CA2340277A1 - Human rna-associated proteins - Google Patents

Abstract

The invention provides human RNA-associated proteins (RNAAP) and polynucleotides which identify and encode RNAAP. 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 the expression of RNAAP.

Description

HUMAN RNA-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human RNA-associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, autoimmune/inflammatory, and infectious disorders.
BACKGROUND OF THE INVENTION
Ribonucleic acid (RNA) is a linear single-stranded polymer of four nucleotides, ATP, GTP, 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.
The unspliced precursors of mature mRNA transcripts are called heterogeneous rruelear RNA (hnRNA) transcripts. hnRNA is generally larger and more unstable than mRNA.
Immediately upon its synthesis, hnRNA is assembled into protein-containing complexes called heterogeneous nuclear ribonucleoprotein particles (hnRNPs). (See, for example, Honore, B. et al.
( 1995) J. Biol. Chem. 270:28780-28789.) hnRNPs associate with small nuclear ribonucleoprotein particles (snRNPs) which are stable RNA-protein complexes that function primarily in splicing introns from hnRNA. Each snRNP contains a single species of RNA and about l0 proteins. The RNA components of snRNPs recognize and base pair with specific sequences of the hnRNA
intron. Five different snRNPs associate at the intron of hnRNA to form the spliceosome, a multicomponent RNP complex which catalyzes the removal of introns and the rejoining of exons.
Also associated with the snRNPs are various accessory factors that stabilize intron-snRNP
interactions. In humans, these factors include spliceosome associated protein 49 (SAP 49) and SAP 145. (Champion-Arnaud, P, and Reed, R. (1994) Genes Dev. 8:1974-1983.) 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 WO 00/11171 PCTlUS99/19361 structural, catalytic, and regulatory purposes. RNA polymerases are proteins that transcribe RNA
from a DNA copy. The HIV Tat protein binds specific sites in the viral RNA to prevent premature transcriptional termination. 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.
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).
The process of splicing may involve more than the removal of an intron from an RNA
transcript. For example, an RNA transcript may be subject to alternative patterns of splicing, resulting in the generation of different species of mRNA from a single primary transcript. In addition, certain transcripts may be subject to traps-splicing, in which an exon from one transcript is joined to an exon of another. Often, specific protein cofactors are required to mediate splicing under these special circumstances. For example, a new splicing factor, PR264, has been implicated in the traps-splicing of a thymus-specific c-myb transcript in humans. (Vellard, M. et al. (1992) Proc. Natl. Acad. Sci. USA 89:2511-2515.) Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified that have roles in splicing, exporting ofthe 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 A 1, 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, supra).
Nascent tRNA transcripts are spliced by unconventional mechanisms that are distinct from those employed by the spliceosome. 1n this case, splicing is carried out by specific endonucleases and ligases that recognize secondary structural features of the tRNA. This process contrasts with the spliceosomal reaction, in which specific nucleotide sequences of the intron are recognized. In addition, tRNAs are further processed by removal of 5' sequences and by chemical modification of some of the nucleotide bases. tRNA processing has been extensively studied in yeast, in which tRNA-specific splicing factors have been identified. (See, for example, Shen, W. C. et al. (1993) J. Biol. Chem. 268:19436-19444.) Proteins are also a part 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 5.85 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 factors, many of which contain multiple subunits, are proteins which are involved in bringing together an initiator tRNA, the mRNA, and the ribosomal 40S subunit. Eukaryotic initiation factor 2 (eIF2), a guanine nucleotide binding protein, recruits the initiator tRNA to the 40S ribosomal subunit. Only when eIF2 is bound to GTP does it associate with the initiator tRNA. elF2B, a guanine nucleotide exchange protein, is responsible for converting eIF2 from the GDP-bound inactive form to the GTP-bound active form. Other initiation factors include eIFIA, elF3, eIF4F (a complex including eIF4E, elF4A, and eIF4G), and eIFS. The elongation factors EFIa, EF1 (3 y, and EF2 are involved in elongating the polypeptide chain following initiation, and the release factor eRF carries out termination of translation. (See V. M. Pain ( 1996) Eur. J. Biochem. 236:747-771.) Other important RNA-associated enzymes with roles in translation are the aminoacyl-transfer RNA (tRNA) synthetases. Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA. The 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is characterized by a distinctive topology of the catalytic domain. Class I
enzymes contain a catalytic domain based on the nucleotide-binding Rossman 'fold'. Class II
enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel 13-sheet motif, as well as -and C- terminal regulatory domains. Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the - and C-terminal regulatory domains (Hartlein, M. and Cusack, S. ( 1995) J.
Mol. Evol. 40:519-530). Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals.
In many cases, mRNA translation, localization, and stability are controlled by regulatory proteins that bind to the 5' and 3' untranslated (UTR) regions of mRNA. An example of such a protein is Spnr, a mouse spermatid perinuclear RNA-binding protein, which may be involved in RNA transport, translational activation, or localization of RNA to cytoplasmic microtubules (Schumacher, J. M. et al. (1995) J. Cell Biol. 129:1023-1032). RNA-associated proteins may alter and regulate RNA conformation and secondary structure. These processes are mediated by RNA
helicases which utilize energy derived from ATP hydrolysis to destabilize and unwind RNA
duplexes. The most well-characterized and ubiquitous family of RNA helicases is the DEAD-box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family. Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Some DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis. All DEAD-box helicases contain several conserved sequence motifs spread out over about 420 amino acids. These motifs include an A-type ATP
binding motif, the DEAD-box/B-type ATP-binding motif, a serine/arginine/threonine tripeptide of unknown function, and a C-terminal glycine-rich motif with a possible role in substrate binding and unwinding. In addition, alignment of divergent DEAD-box helicase sequences has shown that IS 37 amino acid residues are identical among these sequences, suggesting that conservation of these residues is important for helicase function. (Reviewed in Linder, P. et al. ( 1989) Nature 337:121-122.) Overexpression of the DEAD-box 1 protein (DDX 1 ) may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem.
273:21161-21168). These observations suggest that DDX 1 may promote or enhance tumor progression by altering the normal secondary structure and expression levels of RNA in cancer cells. Other DEAD-box helicases have been implicated either directly or indirectly in tumorigenesis. (Discussed in Godbout, supra.) For example, murine p68 is mutated in ultraviolet light-induced tumors, and human DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma. Similarly, a chimeric protein comprised of DDX10 and NUP98, a nucleoporin protein, may be involved in the pathogenesis of certain myeloid malignancies.
Ribonucleases (RNases) are RNA-associated enzymes which catalyze the hydrolysis of phosphodiester bonds in RNA chains, thus cleaving the RNA. For example, RNase P is a ribonucleoprotein enzyme which cleaves the S' 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 N 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.
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.) T'he RRM is about 80 amino acids in length and forms four ~i-strands and two a-helices arranged in an a/~i sandwich.
The RRM contains a core RNP-I 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 which have been identified in lower eukaryotes such as Drosophila melano a~ ster and Caenorhabditis ele ans. 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.) The RRM includes the ribonucleoprotein-1 (RNP-1) RNA binding motifwhich is found in snRNP proteins, hnRNP proteins, splicing factors, mRNA binding proteins, and transcriptional regulatory proteins. Other hallmarks of RNA binding proteins include regions of repeated arginine and serine residues (RS repeats).
The discovery of new human RNA-associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, and infectious disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human RNA-associated proteins, referred to collectively as "RNAAP" and individually as "RNAAP-1,"
"RNAAP-2,"
"RNAAP-3," "RNAAP-4," "RNAAP-5,""RNAAP-6," "RNAAP-7," "RNAAP-8," "RNAAP-9,"
"RNAAP-10," "RNAAP-11," "RNAAP-12," "RNAAP-13," "RNAAP-14," "RNAAP-15,"
"RNAAP-16," "RNAAP-17," "RNAAP-18," "RNAAP-19," "RNAAP-20," "RNAAP-21,""RNAAP-22" "RNAAP-23" "RNAAP-24"and RNAAP-25" In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25 and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ
ID NO:1-25 and fragments thereof. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:I-25 and fragments thereof.
The invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a} hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample. In one aspect, the method further comprises amplifying the polynucleotide prior to hybridization.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:26-50 and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 70% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID N0:26-50 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:26-50 and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-25 and fragments thereof. 1n another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ

ID NO:1-25 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO:1-25 and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of RNAAP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purifed polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:I-25 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of RNAAP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-25 and fragments thereof.
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 RNAAP.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods and algorithms used for identification of RNAAP.
Table 3 shows 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 RNAAP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze RNAAP, 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 to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"RNAAP" refers to the amino acid sequences of substantially purified RNAAP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which, when bound to RNAAP, increases or prolongs the duration of the effect of RNAAP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of RNAAP.
An "allelic variant" is an alternative form of the gene encoding RNAAP.
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. Any given natural or recombinant gene may have none, one, or many allelic forms. 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 atone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding RNAAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as RNAAP or a polypeptide with at least one functional characteristic of RNAAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding RNAAP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding RNAAP. 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 RNAAP. 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 RNAAP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
The terms "amino acid" 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. In this context, "fragments," "immunogenic fragments," or "antigenic fragments" refer to fragments of RNAAP which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of RNAAP. Where "amino acid sequence" is recited to refer to an amino acid 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, when bound to RNAAP, decreases the amount or the duration of the effect of the biological or immunological activity of RNAAP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of RNAAP.
