.
Opin Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of RMEP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). RMEP may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ~i-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl.
Acad. Sci. U.S.A.
89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V.
and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M. V.
and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding RMEP from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID

TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to RMEP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding RMEP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc.
Natl. Acad. Sci. U.S.A. 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference.
Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol.
71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71:4707-4716;
Ranga, U. et al.
(1998) Proc. Natl. Acad. Sci. U.S.A. 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding RMEP to cells which have one or more genetic abnormalities with respect to the expression of RMEP. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E, et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu.
Rev. Nutr. 19:511-544; and Verma, LM. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding RMEP to target cells which have one or more genetic abnormalities with respect to the expression of RMEP. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing RMEP to cells of the central nervous system, for which HSV has a tropism The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al.
(1999) Exp. Eye Res.169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S.
Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant HSV
d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev.
Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding RMEP to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotech. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for RMEP into the alphavirus genome in place of the capsid-coding region results in the production of a large number of RMEP-coding RNAs and the synthesis of high levels of RMEP in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21 ) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A.
et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of RMEP into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, may also be employed to inhibit gene expression.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunolo~ic 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.
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 RMEP.
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 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 RMEP. 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.
An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding RMEP. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased RMEP
expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding RMEP may be therapeutically useful, and in the treament of disorders associated with decreased RMEP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding RMEP may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide;
and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding RMEP is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding RMEP are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding RMEP. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomvces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435; Arndt, G.M. et al.
(2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000} U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. 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. (1997) Nat.
Biotechnol. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the administration of a pharmaceutical composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of ReminQton's Pharmaceutical Sciences (Maack Publishing, Euston PA). Such pharmaceutical compositions may consist of RMEP, antibodies to RMEP, and mimetics, agonists, antagonists, or inhibitors of RMEP.
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, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
Pharmaceutical compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
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.
Specialized forms of pharmaceutical compositions may be prepared for direct intracellular delivery of macromolecules comprising RMEP or fragments thereof. For example, liposome S preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, RMEP or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example RMEP
or fragments thereof, antibodies of RMEP, and agonists, antagonists or inhibitors of RMEP, 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 EDSO (the dose therapeutically effective in 50% of the population) or LDSO (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDSO/EDSO 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 EDso with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.
Long-acting 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 0.1 ~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 RMEP may be used for the diagnosis of disorders characterized by expression of RMEP, or in assays to monitor patients being treated with RMEP or agonists, antagonists, or inhibitors of RMEP. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for RMEP include methods which utilize the antibody and a label to detect RMEP
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 RMEP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of RMEP expression. Normal or standard values for RMEP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to RMEP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of RMEP
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 RMEP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleoddes may be used to detect and quantify gene expression in biopsied tissues in which expression of RMEP may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of RMEP, and to monitor regulation of RMEP levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding RMEP or closely related molecules may be used to identify nucleic acid sequences which encode RMEP. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding RMEP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and may have at least 50%

sequence identity to any of the RMEP encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID
N0:14-26 or from genomic sequences including promoters, enhancers, and introns of the RMEP
gene.
Means for producing specific hybridization probes for DNAs encoding RMEP
include the cloning of polynucleotide sequences encoding RMEP or RMEP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32P or 35S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biodn coupling systems, and the like.
Polynucleotide sequences encoding RMEP may be used for the diagnosis of disorders associated with expression of RMEP. Examples of such disorders include, but are not limited to, a nervous system disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorder of the central nervous system, cerebral palsy, a neuroskeletal disorder, an autonomic nervous system disorder, a cranial nerve disorder, a spinal cord disease, muscular dystrophy and other neuromuscular disorder, a peripheral nervous system disorder, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathy;
myasthenia gravis, periodic paralysis; a mental disorder including mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystids, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthrids, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus . The polynucleotide sequences encoding RMEP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered RMEP expression.
Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding RMEP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding RMEP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding RMEP 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 RMEP, 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 RMEP, 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 RMEP
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 RMEP, or a fragment of a polynucleotide complementary to the polynucleotide encoding RMEP, and will be employed under optimized conditions for identification of a specific gene or condition.
Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding RMEP may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired generic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleodde primers derived from the polynucleotide sequences encoding RMEP are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP
(isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
Methods which may also be used to quantify the expression of RMEP 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. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, incorporated herein by reference. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
In another embodiment, antibodies specific for RMEP, or RMEP or fragments thereof may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
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.
USA 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. USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding RMEP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a mufti-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, 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.
7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, e.g., Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.) Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (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) World Wide Web site. Correlation between the location of the gene encoding RMEP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA
associated with that disorder and thus may further positional cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known.
This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 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 instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, RMEP, 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 RMEP 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 RMEP, or fragments thereof, and washed. Bound RMEP is then detected by methods well known in the art.
Purified RMEP 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 RMEP specifically compete with a test compound for binding RMEP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with RMEP.
In additional embodiments, the nucleotide sequences which encode RMEP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. 60/139,922, 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 Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth 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 S 1000, 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 polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including XL,1-Blue, XL1-BlueMRF, or SOLR
from Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UN1ZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC
Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSICA1V II fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows.
Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ
Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB
2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
Electrophoredc separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or 377 sequencing system (PE Biosystems) in conjunction with standard ABI
protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA
sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VI.
The polynucleotide sequences derived from cDNA 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 tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
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 the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM 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, DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM. HMM
is a probabilistic approach which analyzes consensus primary structures of gene families. (See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID
N0:14-26. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e.g., Sambrook, supra, ch 7; Ausubel, 1995, supra, ch 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics~. This analysis 1S
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:
BLAST Score x Percent Identity 5 x minimum { length(Seq. 1 ), length(Seq. 2) }
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch Two sequences may share more than one HSP (separated by gaps).
If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A
product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding RMEP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation, trauma, cell proliferation, 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- or condition-specific expression are reported in Table 3.

V. Chromosomal Mapping of RMEP Encoding Polynucleotides The cDNA sequences which were used to assemble SEQ ID N0:14 through 26 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID N0:14 through 26 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 5).
Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and G6n~thon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID
NO:, to that map location.
The genetic map locations of SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:17, and SEQ
ID
N0:26 are described in The Invention as ranges, or intervals, of human chromosomes. More than one map location is reported for SEQ ID N0:15, indicating that previously mapped sequences having similarity, but not complete identity, to SEQ ID N0:15 were assembled into their respective clusters.
The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters.
VI. Extension of RMEP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:14-26 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 50% or more, and to anneal to the target sequence at temperatures of about 68 ° C to about 72 ° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NH4)ZS04, and ~i-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, 5 min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p1 PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE
and 0.5 ~1 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 5 ~1 to 10 ~1 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 clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters:
Step l: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA
recoveries were reamplified using the same conditions as described above. Samples were diluted with 20%
dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE
Terminator cycle sequencing ready reaction kit (PE Biosystems).
In like manner, the polynucleotide sequences of SEQ ID N0:14-26 are used to obtain 5' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library.
VII. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:14-26 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments.
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ~Ci of ['y-32P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX
G-25 superfine size exclusion dextran 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, Xba I, 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 conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared VIII. Microarrays The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, su ra , mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), su ra . Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A
typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al.
(1995) Science 270:467-470;
Shalom D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol.
16:27-31.) Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleoddes in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element.
Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.
Tissue or Cell Sample Preparation Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is purified using the oligo-(d'I~ cellulose method. Each poly(A)+
RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/Eil oligo-(d'I~
primer (2lmer), 1X first strand buffer, 0.03 units/~.Q RNase inhibitor, 500 E.iM dATP, 500 ~M dGTP, 500 ~M dTTP, 40 ~M
dCTP, 40 EiM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)+ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH
Laboratories, Inc.
(CLONTECH), Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100%
ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook N~ and resuspended in 14 ~.~1 SX SSC/0.2% SDS.
Microarray Preparation Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 fig. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C

oven.
Array elements are applied to the coated glass substrate using a procedure described in US
Patent No. 5,807,522, incorporated herein by reference. 1 ~.il of the array element DNA, at an average concentration of 100 ng/Erl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 n1 of array element sample per slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microatrays in 0.2%
casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 °C followed by washes in 0.2% SDS and distilled water as before.
Hybridization Hybridization reactions contain 9 ~1 of sample mixture consisting of 0.2 ~g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65 °C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm2 coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 ~1 of SX SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60 °C. The arrays are washed for 10 min at 45 °C in a first wash buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45 °C in a second wash buffer (0.1X SSC), and dried.
Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT 81477, Hamamatsu Photonics Systems, Bridgewater N.>] corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for CyS. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A
specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA
with the two fluorophores and adding identical amounts of each to the hybridization mixture.
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
IX. Complementary Polynucleotides Sequences complementary to the RMEP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring RMEP. 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 RMEP. 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 RMEP-encoding transcript.
X. Expression of RMEP
Expression and purification of RMEP is achieved using bacterial or virus-based expression systems. For expression of RMEP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express RMEP upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of RMEP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant AutoQraphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding RMEP 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 generic modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther.
7:1937-1945.) In most expression systems, RMEP 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 ianonicum, 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 RMEP 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 RMEP obtained by these methods can be used directly in the assays shown in Examples X and XIV.
XI. Demonstration of RMEP Activity RMEP RNA-binding activity is demonstrated by a polyacrylamide gel mobility-shift assay.
In preparation for this assay, RMEP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector containing RMEP cDNA.
The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of RMEP. Extracts containing solubilized proteins can be prepared from cells expressing RMEP by methods well known in the art. Portions of the extract containing RMEP are added to [32P]-labeled RNA. Radioactive RNA can be synthesized in vitro by techniques well known in the art. The mixtures are incubated at 25 °C in the presence of RNase inhibitors under buffered conditions for 5-10 minutes. After incubation, the samples are analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a band on the autoradiogram indicates the formation of a complex between RMEP and the radioactive transcript. A band of similar mobility will not be present in samples prepared using control extracts prepared from untransformed cells.
Alternatively, RMEP, or biologically active fragments thereof, are labeled with "~I
Bolton-Hunter reagent and tested for interaction with candidate RNA metabolism molecules. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled RMEP, washed, and any wells with labeled RMEP
complex are assayed. Data obtained using different concentrations of RMEP are used to calculate values for the number, affinity, and association of RMEP with the candidate molecules.
Alternatively, molecules interacting with RMEP are analyzed using the yeast two-hybrid system as described in Fields, S. and Song, O. (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (CLONTECH).
XII. Functional Assays RMEP function is assessed by expressing the sequences encoding RMEP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ~g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 ~g of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies;
and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G.
(1994) Flow Cvtometry, Oxford, New York NY.
The influence of RMEP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding RMEP and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N~.
mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding RMEP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XIII. Production of RMEP Specific Antibodies RMEP 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 RMEP 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, supra, ch. 11.) Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A
peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH
(Sigma-Aldrich, St.
Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH
complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-RMEP
activity by, for example, binding the peptide or RMEP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIV. Purification of Naturally Occurring RMEP Using Specific Antibodies Naturally occurring or recombinant RMEP is substantially purified by immunoaffinity chromatography using antibodies specific for RMEP. An immunoaffinity column is constructed by covalently coupling anti-RMEP 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 RMEP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of RMEP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/RMEP 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 RMEP is collected.
XV. Identification of Molecules Which Interact with RMEP
RMEP, or biologically active fragments thereof, are labeled with'ZSI Bolton-Hunter reagent.
(See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a mufti-well plate are incubated with the labeled RMEP, washed, and any wells with labeled RMEP complex are assayed. Data obtained using different concentrations of RMEP are used to calculate values for the number, affinity, and association of RMEP with the candidate molecules.
Alternatively, molecules interacting with RMEP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song ( 1989, Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
RMEP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S. Patent No. 6,057,1 O 1 ).
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 certain 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.