The term "antibody" refers to intact molecules as well as to fragments thereof, such as Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind RNAAP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (ICLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation "negative" can refer to the antisense strand, and the designation "positive" can refer to the sense strand.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic RNAAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms "complementary" and "complementarity" refer to the natural binding of polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'." Complementarity between two single-stranded molecules may be "partial," such that only some of the nucleic acids bind, or it may be "complete," such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
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 RNAAP or fragments of RNAAP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucleotide" indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding RNAAP, by northern analysis is indicative of the presence of nucleic acids encoding RNAAP in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding RNAAP.
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.
IS The term "derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
The term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity" may substitute for the word "similarity." A
partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction.
The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30%
similarity or identity).

In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGAL1GN program (DNASTAR, Madison Wl) which creates alignments between two or more sequences according to methods selected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp ( 1988) Gene 73:237-244.) Parameters for each method may be the default parameters provided by MEGALIGN or may be specified by the user. The clustal algorithm groups sequences into clusters by examining the distances between al) pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. ( 1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., C°t or Rat 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, to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on a substrate.
The terms "element" and "array element" in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
i0 The term "modulate" refers to a change in the activity of RNAAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of RNAAP.
The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to I S DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In this context, "fragments" refers to those nucleic acid sequences which comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:26-50, for example, as distinct from any other sequence in the same genome. For 20 example, a fragment of SEQ ID N0:26-50 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:26-50 from related polynucleotide sequences. A fragment of SEQ ID N0:26-50 is at least about 15-20 nucleotides in length. The precise length of the fragment of SEQ ID N0:26-50 and the region of SEQ ID
N0:26-SO to which the fragment corresponds are routinely determinable by one of ordinary skill 25 in the art based on the intended purpose for the fragment. In some cases, a fragment, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding 30 sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously finked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.

The term "oligonucleotide" refers to a nucleic acid sequence of at least about nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. "Oligonucleotide" is substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in iength linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding RNAAP, or fragments thereof, or RNAAP 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, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
The term "stringent conditions" refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60%
free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "variant" of RNAAP polypeptides refers to an amino acid sequence that is altered by IS one or more amino acid residues. The variant may have "conservative"
changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to RNAAP. This definition may also include, for example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding poiypeptide may possess additional functional domains or an absence of domains.
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 poiymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.

THE INVENTION
The invention is based on the discovery of new human RNA-associated proteins (RNAAP), the polynucleotides encoding RNAAP, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, autoimmune/inflammatory, and infectious disorders.
Table I lists the Incyte clones used to assemble full length nucleotide sequences encoding RNAAP. 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 RNAAP 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 clones in column S were used to assemble the consensus nucleotide sequence of each RNAAP 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 I 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 the identity of each protein; and column 7 shows analytical methods used to identify each protein 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 RNAAP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists tissue categories which express RNAAP as a fraction of total tissue categories expressing RNAAP. Column 3 lists diseases, disorders, or conditions associated with those tissues expressing RNAAP. Column 4 lists the vectors used to subclone the cDNA library.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA
libraries from which cDNA clones encoding RNAAP were isolated. Column I
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.
The following fragments of the nucleotide sequences encoding RNAAP are useful, for example, in hybridization or amplification technologies to identify SEQ ID
N0:26-50 and to distinguish between SEQ ID N0:26-50 and related polynucleotide sequences. The useful fragments include the fragment of SEQ ID N0:26 from about nucleotide 586 to about nucleotide 615; the fragment of SEQ ID N0:27 from about nucleotide 399 to about nucleotide 428; the fragment of SEQ ID N0:28 from about nucleotide 234 to about nucleotide 263;
the fragment of SEQ ID N0:29 from about nucleotide 40 to about nucleotide 69; and the Fragment of SEQ ID
N0:30 from about nucleotide 20 to about nucleotide 49, the fragment of SEQ ID
N0:31 from about nucleotide 40 to about nucleotide 80, the fragment of SEQ ID N0:32 from about nucleotide 672 to about nucleotide 713, the fragment of SEQ ID N0:33 from about nucleotide 226 to about nucleotide 276, the fragment of SEQ ID N0:34 from about nucleotide 719 to about nucleotide 761, the fragments of SEQ ID N0:35 from about nucleotide 77 to about nucleotide 167 and from about nucleotide 168 to about nucleotide 259, the fragment of SEQ ID N0:36 from about nucleotide 465 to about nucleotide 506, the fragment of SEQ ID N0:37 from about nucleotide 76 to about nucleotide 117, the fragment of SEQ ID N0:38 from about nucleotide 136 to about nucleotide 180, the fragments of SEQ ID N0:39 from about nucleotide 215 to about nucleotide 262, from about nucleotide 683 to about nucleotide 727, and from about nucleotide 1805 to about nucleotide 1852, the fragment of SEQ ID N0:40 from about nucleotide 162 to about nucleotide 206, the fragment of SEQ ID N0:41 from about nucleotide 379 to about nucleotide 423, the fragment of SEQ ID N0:42 from about nucleotide 164 to about nucleotide 208, the fragment of SEQ ID N0:43 from about nucleotide 1 to about nucleotide 42, the fragments of SEQ ID N0:44 from about nucleotide 249 to about 296 and from about nucleotide 816 to about nucleotide 862, the fragment of SEQ 1D N0:45 from about nucleotide 196 to about nucleotide 240, the fragment of SEQ ID N0:46 from about nucleotide 1 to about nucleotide 54, the fragment of SEQ ID N0:47 from about nucleotide 463 to about nucleotide 507, the fragments of SEQ ID
N0:48 from about nucleotide 551 to about nucleotide 595, from about nucleotide 866 to about nucleotide 910, and from about nucleotide 1406 to about nucleotide 1450, the fragments of SEQ ID
N0:49 from about nucleotide 218 to about nucleotide 263, from about nucleotide 758 to about nucleotide 802, and from about nucleotide I 190 to about nucleotide 1234, and the fragment of SEQ
ID NO:50 from about nucleotide 11 to about nucleotide 70.
The invention alsa encompasses RNAAP variants. A preferred RNAAP variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the RNAAP amino acid sequence, and which contains at least one functional or structural characteristic of RNAAP.
The invention also encompasses polynucleotides which encode RNAAP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:26-S0, which encodes RNAAP.
The invention also encompasses a variant of a polynucieotide sequence encoding RNAAP.

In particular, such a variant polynucleotide sequence will have at least about 70%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding RNAAP. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:26-50 which has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:26-50. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of RNAAP.
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 RNAAP, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring RNAAP, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode RNAAP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring RNAAP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding RNAAP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence encoding RNAAP 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 RNAAP
and RNAAP 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 RNAAP 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
N0:26-50 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M.
and S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM
NaCi and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, SO mM trisodium citrate, 1% SDS, 35% formamide, and 100 /cg/ml denatured salmon sperm DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM
NaCI, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCI, I.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in IS mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Perkin-Elmer), 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 Hamilton MICROLAB 2200 (Hamilton, Reno NV), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the ABI
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI
373 or 377 DNA sequencing systems (Perkin-Elmer), 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 BioloQV, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding RNAAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G.
(1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. ( 1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
1:1 I 1-119.) In this method, multiple restriction enzyme digestions and legations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection ofthe emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode RNAAP may be cloned in recombinant DNA molecules that direct expression of RNAAP, 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 RNAAP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter RNAAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression ofthe 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.
In another embodiment, sequences encoding RNAAP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. ( 1980) Nucl.
Acids Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 225-232.) Alternatively, RNAAP itself or a fragment thereof may be synthesized using chemical methods.
For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of RNAAP, 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 poiypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY.) In order to express a biologically active RNAAP, the nucleotide sequences encoding RNAAP 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 RNAAP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding RNAAP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding RNAAP
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 RNAAP 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 RNAAP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding RNAAP.
For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding RNAAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla CA) or pSPORTI plasmid (Life Technologies). Ligation of sequences encoding RNAAP 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 RNAAP are needed, e.g. for the production of antibodies, vectors which direct high level expression of RNAAP may be used. For example, vectors containing the strong, inducible TS or bacteriophage promoter may be used.
Yeast expression systems may be used for production of RNAAP. 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~astoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Grant et al. ( 1987) Methods Enzymol. 153:5 I 6-54; and Scorer, C. A. et al. ( 1994) Bio/Technoiogy 12:181-184.) Plant systems may also be used for expression of RNAAP. Transcription of sequences encoding RNAAP 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, G. et aI. (1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technoloey (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 RNAAP
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 RNAAP in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat Genet.
15:345-355.) I S For long term production of recombinant proteins in mammalian systems, stable expression of RNAAP in cell lines is preferred. For example, sequences encoding RNAAP 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. Fol lowing the introduction of the vector, cells may be al lowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose ofthe selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk~ or apr cells, respectively.
(See, e.g., Wigler, M. et al. (1977) Cell 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 or pat confer resistance to ehlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. MoI. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan ( 1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), (3 glucuronidase and its substrate (3-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.
Bio1.55:121-131.) 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 RNAAP is inserted within a marker gene sequence, transformed cells containing sequences encoding RNAAP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding RNAAP 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 RNAAP
and that express RNAAP 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 RNAAP
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 RNAAP 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 Immunoloey, 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 RNAAP
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding RNAAP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham 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 RNAAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode RNAAP may be designed to contain signal sequences which direct secretion of RNAAP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, 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 RNAAP 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 RNAAP
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of RNAAP 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 oftheir 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 RNAAP encoding sequence and the heterologous protein sequence, so that RNAAP
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 RNAAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 'SS-methionine.
Fragments of RNAAP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, su ra pp. ~S-60.) Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Various fragments of RNAAP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of RNAAP and RNA-associated proteins. In addition, the expression of RNAAP
is closely associated with cancer, fetal development, cell proliferation, inflammation, and immune response. Therefore, RNAAP appears to play a role in cell proliferative, autoimmune/inflammatory, and infectious disorders. In the treatment of disorders associated with increased RNAAP expression or activity, it is desirable to decrease the expression or activity of RNAAP. In the treatment of the above conditions associated with decreased RNAAP expression or activity, it is desirable to increase the expression or activity of RNAAP.
Therefore, in one embodiment, RNAAP 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 RNAAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankyiosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, 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 an infectious disorder such as infections by viral agents classified as adenovirus, arenavirus, bunyavirus, caiicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus; infections by bacterial agents classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, and campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, and mycoplasma; infections by fungal agents classified as aspergillus, blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, and other fungal agents causing various mycoses; and infections by parasites classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes such as trichinella, intestinal nematodes such as ascaris, lymphatic filaria) nematodes, trematodes such as schistosoma, and cestodes (tapeworm).
In another embodiment, a vector capable of expressing RNAAP 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 RNAAP including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising a substantially purified RNAAP 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 RNAAP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of RNAAP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of RNAAP including, but not limited to, those listed above.
In a further embodiment, an antagonist of RNAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of RNAAP. Examples of such disorders include, but are not limited to, those cell proliferative, autoimmune/inflammatory, and infectious disorders described above. In one aspect, an antibody which specifically binds RNAAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express RNAAP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding RNAAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of RNAAP 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 RNAAP may be produced using methods which are generally known in the art. In particular, purified RNAAP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind RNAAP.
Antibodies to RNAAP
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 especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with RNAAP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to RNAAP have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of RNAAP 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 RNAAP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497;
Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci.
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S.L. et al. ( 1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M.S. et al. ( 1984) Nature 312:604-608; and Takeda, S. et al. ( 1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce RNAAP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad.
Sci. 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. ( 1989) Proc.
Natl. Acad. Sci. 86:
3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for RNAAP 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 ofthe 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 polyclona) or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between RNAAP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering RNAAP epitopes is preferred, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for RNAAP.
Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of RNAAP-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 RNAAP epitopes, represents the average affinity, or avidity, of the antibodies for RNAAP. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular RNAAP epitope, represents a true measure of affinity. High-affinity antibody preparations with K, ranging from about 109 to 10'' L/mole are preferred for use in immunoassays in which the RNAAP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about I OG to 107 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of RNAAP, preferably in active form, from the antibody (Catty, D. ( 1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington, DC;
Liddell, J. E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &
Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least I-2 mg specific antibody/ml, preferably S-10 mg specific antibody/ml, is preferred for use in procedures requiring precipitation of RNAAP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.) In another embodiment of the invention, the polynucleotides encoding RNAAP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding RNAAP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding RNAAP. Thus, complementary molecules or fragments may be used to modulate RNAAP activity, or to achieve regulation of gene function.
Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding RNAAP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacteria! plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding RNAAP. (See, e.g., Sambrook, supra; Ausubel, 1995, s_upra.) Genes encoding RNAAP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding RNAAP.
Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell.
Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, S', or regulatory regions of the gene encoding RNAAP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 from the start site, are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al.
(1994) in Huber, B.E. and B.I. Carr, Molecular and Immunoloaic 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 RNAAP.
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 RNAAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al.
( 1997) Nature Biotechnology 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of RNAAP, antibodies to RNAAP, and mimetics, agonists, antagonists, or inhibitors of RNAAP.
The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduliary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or recta! means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Reminaton's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl I S cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: I mM to 50 mM histidine, 0.1 % to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of RNAAP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example RNAAP or fragments thereof, antibodies of RNAAP, and agonists, antagonists or inhibitors of RNAAP, 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 SO% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDS°/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 EDs° 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
1n another embodiment, antibodies which specifically bind RNAAP may be used for the diagnosis of disorders characterized by expression of RNAAP, or in assays to monitor patients being treated with RNAAP or agonists, antagonists, or inhibitors of RNAAP.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for RNAAP include methods which utilize the antibody and a label to detect RNAAP 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 RNAAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of RNAAP
IS expression. Normal or standard values for RNAAP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to RNAAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of RNAAP 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 RNAAP may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of RNAAP
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of RNAAP, and to monitor regulation of RNAAP
levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding RNAAP or closely related molecules may be used to identify nucleic acid sequences which encode RNAAP.
The specificity of the probe, whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding RNAAP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the RNAAP encoding sequences.
The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:26-50 or from genomic sequences including promoters, enhancers, and introns of the RNAAP gene.
Means for producing specific hybridization probes for DNAs encoding RNAAP
include the cloning of polynucleotide sequences encoding RNAAP or RNAAP 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'zP
or'SS, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
I S Polynucleotide sequences encoding RNAAP may be used for the diagnosis of disorders associated with expression of RNAAP. Examples of such disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MC'TD), 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; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondyiitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, 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 an infectious disorder such as infections by viral agents classified as adenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus, hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus; infections by bacterial agents classified as pneumococcus, staphylococcus, streptococcus, bacillus, corynebacterium, clostridium, meningococcus, gonococcus, listeria, moraxella, kingella, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella, salmonella, and campylobacter, pseudomonas, vibrio, brucella, francisella, yersinia, bartonella, norcardium, actinomyces, mycobacterium, spirochaetale, rickettsia, chlamydia, and mycoplasma; infections by fungal agents classified as aspergillus, btastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia, histoplasma, and other fungal agents causing various mycoses; and infections by parasites classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes such as trichinella, intestinal nematodes such as ascaris, lymphatic filarial nematodes, trematodes such as schistosoma, and cestodes (tapeworm). The polynucleotide sequences encoding RNAAP 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 RNAAP expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding RNAAP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding RNAAP 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 quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding RNAAP 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 RNAAP, 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 RNAAP, 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 RNAAP 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 RNAAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding RNAAP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantify the expression of RNAAP 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 an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of Large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et al. ( 1996) Proc. Natl.
Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc.
Natl. Acad. Sci.
94:2150-2155; and Heller, M.J. et al. ( i 997) U.S. Patent No. 5,605,662.) 1n another embodiment of the invention, nucleic acid sequences encoding RNAAP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., 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.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site.
Correlation between the location of the gene encoding RNAAP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA
associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
In another embodiment of the invention, RNAAP, 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 RNAAP 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 RNAAP, or fragments thereof, and washed. Bound RNAAP is then detected by methods well known in the art. Purified RNAAP 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 IS 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 RNAAP specifically compete with a test compound for binding RNAAP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with RNAAP.
In additional embodiments, the nucleotide sequences which encode RNAAP 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. 60/115,639 and U.S. Ser No. 60/097,550, 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 CsCI cushions or extracted with chloroform. RNA
was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T}-coupled paramagnetic particles (Promega), OLIGOTEX
latex particles (QIAGEN, 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 S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTI plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Paio Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including XL1-BLUE, XL1-BLUEMRF, or SOLR
from Stratagene or DHSa, DH10B, or ELECTROMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector system (Stratagene) or cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, 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 Fluoroskan II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) 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 (Perkin-Elmer). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing systems (Perkin-Eimer) 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 V.
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).
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 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, 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. Str. 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 N0:26-50. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Northern Analysis Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, su ra, ch. 7;
Ausubel, 1995, suara, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Phanmaceuticals).
This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is de#ined as:
% sequence identity x % maximum BLAST score The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a I% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding RNAAP 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. Extension of RNAAP Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:26-50 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 Mgz+, (NH4)zS04, and (3-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 pl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1X TE and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 ~cl to 10 ~cl aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to relegation 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 relegated 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, individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x Garb 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 1: 94°C, 3 min; Step 2: 94°C, IS sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
Samples were diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAM1C
energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABl PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:26-50 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:26-50 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-'ZP] 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 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography and compared.
VII. Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al. ( 1995) Science 270:467-470; Shalom D. et al. ( 1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides Sequences complementary to the RNAAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring RNAAP.
Although use of oligonucleotides comprising from about 1 S 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 RNAAP. 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 RNAAP-encoding transcript.
IX. Expression of RNAAP
Expression and purification of RNAAP is achieved using bacterial or virus-based S expression systems. For expression of RNAAP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the TS or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express RNAAP upon induction with isopropyl beta-D-thiogalactopyranoside (1PTG). Expression of RNAAP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autog-raphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding RNAAP 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;gperda (SP9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci.
USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.) In most expression systems, RNAAP is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be proteolytically cleaved from RNAAP 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 RNAAP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of RNAAP Activity RNAAP activity is demonstrated by the formation of an RNAAP-RNA complex as detected by a polyacrylamide gel mobility-shift assay. In preparation for this assay, RNAAP is expressed by transforming a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector containing RNAAP cDNA. The cells are incubated for 48-72 hours after transformation under conditions which allow expression and accumulation of RNAAP. Extracts containing solubilized proteins can be prepared from cells expressing RNAAP by methods well known in the art. Portions of the extract containing RNAAP are added to ['2P)-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 S-10 minutes. After incubation, the samples are analyzed by polyacrylamide gel electrophoresis followed by autoradiography. The presence of a high molecular weight band on the autoradiogram indicates the formation of a complex between RNAAP and the radioactive transcript. A band of significantly lower molecular weight will be present in samples prepared using control extracts prepared from untransformed cells. The amount of RNAAP-RNA complex can be quantified using phospho-image analysis and is proportional to the activity of RNAAP.
Alternatively, RNAAP, or biologically active fragments thereof, are labeled with 'ZSI
Bolton-Hunter reagent. (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 RNAAP, washed, and any wells with labeled RNAAP complex are assayed. Data obtained using different concentrations of RNAAP are used to calculate values for the number, affinity, and association of RNAAP with the candidate molecules.
XI. Functional Assays RNAAP function is assessed by expressing the sequences encoding RNAAP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.
S-10 ug of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either iiposome formulations or electroporation. I-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 Cytometry, Oxford, New York NY.
The influence of RNAAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding RNAAP and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding RNAAP and other genes of interest can be analyzed by northern analysis or microarray techniques.
XII. Production of ItNAAP Specific Antibodies RNAAP 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 RNAAP amino acid sequence is analyzed using I,ASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies 6y 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 15 residues in length are synthesized using an ABI
431A peptide synthesizer (Perkin-Elmer) 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 activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

XIII. Purification of Naturally Occurring ItNAAP Using Specific Antibodies Naturally occurring or recombinant RNAAP is substantially purified by immunoaffinity chromatography using antibodies specific for RNAAP. An immunoaffinity column is constructed by covalently coupling anti-RNAAP 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 RNAAP are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of RNAAP (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/RNAAP 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 RNAAP is collected.
XIV. Identification of Molecules Which Interact with ItNAAP
RNAAP, or biologically active fragments thereof, are labeled with ''-SI Bolton-Hunter reagent. (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 RNAAP, washed, and any wells with labeled RNAAP complex are assayed. Data obtained using different concentrations of RNAAP are used to calculate values for the number, affinity, and association of RNAAP with the candidate molecules.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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Z s' U .~~n SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
YUE, Henry TANG, Y. Tom CORLEY, Neil C.
GUEGLER, Karl J.
GORGONE, Gina A.
PATTERSON, Chandra BAUGHN, Mariah R.
LAL, Preeti BANDMAN, Olga REDDY, Roopa AZIMZAI, Yalda SHIH, Leo L.
YANG, Junming LU, Dyung Aina M.
<120> HUMAN RNA-ASSOCIATED PROTEINS
<130> PF-0579 PCT
<140> To Be Assigned <141> Herewith <150> 60/097,550; 60/115,639 <151> 1998-08-21; 1999-O1-12 <160> 50 <170> PERL Program <210> 1 <211> 216 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 399781CD1 <400> 1 Met Ser Arg Tyr Leu Arg Pro Pro Asn Thr Ser Leu Phe Val Arg Asn Val Ala Asp Asp Thr Arg Ser Glu Asp Leu Arg Arg Glu Phe Gly Arg Tyr Gly Pro Ile Val Asp Val Tyr Val Pro Leu Asp Phe Tyr Thr Arg Arg Pro Arg Gly Phe Ala Tyr Val Gln Phe Glu Asp Val Arg Asp Ala Glu Asp Ala Leu His Asn Leu Asp Arg Lys Trp Ile Cys Gly Arg Gln Ile Glu Ile Gln Phe Ala Gln Gly Asp Arg Lys Thr Pro Asn Gln Met Lys Ala Lys Glu Gly Arg Asn Val Tyr Ser Ser Ser Arg Tyr Asp Asp Tyr Asp Arg Tyr Arg Arg Ser Arg Ser Arg Ser Tyr Glu Arg Arg Arg Ser Arg Ser Arg Ser Phe Asp Tyr Asn Tyr Arg Arg Ser Tyr Ser Pro Arg Asn Ser Arg Pro Thr Gly Arg Pro Arg Arg Arg Glu Ala Ile Pro Thr Met Ile Asp Gln Thr Ala Ala Gly Ile Pro Ser Thr Val Leu Leu Thr Thr Leu Gln Glu Arg Ser Glu Ser Gly Lys Arg Thr Lys Glu Gly Gln Phe Lys Arg Pro Lys Gly Gly Trp Lys Val Leu Gln Tyr Glu Tyr Cys Thr Asn Ile Leu Thr Leu Val <210> 2 <211> 962 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1806542CD1 <400> 2 Met Asp Glu Gln Ala Leu Leu Gly Leu Asn Pro Asn Ala Asp Ser Asp Phe Arg Gln Arg Ala Leu Ala Tyr Phe Glu Gln Leu Lys Ile Ser Pro Asp Ala Trp Gln Val Cys Ala Glu A1a Leu Ala Gln Arg Thr Tyr Ser Asp Asp His Val Lys Phe Phe Cys Phe Gln Val Leu Glu His