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SEQUENCE LISTING
<110> INCYTE GENOMICS, INC.
BANDMAN, Olga YUE, Henry LAL, Preeti TANG, Y. Tom REDDY, Roopa BAUGHN, Mariah R.
AZIMZAI, Yalda <120> RNA METABOLISM PROTEINS
<130> PF-0712 PCT
<140> To Be Assigned <141> Herewith <150> 60/139,922 <151> 1999-06-17 <160> 26 <170> PERL Program <210> 1 <211> 503 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 046926 <400> 1 Met Glu Tyr Met Ala Glu Ser Thr Asp Arg Ser Pro Gly His Ile Leu Cys Cys Glu Cys Gly Val Pro Ile Ser Pro Asn Pro Ala Asn Ile Cys Val Ala Cys Leu Arg Ser Lys Val Asp Ile Ser Gln Gly Ile Pro Lys Gln Val Ser Ile Ser Phe Cys Lys Gln Cys Gln Arg Tyr Phe Gln Pro Pro Gly Thr Trp Ile Gln Cys Ala Leu Glu Ser Arg Glu Leu Leu Ala Leu Cys Leu Lys Lys Ile Lys Ala Pro Leu Ser Lys Val Arg Leu Val Asp Ala Gly Phe Val Trp Thr Glu Pro His Ser Lys Arg Leu Lys Val Lys Leu Thr Ile Gln Lys Glu Val Met Asn Gly Ala Ile Leu Gln Gln Val Phe Val Val Asp Tyr Val Val Gln Ser Gln Met Cys Gly Asp Cys His Arg Val Glu Ala Lys Asp Phe Trp Lys Ala Val Ile Gln Val Arg Gln Lys Thr Leu His Lys Lys Thr Phe Tyr Tyr Leu Glu Gln Leu Ile Leu Lys Tyr Gly Met His Gln Asri Thr Leu Arg Ile Lys Glu Ile His Asp Gly Leu Asp Phe Tyr Tyr Ser Ser Lys Gln His Ala Gln Lys Met Val Glu Phe Leu Gln Cys Thr Val Pro Cys Arg Tyr Lys Ala Ser Gln Arg Leu Ile Ser Gln Asp Ile His Ser Asn Thr Tyr Asn Tyr Lys Ser Thr Phe Ser Val Glu Ile Val Pro Ile Cys Lys Asp Asn Val Val Cys Leu Ser Pro Lys Leu Ala Gln Ser Leu Gly Asn Met Asn Gln Ile Cys Val Cys Ile Arg Val Thr Ser Ala Ile His Leu Ile Asp Pro Asn Thr Leu Gln Val Ala Asp Ile Asp Gly Ser Thr Phe Trp Ser His Pro Phe Asn Ser Leu Cys His Pro Lys Gln Leu Glu Glu Phe Ile Val Met Glu Cys Ser Ile Val Gln Asp Ile Lys Arg Ala Ala Gly Ala Gly Met Ile Ser Lys Lys His Thr Leu Gly Glu Val Trp Val Gln Lys Thr Ser Glu Met Asn Thr Asp Lys Gln Tyr Phe Cys Arg Thr His Leu Gly His Leu Leu Asn Pro Gly Asp Leu Val Leu Gly Phe Asp Leu Ala Asn Cys Asn Leu Asn Asp Glu His Val Asn Lys Met Asn Ser Asp Arg Val Pro Asp Val Val Leu Ile Lys Lys Ser Tyr Asp Arg Thr Lys Arg Gln Arg Arg Arg Asn Trp Lys Leu Lys Glu Leu Ala Arg Glu Arg Glu Asn Met Asp Thr Asp Asp Glu Arg Gln Tyr Gln Asp Phe Leu Glu Asp Leu Glu Glu Asp Glu Ala Ile Arg Lys Asn Val Asn Ile Tyr Arg Asp Ser Ala Ile Pro Val Glu Ser Asp Thr Asp Asp Glu Gly Ala Pro Arg Ile Ser Leu Ala Glu Met Leu Glu Asp Leu His Ile Ser Gln Asp Ala Thr Gly Glu Glu Gly Ala Ser Met Leu Thr <210> 2 <211> 594 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 618791 <400> 2 Met Ser Ala Gly Glu Val Glu Arg Leu Val Ser Glu Leu Ser Gly Gly Thr Gly Gly Asp Glu Glu Glu Glu Trp Leu Tyr Gly Gly Pro Trp Asp Val His Val His Ser Asp Leu Ala Lys Asp Leu Asp Glu Asn Glu Val Glu Arg Pro Glu Glu Glu Asn Ala Ser Ala Asn Pro Pro Ser Gly Ile Glu Asp Glu Thr Ala Glu Asn Gly Val Pro Lys Pro Lys Val Thr Glu Thr Glu Asp Asp Ser Asp Ser Asp Ser Asp Asp Asp Glu Asp Asp Val His Val Thr Ile Gly Asp Ile Lys Thr Gly Ala Pro Gln Tyr Gly Ser Tyr Gly Thr Ala Pro Val Asn Leu Asn Ile Lys Thr Gly Gly Arg Val Tyr Gly Thr Thr Gly Thr Lys Val Lys Gly Val Asp Leu Asp Ala Pro Gly Ser Ile Asn Gly Val Pro Leu Leu Glu Val Asp Leu Asp Ser Phe Glu Asp Lys Pro Trp Arg Lys Pro Gly Ala Asp Leu Ser Asp Tyr Phe Asn Tyr Gly Phe Asn Glu Asp Thr Trp Lys Ala Tyr Cys Glu Lys Gln Lys Arg Ile Arg Met Gly Leu Glu Val Ile Pro Val Thr Ser Thr Thr Asn Lys Ile Thr Ala Glu Asp Cys Thr Met Glu Val Thr Pro Gly Ala Glu Ile Gln Asp Gly Arg Phe Asn Leu Phe Lys Val Gln Gln Gly Arg Thr Gly Asn Ser Glu Lys Glu Thr Ala Leu Pro Ser Thr Lys Ala Glu Phe Thr Ser Pro Pro Ser Leu Phe Lys Thr Gly Leu Pro Pro Ser Arg Asn Ser Thr Ser Ser Gln Ser Gln Thr Ser Thr Ala Ser Arg Lys Ala Asn Ser Ser Val Gly Lys Trp Gln Asp Arg Tyr Gly Arg Ala Glu Ser Pro Asp Leu Arg Arg Leu Pro Gly Ala Ile Asp Val Ile Gly Gln Thr Ile Thr Ile Ser Arg Val Glu Gly Arg Arg Arg Ala Asn Glu Asn Ser Asn Ile Gln Val Leu Ser Glu Arg Ser Ala Thr Glu Val Asp Asn Asn Phe Ser Lys Pro Pro Pro Phe Phe Pro Pro Gly Ala Pro Pro Thr His Leu Pro Pro Pro Pro Phe Leu Pro Pro Pro Pro Thr Val Ser Thr Ala Pro Pro Leu Ile Pro Pro Pro Gly Phe Pro Pro Pro Pro Gly Ala Pro Pro Pro Ser Leu Ile Pro Thr Ile Glu Ser Gly His Ser Ser Gly Tyr Asp Ser Arg Ser Ala Arg Ala Phe Pro Tyr Gly Asn Val Ala Phe Pro His Leu Pro Gly Ser Ala Pro Ser Trp Pro Ser Leu Val Asp Thr Ser Lys Gln Trp Asp Tyr Tyr Ala Arg Arg Glu Lys Asp Arg Asp Arg Glu Arg Asp Arg Asp Arg Glu Arg Asp Arg Asp Arg Asp Arg Glu Arg Glu Arg Thr Arg Glu Arg Glu Arg Glu Arg Asp His Ser Pro Thr Pro Ser Val Phe Asn Ser Asp Glu Glu Arg Tyr Arg Tyr Arg Glu Tyr Ala Glu Arg Gly Tyr Glu Arg His Arg Ala Ser Arg Glu Lys Glu Glu Arg His Arg Glu Arg Arg His Arg Glu Lys Glu Glu Thr Arg His Lys Ser Ser Arg Ser Asn Ser Arg Arg Arg His Glu Ser Glu Glu Gly Asp Ser His Arg Arg His Lys His Lys Lys Ser Lys Arg Ser Lys Glu Gly Lys Glu Ala Gly Ser Glu Pro Ala Pro Glu Gln Glu Ser Thr Glu Ala Thr Pro Ala Glu <210> 3 <211> 413 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1240366 <400> 3 Met Ser Glu Thr Gln Tyr Ser Ser Leu Thr Gln Thr Leu Ile Met Thr Met Lys Leu Ser Gly Phe Gly Val Ala Asp Ser Met Arg Ile Ser Gly Cys Ser Ile Gln Lys Gln Ser Arg Ile Ile Ile Thr Asp Arg Gln Ala Glu Pro Pro Lys Lys Glu Ala Ala Thr Thr Gly Pro Gln Val Lys Arg Ala Asp Glu Trp Lys Asp Pro Trp Arg Arg Ser Lys Ser Pro Lys Lys Lys Leu Gly Val Ser Val Ser Pro Ser Arg Ala Arg Arg Arg Arg Lys Thr Ser Ala Ser Ser Ala Ser Ala Ser Asn Ser Ser Arg Ser Ser Ser Arg Ser Ser Ser Tyr Ser Gly Ser Gly Ser Ser Arg Ser Arg Ser Arg Ser Ser Ser Tyr Ser Ser Tyr Ser Ser Arg Ser Ser Arg His Ser Ser Phe Ser Gly Ser Arg Ser Arg Ser Arg Ser Phe Ser Ser Ser Pro Ser Pro Ser Pro Thr Pro Ser Pro His Arg Pro Ser Ile Arg Thr Lys Gly Glu Pro Ala Pro Pro Pro Gly Lys Ala Gly Glu Lys Ser Val Lys Lys Pro Ala Pro Pro Pro Ala Pro Pro Gln Ala Thr Lys Thr Thr Ala Pro Val Pro Glu Pro Thr Lys Pro Gly Asp Pro Arg Glu Ala Arg Arg Lys Glu Arg Pro Ala Arg Thr Pro Pro Arg Arg Arg Thr Leu Ser Gly Ser Gly Ser Gly Ser Gly Ser Ser Tyr Ser Gly Ser Ser Ser Arg Ser Arg Ser Leu Ser Val Ser Ser Val Ser Ser Val Ser Ser Ala Thr Ser Ser Ser Ser Ser Ala His Ser Val Asp Ser Glu Asp Met Tyr Ala Asp Leu Ala Ser Pro Val Ser Ser Ala Ser Ser Arg Ser Pro Ala Pro Ala Gln Thr Arg Lys Glu Lys Gly Lys Ser Lys Lys Glu Asp Gly Val Lys Glu Glu Lys Arg Lys Arg Asp Ser Ser Thr Gln Pro Pro Lys Ser Ala Lys Pro Pro Ala Gly Gly Lys Ser Ser Gln Gln Pro Ser Thr Pro Gln Gln Ala Pro Pro Gly Gln Pro Gln Gln Gly Thr Phe Val Ala His Lys Glu Ile Lys Leu Thr Leu Leu Asn Lys Ala Ala Asp Lys Gly Ser Arg Lys Arg Tyr Glu Pro Ser Asp Lys Asp Arg Gln Ser Pro Pro Pro Ala Lys Arg Pro Asn Thr Ser Pro Asp Arg Gly Ser Arg Asp Arg <210> 4 <211> 219 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1295773 <400> 4 Met His Val Gln Leu Ser Thr Ser Arg Leu Arg Thr Ala Pro Gly Met Gly Asp Gln Ser Gly Cys Tyr Arg Cys Gly Lys Glu Gly His Trp Ser Lys Glu Cys Pro Val Asp Arg Thr Gly Arg Val Ala Asp Phe Thr Glu Gln Tyr Asn Glu Gln Tyr Gly Ala Val Arg Thr Pro Tyr Thr Met Gly Tyr Gly Glu Ser Met Tyr Tyr Asn Asp Ala Tyr Gly Ala Leu Asp Tyr Tyr Lys Arg Tyr Arg Val Arg Ser Tyr Glu Ala Val Ala Ala Ala Ala Ala Ala Ser Ala Tyr Asn Tyr Ala Glu Gln Thr Met Ser His Leu Pro Gln Val Gln Ser Thr Thr Val Thr Ser His Leu Asn Ser Thr Ser Val Asp Pro Tyr Asp Arg His Leu Leu Pro Asn Ser Gly Ala Ala Ala Thr Ser Ala Ala Met Ala Ala Ala Ala Ala Thr Thr Ser Ser Tyr Tyr Gly Arg Asp Arg Ser Pro Leu Arg Arg Ala Ala Ala Met Leu Pro Thr Val Gly Glu Gly Tyr Gly Tyr Gly Pro Glu Ser Glu Leu Ser Gln Ala