Gln Val Lys Tyr Lys Tyr Ser Glu Leu Thr Thr Val Gln Gln Gln Leu Ile Arg Glu Thr Leu Ile Ser Trp Leu Gln Ala Gln Met Leu Asn Pro Gln Pro Glu Lys Thr Phe Ile Arg Asn Lys Ala Ala Gln Val Phe Ala Leu Leu Phe Val Thr Glu Tyr Leu Thr Lys Trp Pro Lys Phe Phe Phe Asp Ile Leu Ser Val Val Asp Leu Asn Pro Arg Gly Val Asp Leu Tyr Leu Arg Ile Leu Met Ala Ile Asp Ser Glu Leu Val Asp Arg Asp Val Val His Thr Ser Glu Glu Ala Arg Arg Asn Thr Leu Ile Lys Asp Thr Met Arg Glu Gln Cys Ile Pro Asn Leu Val Glu Ser Trp Tyr Gln Ile Leu Gln Asn Tyr Gln Phe Thr Asn Ser Glu Val Thr Cys Gln Cys Leu Glu Val Val Gly Ala Tyr Val Ser Trp Ile Asp Leu Ser Leu Ile Ala Asn Asp Arg Phe Ile Asn Met Leu Leu Gly His Met Ser Ile Glu Val Leu Arg Glu Glu Ala Cys Asp Cys Leu Phe Glu Val Val Asn Lys Gly Met Asp Pro Val Asp Lys Met Lys Leu Val Glu Ser Leu Cys Gln Val Leu Gln Ser Ala Gly Phe Phe Ser Ile Asp Gln Glu Glu Asp Val 275 280 2$5 Asp Phe Leu Ala Arg Phe Ser Lys Leu Val Asn Gly Met Gly Gln Ser Leu Ile Val Ser Trp Ser Lys Leu Ile Lys Asn Gly Asp Ile Lys Asn Ala Gln Glu Ala Leu Gln Ala Ile Glu Thr Lys Val Ala Leu Met Leu Gln Leu Leu Ile His Glu Asp Asp Asp Ile Ser Ser Asn Ile Ile Gly Phe Cys Tyr Asp Tyr Leu His Ile Leu Lys Gln Leu Thr Val Leu Ser Asp Gln Gln Lys Ala Asn Val Glu Ala Ile Met Leu Ala Val Met Lys Lys Leu Thr Tyr Asp Glu Glu Tyr Asn Phe Glu Asn Glu Gly Glu Asp Glu Ala Met Phe Val Glu Tyr Arg Lys Gln Leu Lys Leu Leu Leu Asp Arg Leu Ala Gln Val Ser Pro Glu Leu Leu Leu Ala Ser Val Arg Arg Val Phe Ser Ser Thr Leu Gln Asn Trp Gln Thr Thr Arg Phe Met Glu Val Glu Val Ala Ile Arg Leu Leu Tyr Met Leu Ala Glu Ala Leu Pro Val Ser His Gly Ala His Phe Ser Gly Asp Val Ser Lys Ala Ser Ala Leu Gln Asp Met Met Arg Thr Leu Val Thr Ser Gly Val Ser Ser Tyr Gln His Thr Ser Val Thr Leu Glu Phe Phe Glu Thr Val Val Arg Tyr Glu Lys Phe Phe Thr Val Glu Pro Gln His Ile Pro Cys Val Leu Met Ala Phe Leu Asp His Arg Gly Leu Arg His Ser Ser Ala Lys Val Arg Ser Arg Thr Ala Tyr Leu Phe Ser Arg Phe Val Lys Ser Leu Asn Lys Gln Met Asn Pro Phe Ile Glu Asp Ile Leu Asn Arg Ile Gln Asp Leu Leu Glu Leu Ser Pro Pro Glu Asn Gly His Gln Ser Leu Leu Ser Ser Asp Asp Gln Leu Phe Ile Tyr Glu Thr Ala Gly Val Leu Ile Val Asn Ser Glu Tyr Pro Ala Glu Arg Lys Gln Ala Leu Met Arg Asn Leu Leu Thr Pro Leu Met Glu Lys Phe Lys Ile Leu Leu Glu Lys Leu Met Leu Ala Gln Asp Glu Glu Arg Gln Ala Ser Leu Ala Asp Cys Leu Asn His Ala Val Gly Phe Ala Ser Arg Thr Ser Lys Ala Phe Ser Asn Lys Gln Thr Val Lys Gln Cys Gly Cys Ser Glu Val Tyr Leu Asp Cys Leu Gln Thr Phe Leu Pro Ala Leu Ser Cys Pro Leu Gln Lys Asp Ile Leu Arg Ser Gly Val Arg Thr Phe Leu His Arg Met Ile Ile Cys Leu Glu Glu Glu Val Leu Pro Phe Ile Pro Ser Ala Ser Glu His Met Leu Lys Asp Cys Glu Ala Lys Asp Leu Gln Glu Phe Ile Pro Leu Ile Asn Gln Ile Thr Ala Lys Phe Lys Ile Gln Val Ser Pro Phe Leu Gln Gln Met Phe Met Pro Leu Leu His Ala Ile Phe Glu Val Leu Leu Arg Pro Ala Glu Glu Asn Asp Gln Ser Ala Ala Leu Glu Lys Gln Met Leu Arg Arg Ser Tyr Phe Ala Phe Leu Gln Thr Val Thr Gly Ser Gly Met Ser Glu Val Ile Ala Asn Gln Gly Ala Glu Asn Val Glu Arg Val Leu Val Thr Val Ile Gln Gly Ala Val Glu Tyr Pro Asp Pro Ile Ala Gln Lys Thr Cys Phe Ile Ile Leu Ser Lys Leu Val Glu Leu Trp Gly Gly Lys Asp Gly Pro Val Gly Phe Ala Asp Phe Val Tyr Lys His Ile Val Pro Ala Cys Phe Leu Ala Pro Leu Lys Gln Thr Phe Asp Leu Ala Asp Ala Gln Thr Val Leu Ala Leu Ser Glu Cys Ala Val Thr Leu Lys Thr Ile His Leu Lys Arg Gly Pro Glu Cys Val Gln Tyr Leu Gln Gln Glu Tyr Leu Pro Ser Leu Gln Val Ala Pro Glu Ile Ile Gln Glu Phe Cys Gln Ala Leu Gln Gln Pro Asp Ala Lys Val Phe Lys Asn Tyr Leu Lys Val Phe Phe Gln Arg Ala Lys Pro <210> 3 <211> 285 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2263514CD1 <400> 3 Met Asp Trp Val Met Lys His Asn Gly Pro Asn Asp Ala Ser Asp Gly Thr Val Arg Leu Arg Gly Leu Pro Phe Gly Cys Ser Lys Glu Glu Ile Val Arg Val Leu Ser Arg Tyr Ile Glu Ile Phe Arg Ser Ser Arg Ser Glu Ile Lys Gly Phe Tyr Asp Pro Pro Arg Arg Leu Leu Gly Gln Arg Pro Gly Pro Tyr Asp Arg Pro Ile Gly Gly Arg Gly Gly Tyr Tyr Gly Ala Gly Arg Gly Ser Tyr Gly Gly Phe Asp Asp Tyr Gly Gly Tyr Asn Asn Tyr Gly Tyr Gly Asn Asp Gly Phe Asp Asp Arg Met Arg Asp Gly Arg Gly Met Gly Gly His Gly Tyr Gly Gly Ala Gly Asp Ala Ser Ser Gly Phe His Gly Gly His Phe Val His Met Arg Gly Leu Pro Phe Arg Ala Thr Glu Asn Ala Ile Ala Asn Phe Phe Ser Pro Leu Asn Pro Ile Arg Val His Ile Asp Ile Gly Ala Asp Gly Arg Ala Thr Gly Glu Ala Asp Val Glu Phe Val Thr His Glu Asp Ala Val Ala Ala Met Ser Lys Asp Lys Asn Asn Met Gln His Arg Tyr Ile Glu Leu Phe Leu Asn Ser Thr Pro Gly Gly Gly Ser Gly Met Gly Gly Ser Gly Met Gly Gly Tyr Gly Arg Asp Gly Met Asp Asn Gln Gly Gly Tyr Gly Ser Val Gly Arg Met Gly Met Gly Asn Asn Tyr Ser Gly Gly Tyr Gly Thr Pro Asp Gly Leu Gly Gly Tyr Gly Arg Gly Gly Gly Gly Ser Gly Gly Tyr Tyr Gly Gln Gly Gly Met Ser Gly Gly Gly Trp Arg Gly Met Tyr <210> 4 <211> 267 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2738270CD1 <400> 4 Met Gly Ala Ala Ala Ala Glu Ala Asp Arg Thr Leu Phe Val Gly Asn Leu Glu Thr Lys Val Thr Glu Glu Leu Leu Phe Glu Leu Phe His Gln Ala Gly Pro Val Ile Lys Val Lys Ile Pro Lys Asp Lys Asp Gly Lys Pro Lys Gln Phe Ala Phe Val Asn Phe Lys His Glu Val Ser Val Pro Tyr Ala Met Asn Leu Leu Asn Gly Ile Lys Leu Tyr Gly Arg Pro Ile Lys Ile Gln Phe Arg Ser Gly Ser Ser His Ala Pro Gln Asp Val Ser Leu Ser Tyr Pro Gln His His Val Gly Asn Ser Ser Pro Thr Ser Thr Ser Pro Ser Ser Arg Tyr Glu Arg Thr Met Asp Asn Met Thr Ser Ser Ala Gln Ile Ile Gln Arg Ser Phe Ser Ser Pro Glu Asn Phe Gln Arg Gln Ala Val Met Asn Ser Ala Leu Arg Gln Met Ser Tyr Gly Gly Lys Phe Gly Ser Ser Pro Leu Asp Gln Ser Gly Phe Ser Pro Ser Val Gln Ser His Ser His Ser Phe Asn Gln Ser Ser Ser Sex Gln Trp Arg Gln Gly Thr Pro Ser Ser Gln Arg Lys Val Arg Met Asn Ser Tyr Pro Tyr Leu Ala Asp Arg His Tyr Ser Arg Glu Gln Arg Tyr Thr Asp His Gly Ser Asp His His Tyr Arg Gly Lys Arg Asp Asp Phe Phe Tyr Glu Asp Arg Asn His Asp Asp Trp Ser His Asp Tyr Asp Asn Arg Arg Asp Ser Ser Arg Asp Gly Lys Trp Arg Ser Ser Arg His <210> 5 <211> 369 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2824412CD1 <400> 5 Met Pro Pro Gln Pro Gln Gly Pro Ala Pro Leu Arg Arg Pro Asp Ser Ser Asp Asp Arg Tyr Val Met Thr Lys His Ala Thr Ile Tyr Pro Thr Glu Glu Glu Leu Gln Ala Val Gln Lys Ile Val Ser Ile Thr Glu Arg Ala Leu Lys Leu Val Ser Asp Ser Leu Ser Glu His Glu Lys Asn Lys Asn Lys Glu Gly Asp Asp Lys Lys Glu Gly Gly Lys Asp Arg Ala Leu Lys Gly Val Leu Arg Val Gly Val Leu Ala Lys Gly Leu Leu Leu Arg Gly Asp Arg Asn Val Asn Leu Val Leu Leu Cys Ser Glu Lys Pro Ser Lys Thr Leu Leu Ser Arg Ile Ala Glu Asn Leu Pro Lys Gln Leu Ala Val Ile Ser Pro Glu Lys Tyr Asp Ile Lys Cys Ala Val Ser Glu Ala Ala Ile Ile Leu Asn Ser Cys Val Glu Pro Lys Met Gln Val Thr Ile Thr Leu Thr Ser Pro Ile Ile Arg Glu Glu Asn Met Arg Glu Gly Asp Val Thr Ser Gly Met Val Lys Asp Pro Pro Asp Val Leu Asp Arg Gln Lys Cys Leu Asp Ala Leu Ala Ala Leu Arg His Ala Lys Trp Phe Gln Ala Arg Ala Asn Gly Leu Gln Ser Cys Val Ile Ile Ile Arg Ile Leu Arg Asp Leu Cys Gln Arg Val Pro Thr Trp Ser Asp Phe Pro Ser Trp Ala Met Glu Leu Leu Val Glu Lys Ala Ile Ser Ser Ala Ser Ser Pro Gln Ser Pro Gly Asp Ala Leu Arg Arg Val Phe Glu Cys Ile Ser Ser Gly Ile Ile Leu Lys Gly Ser Pro Gly Leu Leu Asp Pro Cys Glu Lys Asp Pro Phe Asp Thr Leu Ala Thr Met Thr Asp Gln Gln Arg Glu Asp Ile Thr Ser Ser Ala Gln Phe Ala Leu Arg Leu Leu Ala Phe Arg Gln Ile His Lys Val Leu Gly Met Asp Pro Leu Pro Gln Met Ser Gln Arg Phe Asn Ile His Asn Asn Arg Lys Arg Arg Arg Asp Ser Asp Gly Val Asp Gly Phe Glu Ala Glu Gly Lys Lys Asp Lys Lys Asp Tyr Asp Asn Phe <210> 6 <211> 175 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 002690CD1 <400> 6 Met Arg Leu Ser Val Ala Ala Ala Ile Ser His Gly Arg Val Phe Arg Arg Met Gly Leu Gly Pro Glu Ser Arg Ile His Leu Leu Arg Asn Leu Leu Thr Gly Leu Val Arg His Glu Arg Ile Glu Ala Pro Trp Ala Arg Val Asp Glu Met Arg Gly Tyr Ala Glu Lys Leu Ile Asp Tyr Gly Lys Leu Gly Asp Thr Asn Glu Arg Ala Met Arg Met Ala Asp Phe Trp Leu Thr Glu Lys Asp Leu Ile Pro Lys Leu Phe Gln Val Leu Ala Pro Arg Tyr Lys Asp Gln Thr Gly Gly Tyr Thr Arg Met Leu Gln Ile Pro Asn Arg Ser Leu Asp Arg Ala Lys Met Ala Val Ile Glu Tyr Lys Gly Asn Cys Leu Pro Pro Leu Pro Leu Pro Arg Arg Asp Ser His Leu Thr Leu Leu Asn Gln Leu Leu Gln Gly Leu Arg Gln Asp Leu Arg Gln Ser Gln Glu Ala Ser Asn His Ser Ser His Thr Ala Gln Thr Pro Gly Ile <210> 7 <211> 311 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 041108CD1 <400> 7 Met Leu Gln Phe Val Arg Ala Gly Ala Arg Ala Trp Leu Arg Pro Thr Gly Ser Gln Gly Leu Ser Ser Leu Ala Glu Glu Ala Ala Arg Ala Thr Glu Asn Pro Glu Gln Val Ala Ser Glu Gly Leu Pro Glu Pro Val Leu Arg Lys Val Glu Leu Pro Val Pro Thr His Arg Arg Pro Val Gln Ala Trp Val Glu Ser Leu Arg Gly Phe Glu Gln Glu Arg Val Gly Leu Ala Asp Leu His Pro Asp Val Phe Ala Thr Ala Pro Arg Leu Asp Ile Leu His Gln Val Ala Met Trp Gln Lys Asn Phe Lys Arg Ile Ser Tyr Ala Lys Thr Lys Thr Arg Ala Glu Val Arg Gly Gly Gly Arg Lys Pro Trp Pro Gln Lys Gly Thr Gly Arg Ala Arg His Gly Ser Ile Arg Ser Pro Leu Trp Arg Gly Gly Gly Val Ala His Gly Pro Arg Gly Pro Thr Ser Tyr Tyr Tyr Met Leu Pro Met Lys Val Arg Ala Leu Gly Leu Lys Val Ala Leu Thr Val Lys Leu Ala Gln Asp Asp Leu His Ile Met Asp 5er Leu Glu Leu Pro Thr Gly Asp Pro Gln Tyr Leu Thr Glu Leu Ala His Tyr Arg Arg Trp Gly Asp Ser Val Leu Leu Val Asp Leu Thr His Glu Glu Met Pro Gln Ser Ile Val Glu Ala Thr Ser Arg Leu Lys Thr Phe Asn Leu Ile Pro Ala Val Gly Leu Asn Val His Ser Met Leu Lys His Gln Thr Leu Val Leu Thr Leu Pro Thr Val Ala Phe Leu Glu Asp Lys Leu Leu Trp Gln Asp Ser Arg Tyr Arg Pro Leu Tyr Pro Phe Ser Leu Pro Tyr Ser Asp Phe Pro Arg Pro Leu Pro His Ala Thr Gln Gly Pro Ala Ala Thr Pro Tyr His Cys <210> 8 <211> 330 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 869138CD1 <400> 8 Met Ser Thr Lys Asn Phe Arg Val Ser Asp Gly Asp Trp Ile Cys Pro Asp Lys Lys Cys Gly Asn Val Asn Phe Ala Arg Arg Thr Ser Cys Asn Arg Cys Gly Arg Glu Lys Thr Thr Glu Ala Lys Met Met Lys Ala Gly Gly Thr Glu Ile Gly Lys Thr Leu Ala Glu Lys Ser Arg Gly Leu Phe Ser Ala Asn Asp Trp Gln Cys Lys Thr Cys Ser Asn Val Asn Trp Ala Arg Arg Ser Glu Cys Asn Met Cys Asn Thr Pro Lys Tyr Ala Lys Leu Glu Glu Arg Thr Gly Tyr Gly Gly Gly Phe Asn Glu Arg Glu Asn Val Glu Tyr Ile Glu Arg Glu Glu Ser Asp Gly Glu Tyr Asp Glu Phe Gly Arg Lys Lys Lys Lys Tyr Arg Gly Lys Ala Val Gly Pro Ala Ser Ile Leu Lys Glu Val Glu Asp Lys Glu Ser Glu Gly Glu Glu Glu Asp Glu Asp Glu Asp Leu Ser Lys Tyr Lys Leu Asp Glu Asp Glu Asp Glu Asp Asp Ala Asp Leu Ser Lys Tyr Asn Leu Asp Ala Ser Glu Glu Glu Asp Ser Asn Lys Lys Lys Ser Asn Arg Arg Ser Arg Ser Lys Ser Arg Ser Ser His Ser Arg Ser Ser Ser Arg Ser Ser Ser Pro Ser Ser Ser Arg Ser Arg Ser Arg Ser Arg Ser Arg Ser Ser Ser Ser Ser Gln Ser Arg Ser Arg Ser Ser Ser Arg Glu Arg Ser Arg Ser Arg Gly Ser Lys Ser Arg Ser Ser Ser Arg Ser His Arg Gly Ser Ser Ser Pro Arg Lys Arg Ser Tyr Ser Ser Ser Ser Ser Ser Pro Glu Arg Asn Arg Lys Arg Ser Arg Ser Arg Ser Ser Ser Ser Gly Asp Arg Lys Lys Arg Arg Thr Arg Ser Arg Ser Pro Glu Arg Arg His Arg Ser Ser Ser Gly Ser Ser His Ser Gly Ser Arg Ser Ser Ser Lys Lys Lys <210> 9 <211> 183 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 934406CD1 <400> 9 Met Ser Arg Tyr Leu Arg Pro Pro Asn Thr Ser Leu Phe Val Arg Asn Val Ala Asp Asp Thr Arg Ser Glu Asp Leu Arg Arg Glu Phe Gly Arg Tyr Gly Pro Ile Val Asp Val Tyr Val Pro Leu Asp Phe Tyr Thr Arg Arg Pro Arg Gly Phe Ala Tyr Val Gln Phe Glu Asp Val Arg Asp Ala Glu Asp Ala Leu His Asn Leu Asp Arg Lys Trp Ile Cys Gly Arg Gln Ile Glu Ile Gln Phe Ala Gln Gly Asp Arg Lys Thr Pro Asn Gln Met Lys Ala Lys Glu Gly Arg Asn Val Tyr Ser Ser Ser Arg Tyr Asp Asp Tyr Asp Arg Tyr Arg Arg Ser Arg Ser Arg Ser Tyr Glu Arg Arg Arg Ser Arg Ser Arg Ser Phe Asp Tyr Asn Tyr Arg Arg Ser Tyr Ser Pro Arg Asn Ser Arg Pro Thr Gly Arg Pro Arg Arg Ser Arg Ser His Ser Asp Asn Asp Arg Pro Asn Cys Ser Trp Asn Thr Gln Tyr Ser Ser Ala Tyr Tyr Thr Ser Arg Lys Ile <210> IO