Ser Ala Ala Thr Arg Asn Ser Leu Tyr Asp Met Ala Arg Tyr Glu Arg Glu Gln Tyr Val Asp Arg Ala Arg Tyr Ser Ala Phe <210> 5 <211> 641 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1858421 <400> 5 Met Gly Arg Arg Ser Thr Ser Ser Thr Lys Ser Gly Lys Phe Met Asn Pro Thr Asp Gln Ala Arg Lys Glu Ala Arg Lys Arg Glu Leu Lys Lys Asn Lys Lys Gln Arg Met Met Val Arg Ala Ala Val Leu Lys Met Lys Asp Pro Lys Gln Ile Ile Arg Asp Met Glu Lys Leu Asp Glu Met Glu Phe Asn Pro Val Gln Gln Pro Gln Leu Asn Glu Lys Val Leu Lys Asp Lys Arg Lys Lys Leu Arg Glu Thr Phe Glu Arg Ile Leu Arg Leu Tyr Glu Lys Glu Asn Pro Asp Ile Tyr Lys Glu Leu Arg Lys Leu Glu Val Glu Tyr Glu Gln Lys Arg Ala Gln Leu Ser Gln Tyr Phe Asp Ala Val Lys Asn Ala Gln His Val Glu Val Glu Ser Ile Pro Leu Pro Asp Met Pro His Ala Pro Ser Asn Ile Leu Ile Gln Asp Ile Pro Leu Pro Gly Ala Gln Pro Pro Ser Ile Leu Lys Lys Thr Ser Ala Tyr Gly Pro Pro Thr Arg Ala Val Ser Ile Leu Pro Leu Leu Gly His Gly Val Pro Arg Leu Pro Pro Gly Arg Lys Pro Pro Gly Pro Pro Pro Gly Pro Pro Pro Pro Gln Val Val Gln Met Tyr Gly Arg Lys Val Gly Phe Ala Leu Asp Leu Pro Pro Arg Arg Arg Asp Glu Asp Met Leu Tyr Ser Pro Glu Leu Ala Gln Arg Gly His Asp Asp Asp Val Ser Ser Thr Ser Glu Asp Asp Gly Tyr Pro Glu Asp Met Asp Gln Asp Lys His Asp Asp Ser Thr Asp Asp Ser Asp Thr Asp Lys Ser Asp Gly G1u Ser Asp Gly Asp Glu Phe Val His Arg Asp Asn Gly Glu Arg Asp Asn Asn Glu Glu Lys Lys Ser Gly Leu Ser Val Arg Phe Ala Asp Met Pro Gly Lys Ser Arg Lys Lys Lys Lys Asn Met Lys Glu Leu Thr Pro Leu Gln Ala Met Met Leu Arg Met Ala Gly Gln Glu Ile Pro Glu Glu Gly Arg Glu Val Glu Glu Phe Ser Glu Asp Asp Asp Glu Asp Asp Ser Asp Asp Ser Glu Ala Glu Lys Gln Ser Gln Lys Gln His Lys Glu Glu Ser His Ser Asp Gly Thr Ser Thr Ala Ser Ser Gln Gln Gln Ala Pro Pro Gln Ser Val Pro Pro Ser Gln Ile Gln Ala Pro Pro Met Pro Gly Pro Pro Pro Leu Gly Pro Pro Pro Ala Pro Pro Leu Arg Pro Pro Gly Pro Pro Thr Gly Leu Pro Pro Gly Pro Pro Pro Gly Ala Pro Pro Phe Leu Arg Pro Pro Gly Met Pro Gly Leu Arg Gly Pro Leu Pro Arg Leu Leu Pro Pro Gly Pro Pro Pro Gly Arg Pro Pro Gly Pro Pro Pro Gly Pro Pro Pro Gly Leu Pro Pro Gly Pro Pro Pro Arg Gly Pro Pro Pro Arg Leu Pro Pro Pro Ala Pro Pro Gly Ile Pro Pro Pro Arg Pro Gly Met Met Arg Pro Pro Leu Val Pro Pro Leu Gly Pro Ala Pro Pro Gly Leu Phe Pro Pro Ala Pro Leu Pro Asn Pro Gly Val Leu Ser Ala Pro Pro Asn Leu Ile Gln Arg Pro Lys Ala Asp Asp Thr Ser Ala Ala Thr Ile Glu Lys Lys Ala Thr Ala Thr Ile Ser Ala Lys Pro Gln Ile Thr Asn Pro Lys Ala Glu Ile Thr Arg Phe Val Pro Thr Ala Leu Arg Val Arg Arg Glu Asn Lys Gly Ala Thr Ala Ala Pro Gln Arg Lys Ser Glu Asp Asp Ser Ala Val Pro Leu Ala Lys Ala Ala Pro Lys Ser Gly Pro Ser Val Pro Val Ser Val Gln Thr Lys Asp Asp Val Tyr Glu Ala Phe Met Lys Glu Met Glu Gly Leu Leu <210> 6 <211> 153 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2152431 <400> 6 Met Ala Asp Glu Ile Asp Phe Thr Thr Gly Asp Ala Gly Ala Ser Ser Thr Tyr Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp Val Pro Asn Ile Lys Arg Asn Asp Tyr Gln Leu Ile Cys Ile Gln Asp Gly Tyr Leu Ser Leu Leu Thr Glu Thr Gly Glu Val Arg Glu Asp Leu Lys Leu Pro Glu Gly Glu Leu Gly Lys Glu Ile Glu Gly Lys Tyr Asn Ala Gly Glu Asp Val Gln Val Ser Val Met Cys Ala Met Ser Glu Glu Tyr Ala Val Ala Ile Lys Pro Cys Lys <210> 7 <211> 194 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2641494 <400> 7 Met G1n Ala Val Arg Asn Ala Gly Ser Arg Phe Leu Arg Ser Trp Thr Trp Pro Gln Thr Ala Gly Arg Val Val Ala Arg Thr Pro Ala Gly Thr Ile Cys Thr Gly Ala Arg Gln Leu Gln Asp Ala Ala Ala Lys Gln Lys Val Glu Gln Asn Ala Ala Pro Ser His Thr Lys Phe Ser Ile Tyr Pro Pro Ile Pro Gly Glu Glu Ser Ser Leu Arg Trp Ala Gly Lys Lys Phe Glu Glu Ile Pro Ile Ala His Ile Lys Ala Ser His Asn Asn Thr Gln Ile Gln Val Val Ser Ala Ser Asn Glu Pro Leu Ala Phe Ala Ser Cys Gly Thr Glu Gly Phe Arg Asn Ala Lys Lys Gly Thr Gly Ile Ala Ala Gln Thr Ala Gly Ile Ala Ala Ala Ala Arg Ala Lys Gln Lys Gly Val Ile His Ile Arg Val Val Val Lys Gly Leu Gly Pro Gly Arg Leu Ser Ala Met His Gly Leu Ile Met Gly Gly Leu Glu Val Ile Ser Ile Thr Asp Asn Thr Pro Ile Pro His Asn Gly Cys Arg Pro Arg Lys Ala Arg Lys Leu <210> 8 <211> 629 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3803409 <400> 8 Met Gly Lys Pro Pro Gly Ser Ile Val Arg Pro Ser Ala Pro Pro Ala Arg Ser Ser Val Pro Val Thr Arg Pro Pro Val Pro Ile Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Val Ile Lys Pro Gln Thr Ser Ala Val Glu Gln Glu Arg Trp Asp Glu Asp Ser Phe Tyr Gly Leu Trp Asp Thr Asn Asp Glu Gln Gly Leu Asn Ser Glu Phe Lys Ser Glu Thr Ala Ala Ile Pro Ser Ala Pro Val Leu Pro Pro Pro Pro Val His Ser Ser Ile Pro Pro Pro Gly Pro Val Pro Met Gly Met Pro Pro Met Ser Lys Pro Pro Pro Val Gln Gln Thr Val Asp Tyr Gly His Gly Arg Asp Ile Ser Thr Asn Lys Val Glu Gln Ile Pro Tyr Gly Glu Arg Ile Thr Leu Arg Pro Asp Pro Leu Pro Glu Arg Ser Thr Phe Glu Thr Glu His Ala Gly Gln Arg Asp Arg Tyr Asp Arg Glu Arg Asp Arg Glu Pro Tyr Phe Asp Arg Gln Ser Asn Val Ile Ala Asp His Arg Asp Phe Lys Arg Asp Arg Glu Thr His Arg Asp Arg Asp Arg Asp Arg Gly Val Ile Asp Tyr Asp Arg Asp Arg Phe Asp Arg Glu Arg Arg Pro Arg Asp Asp Arg Ala Gln Ser Tyr Arg Asp Lys Lys Asp His Ser Ser Ser Arg Arg Gly Gly Phe Asp Arg Pro Ser Tyr Asp Arg Lys Ser Asp Arg Pro Val Tyr Glu Gly Pro Ser Met Phe Gly Gly Glu Arg Arg Thr Tyr Pro Glu Glu Arg Met Pro Leu Pro Ala Pro Ser Leu Ser His Gln Pro Pro Pro Ala Pro Arg Val Glu Lys Lys Pro Glu Ser Lys Asn Val Asp Asp Ile Leu Lys Pro Pro Gly Arg Glu Ser Arg Pro Glu Arg Ile Val Val Ile Met Arg Gly Leu Pro Gly Ser Gly Lys Thr His Val Ala Lys Leu Ile Arg Asp Lys Glu Val Glu Phe Gly Gly Pro Ala Pro Arg Val Leu Ser Leu Asp Asp Tyr Phe Ile Thr Glu Val Glu Lys Glu Glu Lys Asp Pro Asp Ser Gly Lys Lys Val Lys Lys Lys Val Met Glu Tyr Glu Tyr Glu Ala Glu Met Glu Glu Thr Tyr Arg Thr Ser Met Phe Lys Thr Phe Lys Lys Thr Leu Asp Asp Gly Phe Phe Pro Phe Ile Ile Leu Asp Ala Ile Asn Asp Arg Val Arg His Phe Asp Gln Phe Trp Ser Ala Ala Lys Thr Lys Gly Phe Glu Val Tyr Leu Ala Glu Met Ser Ala Asp Asn Gln Thr Cys Gly Lys Arg Asn Ile His Gly Arg Lys Leu Lys Glu Ile Asn Lys Met Ala Asp His Trp Glu Thr Ala Pro Arg His Met Met Arg Leu Asp Ile Arg Ser Leu Leu Gln Asp Ala Ala Ile Glu Glu Val Glu Met Glu Asp Phe Asp Ala Asn Ile Glu Glu Gln Lys Glu Glu Lys Lys Asp Ala Glu Glu Glu Glu Ser Glu Leu Gly Tyr Ile Pro Lys Ser Lys Trp Glu Met Asp Thr Ser Glu Ala Lys Leu Asp Lys Leu Asp Gly Leu Arg Thr Gly Thr Lys Arg Lys Arg Asp Trp Glu Ala Ile Ala Ser Arg Met Glu Asp Tyr Leu Gln Leu Pro Asp Asp Tyr Asp Thr Arg Ala Ser Glu Pro Gly Lys Lys Arg Val Arg Trp Ala Asp Leu Glu Glu Lys Lys Asp Ala Asp Arg Lys Arg Ala Ile Gly Phe Val Val Gly Gln Thr Asp Trp Glu Lys Ile Thr Asp Glu Ser Gly His Leu Ala Glu Lys Ala Leu Asn Arg Thr Lys Tyr Ile <210> 9 <211> 445 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3979009 <400> 9 Met Asn Arg His Leu Cys Val Trp Leu Phe Arg His Pro Ser Leu Asn Gly Tyr Leu Gln Cys His Ile Gln Leu His Ser His Gln Phe Arg Gln Ile His Leu Asp Thr Arg Leu Gln Val Phe Arg Gln Asn Arg Asn Cys Ile Leu His Leu Leu Ser Lys Asn Trp Ser Arg Arg Tyr Cys His Gln Asp Thr Lys Met Leu Trp Lys His Lys Ala Leu Gln Lys Tyr Met Glu Asn Leu Ser Lys Glu Tyr Gln Thr Leu Glu Gln Cys Leu Gln His Ile Pro Val Asn Glu Glu Asn Arg Arg Ser Leu Asn Arg Arg His Ala Glu Leu Ala Pro Leu Ala Ala Ile Tyr Gln Glu Ile Gln Glu Thr Glu Gln Ala Ile Glu Glu Leu Glu Ser Met Cys Lys Ser Leu Asn Lys Gln Asp Glu Lys Gln Leu Gln Glu Leu Ala Leu Glu Glu Arg Gln Thr Ile Asp Gln Lys Ile Asn Met Leu Tyr Asn Glu