<211> 670 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1315083CD1 <400> 10 Met Ser His Leu Pro Met Lys Leu Leu Arg Lys Lys Ile Glu Lys Arg Asn Leu Lys Leu Arg Gln Arg Asn Leu Lys Phe Gln Gly Ala Ser Asn Leu Thr Leu Ser Glu Thr Gln Asn Gly Asp Val Ser Glu Glu Thr Met Gly Ser Arg Lys Val Lys Lys Ser Lys Gln Lys Pro Met Asn Val Gly Leu Ser Glu Thr Gln Asn Gly Gly Met Ser Gln Glu Ala Val Gly Asn Ile Lys Val Thr Lys Ser Pro Gln Lys Ser Thr Val Leu Thr Asn Gly Glu Ala Ala Met Gln Ser Ser Asn Ser Glu Ser Lys Lys Lys Lys Lys Lys Lys Arg Lys Met Val Asn Asp Ala Glu Pro Asp Thr Lys Lys Ala Lys Thr Glu Asn Lys Gly Lys Ser Glu Glu Glu Ser Ala Glu Thr Thr Lys Glu Thr Glu Asn Asn Val Glu Lys Pro Asp Asn Asp Glu Asp Glu Ser Glu Val Pro Ser Leu Pro Leu Gly Leu Thr Gly Ala Phe Glu Asp Thr Ser Phe Ala Ser Leu Cys Asn Leu Val Asn Glu Asn Thr Leu Lys Ala Ile Lys Glu Met Gly Phe Thr Asn Met Thr Glu Ile Gln His Lys Ser Ile Arg Pro Leu Leu Glu Gly Arg Asp Leu Leu Ala Ala Ala Lys Thr Gly Ser Gly Lys Thr Leu Ala Phe Leu Ile Pro Ala Val Glu Leu Ile VaI Lys Leu Arg Phe Met Pro Arg Asn Gly Thr Gly Val Leu Ile Leu Ser Pro Thr Arg Glu Leu Ala Met Gln Thr Phe Gly Val Leu Lys Glu Leu Met Thr His His Val His Thr Tyr Gly Leu Ile Met Gly Gly Ser Asn Arg Ser Ala Glu Ala Gln Lys Leu Gly Asn Gly Ile Asn Ile Ile Val Ala Thr Pro Gly Arg Leu Leu Asp His Met Gln Asn Thr Pro Gly Phe Met Tyr Lys Asn Leu Gln Cys Leu Val Ile Asp Glu Ala Asp Arg Ile Leu Asp Val Gly Phe Glu Glu Glu Leu Lys Gln Ile Ile Lys Leu Leu Pro Thr Arg Arg Gln Thr Met Leu Phe Ser Ala Thr Gln Thr Arg Lys Val Glu Asp Leu Ala Arg Ile Ser Leu Lys Lys Glu Pro Leu Tyr Val Gly Val Asp Asp Asp Lys Ala Asn Ala Thr Val Asp Gly Leu Glu Gln Gly Tyr Val Val Cys Pro Ser Glu Lys Arg Phe Leu Leu Leu Phe Thr Phe Leu Lys Lys Asn Arg Lys Lys Lys Leu Met Val Phe Phe Ser Ser Cys Met Ser Val Lys Tyr His Tyr Glu Leu Leu Asn Tyr Ile Asp Leu Pro Val Leu Ala Ile His Gly Lys Gln Lys Gln Asn Lys Arg Thr Thr Thr Phe Phe Gln Phe Cys Asn Ala Asp Ser Gly Thr Leu Leu Cys Thr Asp Val Ala Ala Arg Gly Leu Asp Ile Pro Glu Val Asp Trp Ile Val Gln Tyr Asp Pro Pro Asp Asp Pro Lys Glu Tyr Ile His Arg Val Gly Arg Thr Ala Arg Gly Leu Asn Gly Arg Gly His Ala Leu Leu Ile Leu Arg Pro Glu Glu Leu Gly Phe Leu Arg Tyr Leu Lys Gln Ser Lys Val Pro Leu Ser Glu Phe Asp Phe Ser Trp Ser Lys Ile Ser Asp Ile Gln Ser Gln Leu Glu Lys Leu Ile Glu Lys Asn Tyr Phe Leu His Lys Ser Ala Gln Glu Ala Tyr Lys Ser Tyr Ile Arg Ala Tyr Asp Ser His Ser Leu Lys Gln Ile Phe Asn Val Asn Asn Leu Asn Leu Pro Gln Val Ala Leu Ser Phe Gly Phe Lys Val Pro Pro Phe Val Asp Leu Asn Val Asn Ser Asn Glu Gly Lys Gln Lys Lys Arg Gly Gly Gly Gly Gly Phe Gly Tyr Gln Lys Thr Lys Lys Val Glu Lys Ser Lys Ile Phe Lys His Ile Ser Lys Lys Ser Ser Asp Ser Arg Gln Phe Ser His <210> 11 <211> 452 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte Identification No.: 1444908CD1 <400> 11 Met Glu Phe Gln Ala Val Val Met Ala Val Gly Gly Gly Ser Arg Met Thr Asp Leu Thr Ser Ser Ile Pro Lys Pro Leu Leu Pro Val Gly Asn Lys Pro Leu Ile Trp Tyr Pro Leu Asn Leu Leu Glu Arg Val Gly Phe Glu Glu Val Ile Val Val Thr Thr Arg Asp Val Gln Lys Ala Leu Cys Ala Glu Phe Lys Met Lys Met Lys Pro Asp Ile Val Cys Ile Pro Asp Asp Ala Asp Met Gly Thr Ala Asp Ser Leu Arg Tyr Ile Tyr Pro Lys Leu Lys Thr Asp Val Leu Val Leu Ser Cys Asp Leu Ile Thr Asp Val Ala Leu His Glu Val Val Asp Leu Phe Arg Ala Tyr Asp Ala Ser Leu Ala Met Leu Met Arg Lys Gly Gln Asp Ser Ile Glu Pro Val Pro Gly Gln Lys Gly Lys Lys Lys Ala Val Glu Gln Arg Asp Phe Ile Gly Val Asp Ser Thr Gly Lys Arg Leu Leu Phe Met Ala Asn Glu Ala Asp Leu Asp Glu Glu Leu Val Ile Lys Gly Ser Ile Leu Gln Lys His Pro Arg Ile Arg Phe His Thr Gly Leu Val Asp Ala His Leu Tyr Cys Leu Lys Lys Tyr Ile Val Asp Phe Leu Met Glu Asn Gly Ser Ile Thr Ser Ile Arg Ser Glu Leu Ile Pro Tyr Leu Val Arg Lys Gln Phe Ser Ser Ala Ser Ser Gln Gln Gly Gln Glu Glu Lys Glu Glu Asp Leu Lys Lys Lys Glu Leu Lys Ser Leu Asp Ile Tyr Ser Phe Ile Lys Glu Ala Asn Thr Leu Asn Leu Ala Pro Tyr Asp Ala Cys Trp Asn Ala Cys Arg Gly Asp Arg Trp Glu Asp Leu Ser Arg Ser Gln Val Arg Cys Tyr Val His Ile Met Lys Glu Gly Leu Cys Ser Arg Val Ser Thr Leu Gly Leu Tyr Met Glu Ala Asn Arg Gln Val Pro Lys Leu Leu Ser Ala Leu Cys Pro Glu Glu Pro Pro Val His Ser Ser Ala Gln Ile Val Ser Lys His Leu Val Gly Val Asp Ser Leu Ile Gly Pro Glu Thr Gln Ile Gly Glu Lys Ser Ser Ile Lys Arg Ser Val Ile Gly Ser Ser Cys Leu Ile Lys Asp Arg Val Thr Ile Thr Asn Cys Leu Leu Met Asn Ser Val Thr Val Glu Glu Gly Ser Asn Ile Gln Gly Ser Val Ile Cys Asn Asn Ala Val Ile Glu Lys Gly Ala Asp Ile Lys Asp Cys Leu Ile Gly Ser Gly Gln Arg Ile Glu Ala Lys Ala Lys Arg Val Asn Glu Val Ile Val Gly Asn Asp Gln Leu Met Glu IIe <210> 12 <211> ?48 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 15574BICD1 <400> 12 Met Ala Asp Ser Ser Gly Gln Gln Gly Lys Gly Arg Arg Val Gln Pro Gln Trp Ser Pro Pro Ala Gly Thr Gln Pro Cys Arg Leu His Leu Tyr Asn Ser Leu Thr Arg Asn Lys Glu Val Phe Ile Pro Gln Asp Gly Lys Lys Val Thr Trp Tyr Cys Cys Gly Pro Thr Val Tyr Asp Ala Ser His Met Gly His Ala Arg Ser Tyr Ile Ser Phe Asp Ile Leu Arg Arg Val Leu Lys Asp Tyr Phe Lys Phe Asp Val Phe Tyr Cys Met Asn Ile Thr Asp Ile Asp Asp Lys Ile Ile Lys Arg Ala Arg Gln Asn His Leu Phe Glu Gln Tyr Arg Glu Lys Arg Pro Glu Ala Ala Gln Leu Leu Glu Asp Val Gln Ala Ala Leu Lys Pro Phe Ser Val Lys Leu Asn Glu Thr Thr Asp Pro Asp Lys Lys Gln Met Leu Glu Arg Ile Gln His Ala Val Gln Leu Ala Thr Glu Pro Leu Glu