Leu Phe Gln Ser Leu Val Pro Lys Glu Lys Tyr Asp Lys Asn Asp Val Ile Leu Glu Val Thr Ala Gly Arg Thr Thr Gly Gly Asp Ile Cys Gln Gln Phe Thr Arg Glu Ile Phe Asp Met Tyr Gln Asn Tyr Ser Cys Tyr Lys His Trp Gln Phe Glu Leu Leu Asn Tyr Thr Pro Ala Asp Tyr Gly Gly Leu His His Ala Ala Ala Arg Ile Ser Gly Asp Gly Val Tyr Lys His Leu Lys Tyr Glu Gly Gly Ile His Arg Val Gln Arg Ile Pro Glu Val Gly Leu Ser Ser Arg Met Gln Arg Ile His Thr Gly Thr Met Ser Val Ile Val Leu Pro Gln Pro Asp Glu Val Asp Val Lys Leu Asp Pro Lys Asp Leu Arg Ile Asp Thr Phe Arg Ala Lys Gly Ala Gly Gly Gln His Val Asn Lys Thr Asp Ser Ala Val Arg Leu Val His Ile Pro Thr Gly Leu Val Val Glu Cys Gln Gln Glu Arg Ser Gln Ile Lys Asn Lys Glu Ile Ala Phe Arg Val Leu Arg Ala Arg Leu Tyr Gln Gln Ile Ile Glu Lys Asp Lys Arg Gln Gln Gln Ser Ala Arg Lys Leu Gln Val Gly Thr Arg Ala Gln Ser Glu Arg Ile Arg Thr Tyr Asn Phe Thr Gln Asp Arg Val Ser Asp His Arg Ile Ala Tyr Glu Val Arg Asp Ile Lys Glu Phe Leu Cys Gly Gly Lys Gly Leu Asp Gln Leu Ile Gln Arg Leu Leu Gln Ser Ala Asp Glu Glu Ala Ile Ala Glu Leu Leu Asp Glu His Leu Lys Ser Ala Lys <210> 10 <211> 280 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3992058 <400> 10 Met Val Ala Arg Arg Arg Lys Cys Ala Ala Arg Asp Pro Glu Asp Arg Ile Pro Ser Pro Leu Gly Tyr Ala Ala Ile Pro Ile Lys Phe Ser Glu Lys Gln Gln Ala Ser His Tyr Leu Tyr Val Arg Ala His Gly Val Arg Gln Gly Thr Lys Ser Thr Trp Pro Gln Lys Arg Thr Leu Phe Val Leu Asn Val Pro Pro Tyr Cys Thr Glu Glu Ser Leu Ser Arg Leu Leu Ser Thr Cys Gly Leu Val Gln Ser Ile Glu Leu Gln Glu Lys Pro Asp Leu Ala Glu Ser Pro Lys Glu Ser Arg Ser Lys Phe Phe His Pro Lys Pro Val Pro Gly Phe Gln Val Ala Tyr Val Val Phe Gln Lys Pro Ser Gly Val Ser Ala Ala Leu Ala Leu Lys Gly Pro Leu Leu Val Ser Thr Glu Ser His Pro Val Lys Ser Gly Ile His Lys Trp Ile Ser Asp Tyr Ala Asp Ser Val Pro Asp Pro Glu Ala Leu Arg Val Glu Val Asp Thr Phe Met Glu Ala Tyr Asp Gln Lys Ile Ala Glu Glu Glu Ala Lys Ala Lys Glu Glu Glu Gly Val Pro Asp Glu Glu Gly Trp Val Lys Val Thr Arg Arg Gly Arg Arg Pro Val Leu Pro Arg Thr Glu Ala Ala Ser Leu Arg Val Leu Glu Arg Glu Arg Arg Lys Arg Ser Arg Lys Glu Leu Leu Asn Phe Tyr Ala Trp Gln His Arg Glu Ser Lys Met Glu His Leu Ala Gln Leu Arg Lys Lys Phe Glu Glu Asp Lys Gln Arg Ile Glu Leu Leu Arg Ala Gln Arg Lys Phe Arg Pro Tyr <210> 11 <211> 130 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 4011179 <400> 11 Met Ala Arg Gly Val Val Ser Ala Lys Gly Gly Ala Val Ala Gly Lys Lys Lys Gly Ser Val Ser Phe Thr Ile Asp Cys Thr Lys Pro Val Glu Asp Lys Ile Met Glu Val Ala Ser Leu Glu Lys Phe Leu Gln Glu Arg Ile Lys Val Ala Gly Gly Lys Ala Gly Asn Leu Gly Asp Ser Val Thr Ile Ser Arg Glu Lys Thr Lys Val Thr Val Thr Ser Asp Gly Pro Phe Ser Lys Arg Tyr Leu Lys Tyr Leu Thr Lys Lys Tyr Leu Lys Lys His Asn Val Arg Asp Trp Leu Arg Val Val Ala Ala Asn Lys Asp Arg Asn Val Tyr Glu Leu Arg Tyr Phe Asn Ile Ala Glu Asn Glu Gly Glu Glu Glu Asp <210> 12 <211> 226 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 5425219 <400> 12 Met Ser Asn Tyr Val Asn Asp Met Trp Pro Gly Ser Pro Gln Glu Lys Asp Ser Pro Ser Thr Ser Arg Ser Gly Gly Ser ser Arg Leu Ser Ser Arg Ser Arg Ser Arg Ser Phe Ser Arg Ser Ser Arg Ser His Ser Arg Val Ser Ser Arg Phe Ser Ser Arg Ser Arg Arg Ser Lys Ser Arg Ser Arg Ser Arg Arg Arg His Gln Arg Lys Tyr Arg Arg Tyr Ser Arg Ser Tyr Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Arg Tyr Arg Glu Arg Arg Tyr Gly Phe Thr Arg Arg Tyr Tyr Arg Ser Pro Ser Arg Tyr Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Gly Arg Ser Tyr Cys Gly Arg Ala Tyr Ala Ile Ala Arg Gly Gln Arg Tyr Tyr Gly Phe Gly Arg Thr Val Tyr Pro Glu Glu His Ser Arg Trp Arg Asp Arg Ser Arg Thr Arg Ser Arg Ser Arg Thr Pro Phe Arg Leu Ser Glu Lys Asp Arg Met Glu Leu Leu Glu Ile Ala Lys Thr Asn Ala Ala Lys Ala Leu Gly Thr Thr Asn Ile Asp Leu Pro Ala Ser Leu Arg Thr Val Pro Ser Ala Lys Glu Thr Ser Arg Gly Ile Gly Val Ser Ser Asn Gly Ala Lys Pro Glu Lys Ser <210> 13 <211> 296 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 5522684 <400> 13 Met Ala Gly Pro Leu Gln Gly Gly Gly Ala Arg Ala Leu Asp Leu Leu Arg Gly Leu Pro Arg Val Ser Leu Ala Asn Leu Lys Pro Asn Pro Gly Ser Lys Lys Pro Glu Arg Arg Pro Arg Gly Arg Arg Arg Gly Arg Lys Cys Gly Arg Gly His Lys Gly Glu Arg Gln Arg Gly Thr Arg Pro Arg Leu Gly Phe Glu Gly Gly Gln Thr Pro Phe Tyr Ile Arg Ile Pro Lys Tyr Gly Phe Asn Glu Gly His Ser Phe Arg Arg Gln Tyr Lys Pro Leu Ser Leu Asn Arg Leu Gln Tyr Leu Ile Asp Leu Gly Arg Val Asp Pro Ser Gln Pro Ile Asp Leu Thr Gln Leu Val Asn Gly Arg Gly Val Thr Ile Gln Pro Leu Lys Arg Asp Tyr Gly Val Gln Leu Val Glu Glu Gly Ala Asp Thr Phe Thr Ala Lys Val Asn Ile Glu Val Gln Leu Ala Ser Glu Leu Ala Ile Ala Ala Ile Glu Lys Asn Gly Gly Val Val Thr Thr Ala Phe Tyr Asp Pro Arg Ser Leu Asp Ile Val Cys Lys Pro Val Pro Phe Phe Leu Arg Gly Gln Pro Ile Pro Lys Arg Met Leu Pro Pro Glu Glu Leu Val Pro Tyr Tyr Thr Asp Ala Lys Asn Arg Gly Tyr Leu Ala Asp Pro Ala Lys Phe Pro Glu Ala Arg Leu Glu Leu Ala Arg Lys Tyr Gly Tyr Ile Leu Pro Asp Ile Thr Lys Asp Glu Leu Phe Lys Met Leu Cys Thr Arg Lys Asp Pro Arg Gln Ile Phe Phe Gly Leu Ala Pro Gly Trp Val Val Asn Met Ala Asp Lys Lys Ile Leu Lys Pro Thr Asp Glu Asn Leu Leu Lys Tyr Tyr Thr Ser <210> 14 <211> 2297 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 046926 <400> 14 ttctctgtgg cggagacagc caggttggca gctgacggga cagccggggt ctattttgtt 60 gcgggttttc agcaaatcca gggctggtct ggaggcgcga aaacttaagg catacagaac 120 gatggagtat atggcagaat ccaccgaccg cagccctgga cacatcttgt gctgtgagtg 180 tggtgttccg ataagtccaa atcctgccaa tatttgtgtg gcctgtttgc gaagtaaagt 240 ggacatcagc caaggtattc cgaaacaagt ctcgatttcg ttctgcaaac aatgtcaaag 300 gtattttcaa ccaccaggaa cttggataca gtgtgcttta gaatccaggg aacttcttgc 360 tttgtgcttg aaaaaaatca aagcccctct gagtaaggta cggcttgtag atgcaggctt 420 tgtttggact gagcctcatt ctaagagact taaagttaaa ctgactattc agaaagaggt 480 gatgaatggt gctatccttc aacaagtgtt tgtggtggat tatgttgttc agtcccaaat 540 gtgtggagat tgccatagag tagaagctaa ggatttctgg aaggctgtga ttcaagtgag 600 gcaaaagact ttgcataaaa aaactttcta ctatctggaa cagttaattc tgaaatatgg 660 aatgcatcag aatacacttc gtatcaaaga gattcatgat ggtctggatt tttattattc 720 ctcaaaacaa catgctcaga agatggtcga atttcttcag tgtacagttc cctgtagata 780 caaagcatca caaagactga tctctcaaga tatccatagt aacacataca attacaaaag 840 cactttttct gtggaaattg ttccaatatg caaggataat gttgtctgtc tgtctccaaa 900 actggcacaa agcctgggaa atatgaacca gatttgtgtg tgtattcgag taaccagtgc 960 cattcacctc attgatccaa acaccctaca agtggcagat attgatggga gcactttctg 1020 gagtcaccct ttcaatagtt tatgtcatcc caaacagcta gaggagttta ttgtgatgga 1080 atgcagcata gtccaagata taaaacgtgc tgcaggtgct ggaatgatat caaaaaagca 1140 taccctcggg gaagtctggg tacagaagac atctgaaatg aatacagata aacagtattt 1200 ttgtcgtact catttgggac atcttctaaa tcccggagac ctggtgttag ggtttgattt 1260 ggccaactgt aacttaaatg atgagcatgt caacaaaatg aactcagata gagttccaga 1320 tgtggtatta atcaagaaga gctatgaccg gaccaaacgt cagcgtcgta gaaactggaa 1380 attgaaagag cttgcaagag agagagaaaa catggataca gatgatgaaa ggcaatacca 1440 agattttctt gaagatcttg aagaagatga ggcaattcga aaaaatgtca acatttacag 1500 agattcagcc atccctgtgg aaagtgacac cgatgatgaa ggagcacctc gaattagtct 1560 ggctgagatg cttgaagacc ttcatatttc ccaagatgcc actggtgaag aaggtgcatc 1620 aatgctgaca taatgagatg ttgtagactg tttccataca tgggcttaag aagttggaca 1680 gagttacctt aagtgtctct actatctttg cctccagatt tcaagaggag aaatttagtt 1740 ttaaacctga ataaacatgt ttgttttcag tgctcactca aaccactaaa acagatggat 1800 agctttgagg ttttagataa ggaaagatta tggagaatgt agttgttatt gatttttggc 1860 aattttacat ttggaatttt atcactgtgc ttttttatat gaggcactgt agtattttca 1920 catagtatag tactctggat gtaaaagctc aaaaattgtg attccttgaa cgttcactaa 1980 atcttcaagc aaaaacacat ttttacatta tttttacgtt gattatttta gtgaaagacc 2040 atatgaagaa gcatttttaa tattaacttg ttacatactt tgatccactt tacatcattt 2100 ttatgttgtt gaggtaggga aattagggtt cagtttatca ctggacattc aggaggcaag 2160 tcaatctttt ttatttcctt ataaaattaa ctcttcaaaa gctgttaaac agagagttat 2220 cttaattttt attgcagtag gaggaaatat atttaaaata tttgtagatt tatagcaaat 2280 agagactcgt tatttaa 2297 <210> 15 <211> 2144 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 618791 <400> 15 gacctgcgct ggaggcttca tctttgccgc cgctgccgtc gccttcctgg gattggagtc 60 tcgagctttc ttcgttcgtt cgtcggcggg ttcgcgccct tctcgcgcct cggggctgcg 120 aggctgggga aggggttgga gggggctgtt gatcgccgcg tttaagttgc gctcggggcg 180 gccatgtcgg ccggcgaggt cgagcgccta gtgtcggagc tgagcggcgg gaccggaggg 240 gatgaggagg aagagtggct ctatggcggc ccatgggacg tgcatgtgca cagtgatttg 300 gcaaaggacc tagatgaaaa tgaagttgaa aggccagaag aagaaaatgc cagtgctaat 360 cctccatctg gaattgaaga tgaaactgct gaaaatggtg taccaaaacc gaaagtgact 420 gagaccgaag atgatagtga tagtgacagc gatgatgatg aagatgatgt tcatgtcact 480 ataggagaca ttaaaacggg agcaccacag tatgggagtt atggtacagc acctgtaaat 540 cttaacatca agacaggggg aagagtttat ggaactacag ggacaaaagt caaaggagta 600 gaccttgatg cacctggaag cattaatgga gttccactct tagaggtaga tttggattct 660 tttgaagata aaccatggcg taaacctggt gctgatcttt ctgattattt taattatggg 720 tttaatgaag atacctggaa agcttactgt gaaaaacaaa agaggatacg aatgggactt 780 gaagttatac cagtaacctc tactacaaat aaaattacgg ccgaagactg tactatggaa 840 gttacaccag gtgcagagat ccaagatggc agattcaatc tttttaaggt acagcaggga 900 agaactggaa actcagagaa agaaactgcc cttccatcta caaaagctga gtttacttct 960 cctccttctt tgttcaagac tgggcttcca ccgagcagaa acagcacttc ttctcagtct 1020 cagacaagta ctgcctccag aaaagccaat tcaagcgttg ggaagtggca ggatcgatat 1080 gggagggccg aatcacctga tctaaggaga ttacctgggg caattgatgt tatcggtcag 1140 actataacta tcagccgagt agaaggcagg cgacgggcaa atgagaacag caacatacag 1200 gtcctttctg aaagatctgc tactgaagta gacaacaatt ttagcaaacc acctccgttt 1260 ttccctccag gagctcctcc cactcacctt ccacctcctc catttcttcc acctcctccg 1320 actgtcagca ctgctccacc tctgattcca ccaccgggtt ttcctcctcc accaggcgct 1380 ccacctccat ctcttatacc aacaatagaa agtggacatt cctctggtta tgatagtcgt 1440 tctgcacgtg catttccata tggcaatgtt gcctttcccc atcttcctgg ttctgctcct 1500 tcgtggccta gtcttgtgga caccagcaag cagtgggact attatgccag aagagagaaa 1560 gaccgagata gagagagaga cagagacaga gagcgagacc gtgatcggga cagagaaaga 1620 gaacgcacca gagagagaga gagggagcgt gatcacagtc ctacaccaag tgttttcaac 1680 agcgatgaag aacgatacag atacagggaa tatgcagaaa gaggttatga gcgtcacaga 1740 gcaagtcgag aaaaagaaga acgacataga gaaagacgac acagggagaa agaggaaacc 1800 agacataagt cttctcgaag taatagtaga cgtcgccatg aaagtgaaga aggagatagt 1860 cacaggagac acaaacacaa aaaatctaaa agaagcaaag aaggaaaaga agcgggcagt 1920 gagcctgccc ctgaacagga gagcaccgaa gctacacctg cagaataggc atggttttgg 1980 ccttttgtgt atattagtac cagaagtaga tactataaat cttgttattt ttctggataa 2040 tgtttaagaa atttacctta aatcttgttc tgtttgttag tatgaaaagt taactttttt 2100 tccaaaataa aagagtgaat ttttcatgtt aagttaaaaa aaaa 2144 <210> 16 <211> 1343 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1240366 <400> 16 cggacgcgtg ggttggaatt ctcggatccc gagagatgtc agagacacag tactcgagcc 60 ttacgcagac ccttattatg actatgaaat tgagcggttt tggcgtggcg gacagtatga 120 gaatttcagg gtgcagtata cagaaacaga gccgtatcat aattaccgac cgccaagctg 180 agccaccaaa gaaggaggct gccaccacgg ggccgcaggt gaagagagca gatgagtgga 240 aggacccttg gcgccgatcc aagtctccca agaagaaact cggggtgtcg gtctccccga 300 gccgggctcg aaggcgtcgg aaaacatcag cctcgtcagc ctctgcctct aattcctcca 360 ggtcgtcttc gcggtcatcg tcctactctg gctccggctc ctcccggtcg cgatcccggt 420 cttcatccta cagctcctac tccagccgct cttccagaca cagctcgttc tcaggaagcc 480 ggtccaggtc ccggtccttc tcttcgtccc cgtccccgtc cccaacacct tccccacata 540 gaccttccat cagaaccaag ggagagccgg ccccgccgcc cgggaaagca ggagagaagt 600 cagtgaagaa gccggccccg cctccagccc caccacaggc caccaaaacc actgctcctg 660 tccccgagcc caccaagcca ggagaccctc gggaagccag gaggaaggag cggccagcca 720 ggaccccccc caggaggcgg acgctaagcg gcagcggcag tggcagtggt agcagctata 780 gtggttccag ctcccgatcc aggtccctga gcgtgagcag cgtctcctca gtgtccagtg 840 ctacgtcgag cagcagctct gcacacagcg tggactcgga ggacatgtac gcagacctgg 900 ctagccccgt gtcctcagcc agctctcggt ccccggcccc agcccagacc aggaaggaga 960 aaggaaaatc taagaaagaa gacggtgtta aagaggaaaa gcggaaaagg gattcgtcca 1020 cacaaccacc caaatctgca aaacctccag caggggggaa gtcctcccag cagccctcga 1080 caccccagca ggcacccccc gggcagcccc agcagggcac atttgtggcc cacaaggaga 1140 tcaagttgac actgttgaat aaggcggctg ataaaggaag caggaagcgc tatgaaccat 1200 cagacaagga caggcagagc cctcctccag ccaagcggcc caacacatcc ccagaccgag 1260 gttctcggga ccgatagtca ggtgggagac tgggctcccc gaagccagag cggcagcaag 1320 cttattccct ttagtgaggg gac <210> 17 <211> 1346 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1295773 <400> 17 atctaggacc ttgacagcac tgagtatcaa ggcaaaagaa tgcatgtgca gttgtccaca 60 agccggcttc ggactgcccc tggtatggga gaccagagtg gctgctatcg gtgtgggaaa 120 gaagggcact ggtccaaaga gtgcccagta gatcgtacgg gtcgtgtggc agactttact 180 gagcagtata atgaacaata tggagcagtt cgaacacctt acaccatggg ctacggggaa 240 tccatgtatt acaacgatgc atatggagca ctcgactact ataagcgata ccgggtccgc 300 tcttatgagg cagtagcagc ggcggcagcg gcttctgcat acaactacgc agagcagacc 360 atgtcccatc tgcctcaagt ccaaagcaca actgtgacca gccacctcaa ctctacttct 420 gttgatccct atgacagaca cctattgcca aactctggcg ctgctgccac ttcagctgct 480 atggctgctg ctgcagccac cacttcctcc tactatggaa gggacaggag cccactgcgt 540 cgtgctgcag ccatgctccc cacagttgga gagggctacg gttatgggcc agagagtgaa 600 ttatctcagg cttccgcagc tacacggaat tctctgtatg acatggcccg gtatgaacgg 660 gagcagtatg tggaccgagc ccggtactca gccttttaaa aactggaggt aggataattg 720 cggactgaac cctcgggctg cggtcatata tgagaacttg ctccgcgcgg tcccctttgc 780 cgggatgttt ccattgcttc atgtttcagt aaacaaaagg agtttgtgac caactatgtt 840 ttctttctta attcttctaa gttgactttt ctttcctcct gaaactagtc tctgtagcct 900 ttcactctgt tccttatatt ctcagcctct gagcagccct aggtaaggat tatgctggca 960 tccccttttt cctgtgcagt ggaacccctc ttatcttgct ttccctagga gttgaatcct 1020 tctccctgcc tacctgcagc atctcctttc cctttaaaat gaccatgtag tggcaagcag 1080 ccttttactc ttctgttagc tctggactct taacacttaa gttactcttc tgaaattgct 1140 aggaccattg ggggttttgt tgttttgttt gttttttatg tccgacctgt gatcgtggta 1200 cagcattagc tgaaatttac ccttgtttta ctccactcct ccctttttta aaaaaatttt 1260 ttgacaaata aatgtttcta acacttaaaa aaaaaaatga agaataaaca aagaaaaaat 1320 ccaagtacat aacagaaaaa aaaaaa 1346 <210> 18 <211> 2720 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1858421 <400> 18 gtgaggagtg cggaggggcg cgaggtttca agatggcggt agctgagggg ttgaccgaga 60 gacccagttg aaggccttta cgaagtgaaa gaggccggga atcgccccct acccgcttct 120 cgtagtcctg ggagcacagc agaagtgttt ttcttttttt aatgaacaag taaaccatac 180 aaattgtcaa catgggacgg agatctacat catccaccaa gagtggaaaa tttatgaacc 240 ccacagacca agcccgaaag gaagcccgga agagagaatt aaagaagaac aaaaaacagc 300 gcatgatggt tcgagctgca gttttaaaga tgaaggatcc aaaacagata atccgagaca 360 tggagaaatt ggatgaaatg gagtttaacc cagtgcaaca gccacaatta aatgagaaag 420 tactgaaaga caagcgtaaa aagctgcgtg aaacctttga acgtattcta cgactctatg 480 aaaaagagaa tccagatatt tacaaagaat tgagaaagct agaagtagaa tatgaacaga 540 agagggctca acttagccaa tattttgatg ctgtcaagaa tgctcagcat gtggaagtgg 600 agagtattcc tttgccagat atgccacatg ctccttccaa cattttgatc caggacattc 660 cacttcctgg tgcccagcca ccctctatcc taaagaaaac ctcagcctat ggacctccaa 720 ctcgggcagt ttctatcctt cctcttcttg gacatggtgt tccacgtttg ccccctggca 780 gaaaacctcc tggccctccc cctggtccac ctcctcctca agtcgtgcag atgtatggcc 840 gtaaagtggg ttttgcccta gatcttcccc ctcgtaggcg agatgaagac atgttatata 900 gtcctgaact tgcccagcga ggtcatgatg atgatgtttc tagcaccagt gaagatgatg 960 gctatcctga ggacatggat caagataagc atgatgacag tactgatgac agtgacaccg 1020 acaaatcaga tggagaaagt gacggggatg aatttgtgca ccgtgataat ggtgagagag 1080 acaacaatga agaaaagaag tcaggtctga gtgtacggtt tgcagatatg cctggaaaat 1140 caaggaagaa aaagaagaac atgaaggaac tgactcctct tcaagccatg atgcttcgta 1200 tggcaggtca agaaatccct gaggagggac gggaagtaga ggaattttca gaggacgatg 1260 atgaagatga ttctgatgac tctgaagcag aaaagcaatc acaaaagcag cataaagagg 1320 aatcccattc tgatggcaca tccactgctt cttcacagca gcaggctccg ccgcagtctg 1380 ttcctccttc tcagatacaa gcacctccca tgccaggacc accacctctt ggaccaccac 1440 ctgctccacc attacggcct cctgggccac ctacaggcct tcctcctggt ccacctccag 1500 gagctcctcc attcctgaga ccacctggaa tgccaggact ccgagggccc ttaccccgac 1560 ttttacctcc aggaccacca ccaggccgac cccctggccc tcccccaggt ccacctccag 1620 gtctgcctcc tggtccccct cctcgtggac ccccaccaag gctacctccc cctgcacctc 1680 caggtattcc tccacctcgt cctggcatga tgcgcccacc tttggtgcct ccccttggac 1740 ctgccccccc tgggctgttc ccaccagctc ccttgccaaa ccctggggtt ttaagtgccc 1800 cacccaactt gattcagcga cccaaggcgg atgatacaag tgcagccacc attgagaaga 1860 aagccacagc aaccatcagt gccaagccac agatcactaa tcccaaggca gagattactc 1920 gatttgtgcc cactgcactg agagtacgtc gggagaataa aggggctact gctgctcccc 1980 aaagaaagtc agaggatgat tctgctgtgc ctcttgccaa agcagcaccc aaatctggtc 2040 cttctgttcc tgtctcagta caaactaagg atgatgtcta tgaggctttc atgaaagaga 2100 tggaagggct actgtgacag cttttgatgc cagaaaaggc ttctgttcac aacagtggcc 2160 catggagaaa gaggctctta ttaaacttag atgaaagagc tgcttccatt gtcagggtat 2220 tttctaattt cagttcaagg aatatcctaa aatttagcct tgttcagaat ttactgcaca 2280 taaaaaaggg tatttcatcc agaatagatc agttattgaa gcagtgctgc taacatccat 2340 tccctttcat accaccattt tcaccctgtt tcttcccctc ctccagttct ttggaaattt 2400 gtgatcgggg gatcttagtt gcttatttgt tttgactctt gtgtgctgtg ggcactggag 2460 tagagatttc tggagaaaaa aaaacagttt atttcatctt gccttttgtg tttgagttat 2520 ttttaatatt ttcctgtaaa tattttgtaa tattttactt gtaatgaaat ggatcacaat 2580 gtcatttcct aatacaaggc aggatatgtg ggaagaatat gtacaattat ttgattaaaa 2640 ttatttccca ctgacctaaa ctttcagtga tttgtgggaa aaataaataa atgttctaca 2700 ccaagaaaaa aaaaaaaaaa 2720 <210> 19 <211> 676 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2152431 <400> 19 gggaccagcg cgggtgcgca gacgaaaggc gctctttgcc agctgaaagt tcccacggaa 60 aaactaccat ctcccctgcc caccatggca gacgaaattg atttcactac tggagatgcc 120 ggggcttcca gcacttaccc tatgcagtgc tcggccttgc gcaaaaacgg cttcgtggtg 180 ctcaaaggac gaccatgcaa aatagtggag atgtcaactt ccaaaactgg aaagcatggt 240 catgccaagg ttcaccttgt tggaattgat attttcacgg gcaaaaaata tgaagatatt 300 tgtccttcta ctcacaacat ggatgttcca aatattaaga gaaatgatta tcaactgata 360 tgcattcaag atggttacct ttccctgctg acagaaactg gtgaagttcg tgaggatctt 420 aaactgccag aaggtgaact aggcaaagaa atagagggaa aatacaatgc aggtgaagat 480 gtacaggtgt ctgtcatgtg tgcaatgagt gaagaatatg ctgtagccat aaaaccctgc 540 aaataaacgg aaacatcagg catgaacact gtttatgtct gaatcaactg cagatctaat 600 ttggttctaa gttgtcacca aagctatagc cttcataagc aacctcattt ctttttttaa 660 ttgttttcag attgtg 676 <210> 20 <211> 909 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2641494 <400> 20 acggaaactg actggggtca attcaagtca tgcaggctgt gagaaacgcg gggtcgcggt 60 tcctgcggtc ctggacttgg ccccagacag ccggcagggt cgtggccaga acgccggccg 120 ggaccatctg cacaggcgct cgacagctcc aagacgctgc ggccaagcag aaagttgaac 180 agaacgcggc tcccagccac accaagttca gcatttaccc tcccattcca ggagaggaga 240 gctctctgag gtgggcagga aagaaatttg aggagatccc aattgcacac attaaagcat 300 cccacaacaa cacacagatc caggtagtct ctgctagtaa tgagcccctt gcctttgctt 360 cctgtggcac agagggattt cggaatgcca agaagggcac aggcatcgca gcacagacag 420 caggcatagc cgcagcggcg agagctaaac aaaagggcgt gatccacatc cgagttgtgg 480 tgaaaggcct ggggccagga cgcttgtctg ccatgcacgg actgatcatg ggcggcctgg 540 aagtgatctc aatcacagac aacaccccaa tcccacacaa cggctgccgc cccaggaagg 600 ctcggaagct gtgatgggaa ggaggcctgc acttggacct gacctcaagc ctcagctcca 660 gtgggacctt gtaaaatgct ccctgtcaga gctctccaga atatgcttgt tggagatcct 720 tcaggcagta agggagagtt ttgcctcctt acacagtggc ctttgcttgc acctccagct 780 ggagatgggt gtgccccaga agtaagcttt gcatctctta caagagggga gctacagggg 840 cagccgtggc ctaggcccaa actctgctct gagaaaataa atatctgtac cacctgtcaa 900 aaaaaaaaa gpg <210> 21 <211> 2405 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3803409 <400> 21 cttcaagacc tggaatgtat ccgcctccag ggtcgtatag acctaccccc tcctatgggc 60 aaaccaccag gttcaattgt aagaccctct gctccaccag caagatcatc tgttcctgtg 120 accaggccac ctgtcccaat accaccacct ccacctcctc cacctctacc tcctcctcct 180 ccagtgataa agccacaaac ttcagctgta gaacaggaac gatgggatga agattctttc 240 tatgggctct gggatacaaa tgatgaacaa ggactgaatt cagaatttaa gtcagaaact 300 gcagcaattc catctgctcc agtattacca cccccacctg ttcactcttc cattccccct 360 cctggcccag tgcctatggg tatgccacca atgtccaagc caccaccagt acaacagact 420 gttgattatg gccatggccg agatatatcc actaataaag ttgaacagat accttatgga 480 gaaagaataa ctctacgccc agatccacta cctgaaagat caacttttga gacagagcat 540 gcaggccaac gtgatcgtta tgatagagaa agagatcgtg agccttattt tgatcgtcaa 600 agtaatgtca tagcagatca tcgagatttt aaaagggatc gtgagacaca tagagatcga 660 gaccgggatc gtggtgttat tgactatgac cgggatcgat ttgacagaga acgccgaccc 720 cgagatgata gagctcagtc atatcgagac aaaaaagacc attcctcatc cagaagaggg 780 ggttttgata ggccatccta tgaccggaag tctgaccgac cagtctatga aggaccatcc 840 atgtttggag gagaacgaag gacttatcct gaggagcgaa tgcctctgcc agctccttca 900 ctgagccacc agccacctcc agctccacga gtcgagaaga agcctgaatc aaagaatgtg 960 gacgatattt tgaaaccacc gggccgggag agcagacctg agagaattgt tgttataatg 1020 agaggattac ctggcagtgg aaagacacat gttgcaaaac ttattcgaga taaggaggta 1080 gaatttggag gacctgcacc cagagttcta agcctggatg attacttcat cactgaagtg 1140 gaaaaagaag aaaaagatcc agattctgga aagaaagtga aaaagaaggt aatggaatat 1200 gaatatgaag ctgagatgga ggagacttac cgcaccagca tgttcaaaac tttcaaaaag 1260 actctggatg atggcttttt tcccttcatc atcctggatg ccatcaatga cagagttagg 1320 cattttgacc agttttggag tgcagcaaaa accaagggat ttgaggtata tttggctgaa 1380 atgagtgcag ataaccagac ttgtggcaag agaaatattc atggaagaaa gcttaaagaa 1440 ataaataaga tggctgatca ctgggaaact gcacctcgtc acatgatgcg tctagatatt 1500 cgttctttgc tgcaagatgc tgctattgaa gaggtagaga tggaagattt tgatgcaaat 1560 atcgaagaac agaaagaaga aaagaaagat gcagaggaag aggaaagcga actgggttac 1620 attccgaaaa gcaaatggga gatggacaca tctgaggcaa agctagacaa gttggatggc 1680 ttgaggactg gtactaaaag gaaacgtgac tgggaggcca ttgccagcag aatggaggat 1740 tatcttcagc tccccgatga ttatgatact cgtgcttctg agcctgggaa gaagagggtc 1800 agatgggcag acctggaaga gaagaaggat gcagatagga aaagggccat aggttttgtg 1860 gtcggacaga ctgattggga gaagatcaca gatgaaagtg gtcacctggc tgaaaaagcc 1920 ctcaatcgaa ccaaatatat atgagactta gtttttgaac ggagtcatta ttcctctaag 1980 gtggttcgct ttgaggtggt ctgaagccaa ggcctcgcgg agcttctttg tgtgtcacct 2040 tgcttccacg tttcagttct tgttttgttt ctactgcttt agtttttttt aaagttctcc 2100 agtgtcccca agaggtatta gaatcttgct gtacccaagc aagacgttaa tttttctttt 2160 aactgttttg gggagggagg gagtgatagc ttaactgctg aagccaggcg ggggtctgct 2220 ggaggattcc aacagagagt atttcctcca ctgtacaatg tcacagacta tctctatcat 2280 cattgctttg tggctgtttc tgttttttac tgtatgtaac tggtagctga ttgtactagg 2340 attaaaaaca ataaactttc atgataaagc cgatgagatt catgggctat acagaaaaaa 2400 aaaaa 2405 <210> 22 <211> 1754 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3979009 <400> 22 cgggtttgtc gggctgaaat gtggcgggtc tcggaaggtt ccgacctcag taaagagagc 60 taacgtgtat tcttcttttt cttagatgct gagatgaatc gtcacctgtg tgtttggctt 120 tttagacatc catctcttaa tggttacctc cagtgtcaca tccagctcca ttctcatcaa 180 tttagacaga tacatcttga tacaaggctg caagttttta gacaaaacag gaattgcatt 240 cttcatctgt taagtaagaa ttggtccagg agatattgcc atcaagacac caagatgctc 300 tggaagcata aagcactaca gaaatatatg gagaacctga gtaaggagta ccaaacactt 360 gagcaatgtc tgcagcatat ccctgtgaat gaagaaaacc gaaggtcctt gaacagaagg 420 catgctgagt tggcacctct tgcagccatt taccaagaaa ttcaggagac tgaacaagca 480 attgaagaat tagaatcaat gtgtaaaagc ctaaataaac aagatgaaaa gcagttacaa 540 gaacttgcac tggaagaaag gcaaaccatt gatcaaaaaa tcaacatgtt gtacaatgag 600 cttttccaga gccttgtgcc aaaggagaaa tatgacaaaa atgatgttat tttagaggtg 660 acagctggaa ggactactgg aggtgacatc tgccaacaat ttacccgaga aatatttgac 720 atgtaccaga attattcgtg ctataaacac tggcaatttg aacttctgaa ttatacacca 780 gcagattatg gtggactaca tcatgcagcc gcccgaattt ccggtgacgg tgtctataag 840 catttgaagt atgagggtgg gattcaccga gttcagcgca tccccgaggt gggcctgtcc 900 tcaaggatgc agcgcattca cacaggaacg atgtcggtta ttgtccttcc tcagccagat 960 gaggtggatg tgaaattgga ccccaaggat ttgcgaatag atacatttcg agccaaagga 1020 gcaggagggc agcatgttaa taaaactgat agtgccgtca gacttgtcca catccccaca 1080 gggctagtag tagaatgcca acaagaaaga tcacagataa aaaataaaga aatagccttt 1140 cgtgtgttga gagctagact ctaccagcag attattgaga aagacaagcg tcagcaacaa 1200 agtgctagaa aactgcaggt gggaacaaga gcccagtcag agcgaattcg gacatataat 1260 ttcacccagg atagagtcag tgaccacagg atagcatatg aagttcgtga tattaaggaa 1320 tttttatgtg gtgggaaggg cctggatcag ctaattcaga gactgcttca atcagcagat 1380 gaagaagcca ttgctgaact tttggatgaa caccttaaat cagcaaaata aatactaact 1440 tattattatt tatgattata taaatgaaat ggacctatat caagaggcag actgaagctt 1500 ggaaatcatt atgaatattt gtaaattaca gctttaagaa cacattacac ataaatatat 1560 gttttgtaat taatcgaagt cacatttcct gacctaagaa tttattttag gtttcctgta 1620 aagtacaatc caactcatca agtagaaaat aagcatgcat cattgaaaag ggaaagtatt 1680 gagaattgat tgtgtcattt aggacaagtc acttgttctc ttaaaatgcc ttttttcccc 1740 agccatctat gaat 1754 <210> 23 <211> 1221 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3992058 <400> 23 ccgcgctccc gggtggcaag atggtggcgc gcaggaggaa gtgcgccgcg cgggacccgg 60 aggaccgtat ccccagccca ctgggctacg cagctattcc aatcaagttc tctgaaaagc 120 aacaggcttc tcactacctc tatgtgagag cacacggcgt tcgacaaggc accaagtcca 180 cctggcctca gaagaggact ctttttgtcc tcaatgtgcc cccatactgc acagaggaga 240 gcctgtcccg cctcctgtcc acctgtggcc tcgtccagtc tatagagttg caggagaagc 300 cggacctggc tgagagccca aaggagtcaa ggtcgaagtt ttttcatccc aagccagttc 360 cgggtttcca ggtagcctac gtggtgttcc agaagccaag tggggtgtca gcggccttgg 420 ccctgaaggg ccccctgctg gtgtccacag agagccaccc tgtgaagagt ggcattcaca 480 agtggatcag tgactacgca gactctgtgc ccgaccctga ggccctgagg gtggaagtgg 540 acacgttcat ggaggcatat gaccagaaga tcgctgagga agaagctaag gccaaggagg 600 aggagggggt ccctgacgag gagggctggg tgaaggtgac ccgccggggc cggcggcctg 660 tgctcccccg gactgaggca gccagcttgc gggtgctgga gagggagaga cggaagcgca 720 gccgaaaaga gctgctcaac ttctacgcct ggcagcatcg agagagcaag atggagcatc 780 tagcgcagct gcgcaagaag ttcgaggagg acaagcagag gatcgagctg ctgcgggccc 840 agcgcaaatt ccgaccgtac tgagctgtga gagccgcagt gaatggctgg aggtgcaggg 900 ccaggaggag gcgaggcagg gcctgcagcg gtctctgaga ggccgagctc tggccaacgg 960 gccccaggtt gaaggccacc gcgtccaaca gccccatcag agtccacaca ggccaggagg 1020 gaaggaccag gccacccctc gggtcttgtg cttcagcagt cctggggacc caggcgtgcc 1080 gagaggagga cttgtccttc ctgcttcttg cctccacacc ctcctctcca ggaccctgga 1140 tgaatccgtt ctgtgcttcc ttttccctca atgcaaaagc ccttgctggc aacgaaaaag 1200 cctcaaaagc aaaaaaaaaa a 1221 <210> 24 <211> 628 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 4011179 <400> 24 ggaaacccta gccgccatat ctcacgatcc actcgagcac caagccgagg gaaggtgagg 60 agcgatggcg cgcggcgtgg tgtcggcgaa gggcggcgcg gtcgcgggca agaagaaggg 120 gtcggtttcc ttcacgatcg actgcaccaa gccagtggag gacaagatca tggaggtcgc 180 ctcgctcgag aagttcctgc aggagcgcat caaggtcgcc ggcggcaagg ctggcaacct 240 cggcgactcc gtcaccatct ctcgcgagaa gaccaaggtc accgtcacct ctgacggacc 300 cttctccaag aggtacctga agtacttgac caagaagtac ttgaagaagc acaacgtgcg 360 ggattggcta cgcgtggttg ctgccaacaa ggaccgaaac gtctatgagc tccgctactt 420 caacattgct gagaacgagg gcgaggaaga agattagatt gcactacgct tatattttag 480 tattgaactc gttgcatttt gatacctgta cccgtagttt cgcaaatgtc ccatgttatg 540 gtgtggtatg gttaatttga agaatcctta tgtactgaat ctctgcaaaa agctatgttg 600 tggacagaag tgtaacgtgc cagatttt 628 <210> 25 <211> 1500 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 5425219 <400> 25 gtctgctaac gtagtccctc agtgcgcatc cggacgtagg aggtggaggt tgtggaattc 60 gccgttcgaa agcagggact aaaagcccca cttcgtctta cgttccgaaa ggaaggcgtc 120 tgttgagcct ttctctcagt cgtgagggag gcgtcgacgg cgtgcggaag tcctgagttg 180 aggcttgcgg gatcctttcc ggagaaagcg caggctaaag ccgcaggtga agatgtccaa 240 ctacgtgaac gacatgtggc cgggctcgcc gcaggagaag gattcgccct cgacctcgcg 300 gtcgggcggg tccagccggc tgtcgtcgcg gtctaggagc cgctcttttt ccagaagctc 360 tcggtcccat tcccgcgtct cgagccggtt ttcgtccagg agtcggagga gcaagtccag 420 gtcccgttcc cgaaggcgcc accagcggaa gtacaggcgc tactcgcggt catactcgcg 480 gagccggtcg cgatcccgca gccgccgtta ccgagagagg cgctacgggt tcaccaggag 540 atactaccgg tctccttcgc ggtaccggtc ccggtcccgt agcaggtcgc gctctcgggg 600 aaggtcgtac tgcggaaggg cgtacgcgat cgcgcgggga cagcgctact acggctttgg 660 tcgcacagtg tacccggagg agcacagcag atggagggac agatccagga cgaggtcgcg 720 gagcagaacc ccctttcgct taagtgaaaa agatcgaatg gagctgttag aaatagcaaa 780 aaccaatgca gcgaaagctc taggaacaac caacattgac ttgccagcta gtctcagaac 840 tgttccttca gccaaagaaa caagccgtgg aataggtgta tcaagtaatg gtgcaaagcc 900 tgaaaaatca tgaatgtggt ctgcagacat tgatgaagaa aatctgttgc tgtcggaaaa 960 ggtaacagaa gatggaactc gaaatcccaa tgaaaaacct acccagcaaa gaagcatagc 1020 ttttagctct aataattctg tagcaaagcc aatacaaaaa tcagctaaag ctgccacaga 1080 agaggcatct tcaagatcac caaaaataga tcagaaaaaa agtccatatg gactgtggat 1140 acctatctaa aagaagaaaa ctgatggcta agtttgcatg aaaactgcac tttattgcaa 1200 gttagtgttt ctagcattat cccatccctt tgagccattc aggggtactt gtgcatttaa 1260 aaaccaacac aaaaagatgt aaatacttaa cactcaaata ttaacatttt aggtttctct 1320 tgcagatatg agagatagca cagatggacc aaaggttatg cacaggtggg agtcttttgt 1380 atatagttgt aaatattgtc ttggttatgt aaaaatgaaa ttttttagac acagtaattg 1440 aactgtattc ctgttttgta tatttaataa atttcttgtt ttcattctta aaaaaaaaaa 1500 <210> 26 <211> 1143 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 5522684 <400> 26 gaccacgtgg cctccgagca gctcagggcg cccttgaaag ttcttggatc tgcgggttat 60 ggccggtccc ttgcagggcg gtggggcccg ggccctggac ctactccggg gcctgccgcg 120 tgtgagcctg gccaacttaa agccgaatcc cggctccaag aaaccggaga gaagaccaag 180 aggtcggaga agaggtagaa aatgtggcag aggccataaa ggagaaaggc aaagaggaac 240 ccggccccgc ttgggctttg agggaggcca gactccattt tacatccgaa tcccaaaata 300 cgggtttaac gaaggacata gtttcagacg ccagtataag cctttgagtc tcaatagact 360 gcagtatctt attgatttgg gtcgtgttga tcctagtcaa cctattgact taacccagct 420 tgtcaatggg agaggtgtga ccatccagcc acttaaaagg gattatggtg tccagctggt 480 tgaggagggt gctgacacct ttacggcaaa agttaatatt gaagtacagt tggcttcaga 540 actagctatt gctgccattg aaaaaaatgg tggtgttgtt actacagcct tctatgatcc 600 aagaagtctg gacattgtat gcaaacctgt tccattcttt cttcgtggac aacccattcc 660 aaaaagaatg cttccaccag aagaactggt accatattac actgatgcaa agaaccgtgg 720 gtacctggcg gatcctgcca aatttcctga agcacgactt gaactcgcca ggaagtatgg 780 ttatatctta cctgatatca ctaaagatga actcttcaaa atgctctgta ctaggaagga 840 tccaaggcag attttctttg gtcttgctcc aggatgggtg gtgaatatgg ccgataagaa 900 aatcctaaaa cctacagatg aaaatctcct taagtattat acctcatgaa ttcccgtcca 960 aggaagcaga gttgttaaag agtactggaa taggggctga aggatctata ttcccttatt 1020 gcattttcct tatgtataat tttccagatg gtgatgttac ttttcagtgt actcatatgt 1080 ctcattttca tctaaaatta aatggcagga aacaaggact gcatagagaa aaaaaaaaaa 1140 aaa

Claims (27)

What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
a) 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:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, b) a naturally occurring amino acid sequence having at least 90% sequence identity to 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:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, c) a biologically active fragment of 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:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID
NO:13, and d) an immunogenic fragment of 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:6, SEQ ID
NO:7, SEQ
ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID
NO:13.

2. An isolated polypeptide of claim 1 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:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

3. An isolated polynucleotide encoding a polypeptide of claim 1.

4. An isolated polynucleotide encoding a polypeptide of claim 2.

5. An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID
NO:26.

6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.

7. A cell transformed with a recombinant polynucleotide of claim 6.

8. A transgenic organism comprising a recombinant polynucleotide of claim 6.

9. A method for producing a polypeptide of claim 1, the method comprising:
a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.

10. An isolated antibody which specifically binds to a polypeptide of claim 1.

11. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
a) a polynucleotide sequence selected from the group consisting of SEQ ID
NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, b) a naturally occurring polynucleotide sequence having at least 90% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ
ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).

12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim 11.

13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising:
a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.

14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.

15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising:

a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

16. A pharmaceutical composition comprising an effective amount of a polypeptide of claim 1 and a pharmaceutically acceptable excipient.

17. A pharmaceutical composition of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID N0:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12 and SEQ ID NO:13.

18. A method for treating a disease or condition associated with decreased expression of functional RMEP, comprising administering to a patient in need of such treatment the pharmaceutical composition of claim 16.

19. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising:
a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.

20. A pharmaceutical composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.

21. A method for treating a disease or condition associated with decreased expression of functional RMEP, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 20.

22. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising:

a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.

23. A pharmaceutical composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.

24. A method for treating a disease or condition associated with overexpression of functional RMEP, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 23.

25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of:
a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1.

26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising:
a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1.

27. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising:
a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.