Lys Ala Val Gln Ser Arg Leu Thr Gly Glu Glu Val Asn Ser Cys Val Glu Val Leu Leu Glu Glu Ala Lys Asp Leu Leu Ser Asp Trp Leu Asp Ser Thr Leu Gly Cys Asp Val Thr Asp Asn Ser Ile Phe Ser Lys Leu Pro Lys Phe Trp Glu Gly Asp Phe His Arg Asp Met Glu Ala Leu Asn Val Leu Pro Pro Asp Val Leu Thr Arg Val Ser Glu Tyr Val Pro Glu Ile Val Asn Phe Val Gln Lys Ile Val Asp Asn Gly Tyr Gly Tyr Val Ser Asn Gly Ser Val Tyr Phe Asp Thr Ala Lys Phe Ala Ser Ser Glu Lys His Ser Tyr Gly Lys Leu Val Pro Glu Ala Val Gly Asp Gln Lys Ala Leu Gln Glu Gly Glu Gly Asp Leu Ser Ile Ser Ala Asp Arg Leu Ser Glu Lys Arg Ser Pro Asn Asp Phe Ala Leu Trp Lys Ala Ser Lys Pro Gly Glu Pro Ser Trp Pro Cys Pro Trp GIy Lys Gly Arg Pro Gly Trp His Ile Glu Cys Ser Ala Met Ala Gly Thr Leu Leu Gly Ala Ser Met Asp Ile His Gly Gly Gly Phe Asp Leu Arg Phe Pro His His Asp Asn Glu Leu Ala Gln Ser Glu Ala Tyr Phe Glu Asn Asp Cys Trp Val Arg Tyr Phe Leu His Thr Gly His Leu Thr Ile Ala Gly Cys Lys Met Ser Lys Ser Leu Lys Asn Phe Ile Thr Ile Lys Asp Ala Leu Lys Lys His Ser Ala Arg Gln Leu Arg Leu Ala Phe Leu Met His Ser Trp Lys Asp Thr Leu Asp Tyr Ser Ser Asn Thr Met Glu Ser Ala Leu Gln Tyr Glu Lys Phe Leu Asn Glu Phe Phe Leu Asn Val Lys Asp Ile Leu Arg Ala Pro Val Asp Ile Thr Gly Gln Phe Glu Lys Trp Gly Glu Glu Glu Ala Glu Leu Asn Lys Asn Phe Tyr Asp Lys Lys Thr Ala Ile His Lys Ala Leu Cys Asp Asn Val Asp Thr Arg Thr Val Met Glu Glu Met Arg Ala Leu Val Ser Gln Cys Asn Leu Tyr Met Ala Ala Arg Lys Ala Val Arg Lys Arg Pro Asn Gln Ala Leu Leu Glu Asn Ile Ala Leu Tyr Leu Thr His Met Leu Lys IIe Phe Gly Ala Val Glu Glu Asp Ser Ser Leu Gly Phe Pro Val Gly Gly Pro Gly Thr Ser Leu Ser Leu Glu Ala Thr Val Met Pro Tyr Leu Gln Val Leu Ser Glu Phe Arg Glu Gly Val Arg Lys Ile Ala Arg Glu Gln Lys Val Pro Glu Ile Leu Gln Leu Ser Asp Ala Leu Arg Asp Asn Ile Leu Pro Glu Leu Gly Val Arg Phe Glu Asp His Glu Gly Leu Pro Thr Val Val Lys Leu Val Asp Arg Asn Thr Leu Leu Lys Glu Arg Glu Glu Lys Arg Arg Val Glu Glu Glu Lys Arg Lys Lys Lys Glu Glu Ala Ala Arg Arg Lys Gln Glu Gln Glu Ala Ala Lys Leu Ala Lys Met Lys Ile Pro Pro Ser Glu Met Phe Leu Ser Glu Thr Asp Lys Tyr Ser Lys Phe Asp Glu Asn Gly Leu Pro Thr His Asp Met Glu Gly Lys Glu Leu Ser Lys Gly Gln Ala Lys Lys Leu Lys Lys Leu Phe Glu Ala Gln Glu Lys Leu Tyr Lys Glu Tyr Leu Gln Met Ala Gln Asn Gly Ser Phe Gln <210> 13 <211> 328 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1747456CD1 <400> 13 Met Glu Ala Asn Gly Ser Gln Gly Thr Ser Gly Ser Ala Asn Asp Ser Gln His Asp Pro Gly Lys Met Phe Ile Gly Gly Leu Ser Trp Gln Thr Ser Pro Asp Ser Leu Arg Asp Tyr Phe Ser Lys Phe Gly Glu Ile Arg Glu Cys Met Val Met Arg Asp Pro Thr Thr Lys Arg Ser Arg Giy Phe Gly Phe Val Thr Phe Ala Asp Pro Ala Ser Val Asp Lys Val Leu Gly Gln Pro His His Glu Leu Asp Ser Lys Thr Ile Asp Pro Lys Val Ala Phe Pro Arg Arg Ala Gln Pro Lys Met Val Thr Arg Thr Lys Lys Ile Phe Val Gly Gly Leu Ser Ala Asn Thr Val Val Glu Asp Val Lys Gln Tyr Phe Glu Gln Phe Gly Lys Val Glu Asp Ala Met Leu Met Phe Asp Lys Thr Thr Asn Arg His Arg Gly Phe Gly Phe Val Thr Phe Glu Asn Glu Asp Val Val Glu Lys Val Cys Glu Ile His Phe His Glu Ile Asn Asn Lys Met Val Glu Cys Lys Lys Ala Gln Pro Lys Glu Val Met Phe Pro Pro Gly Thr Arg Gly Arg Ala Arg Gly Leu Pro Tyr Thr Met Asp Ala Phe Met Leu Gly Met Gly Met Leu Gly Tyr Pro Asn Phe Val Ala Thr Tyr Gly Arg Gly Tyr Pro Gly Phe Ala Pro Ser Tyr Gly Tyr Gln Phe Pro Gly Phe Pro Ala Ala Ala Tyr Gly Pro Val Ala Ala Ala Ala Val Ala Ala Ala Arg Gly Ser Gly Ser Asn Pro Ala Arg Pro Gly Gly Phe Pro Gly Ala Asn Ser Pro Gly Pro Val Ala Asp Leu Tyr Gly Pro Ala Ser Gln Asp Ser Gly Val Gly Asn Tyr Ile Ser Ala Ala Ser Pro Gln Pro Gly Ser Gly Phe Gly His Gly Ile Ala Gly Pro Leu Ile Ala Thr Ala Phe Thr Asn Gly Tyr His <210> 14 <211> 563 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1748626CD1 <400> 14 Met Pro Glu Asp Asp Gln Arg Ala Thr Arg Asn Leu Phe Ile Gly Asn Leu Asp His Ser Val Ser Glu Val Glu Leu Arg Arg Ala Phe Glu Lys Tyr Gly Ile Ile Glu Glu Val Val Ile Lys Arg Pro Ala Arg Gly Gln Gly Gly Ala Tyr Ala Phe Leu Lys Phe Gln Asn Leu Asp Met Ala His Arg Ala Lys Val Ala Met Ser Gly Arg Val Ile Gly Arg Asn Pro Ile Lys Ile Gly Tyr Gly Lys Ala Asn Pro Thr Thr Arg Leu Trp Val Gly Gly Leu Gly Pro Asn Thr Ser Leu Ala Ala Leu Ala Arg Glu Phe Asp Arg Phe Gly Ser Ile Arg Thr Ile Asp His Val Lys Gly Asp Ser Phe Ala Tyr Ile Gln Tyr Glu Ser Leu Asp Ala Ala Gln Ala Ala Cys Ala Lys Met Arg Gly Phe Pro Leu Gly Gly Pro Asp Arg Arg Leu Arg Val Asp Phe Ala Lys Ala Glu Glu Thr Arg Tyr Pro Gln Gln Tyr Gln Pro Ser Pro Leu Pro Val His Tyr Glu Leu Leu Thr Asp Gly Tyr Thr Arg His Arg Asn Leu Asp Ala Asp Leu Val Arg Asp Arg Thr Pro Pro His Leu Leu Tyr Ser Asp Arg Asp Arg Thr Phe Leu Glu Gly Asp Trp Thr Ser Pro Ser Lys Ser Ser Asp Arg Arg Asn Ser Leu Glu Gly Tyr Ser Arg Ser Val Arg Ser Arg Ser Gly Glu Arg Trp Gly Ala Asp Gly Asp Arg Gly Leu Pro Lys Pro Trp Glu Glu Arg Arg Lys Arg Arg Ser Leu Ser Ser Asp Arg Gly Arg Thr Thr His Ser Pro Tyr Glu Glu Arg Ser Arg Thr Lys Gly Ser Gly Gln Gln Ser Glu Arg Gly Ser Asp Arg Thr Pro Glu Arg Ser Arg Lys Glu Asn His Ser Ser Glu Gly Thr Lys Glu Ser Ser Ser Asn Ser Leu Ser Asn Ser Arg His Gly Ala Glu Glu Arg Gly His His His His His His Glu Ala Ala Asp Ser Ser His Gly Lys Lys Ala Arg Asp Ser Glu Arg Asn His Arg Thr Thr Glu Ala Glu Pro Lys Pro Leu Glu Glu Pro Lys His Glu Thr Lys Lys Leu Lys Asn Leu Ser Glu Tyr Ala Gln Thr Leu Gln Leu Gly Trp Asn Gly Leu Leu Val Leu Lys Asn Ser Cys Phe Pro Thr Ser Met His Ile Leu Glu Gly Asp Gln Gly Val Ile Ser Ser Leu Leu Lys Asp His Thr Ser Gly Ser Lys Leu Thr Gln Leu Lys Ile Ala Gln Arg Leu Arg Leu Asp Gln Pro Lys Leu Asp Glu Val Thr Arg Arg Ile Lys Gln Gly Ser Pro Asn Gly Tyr Ala Val Leu Leu Ala Thr Gln Ala Thr Pro Ser Gly Leu Gly Thr Glu Gly Met Pro Thr Val Glu Pro Gly Leu Gln Arg Arg Leu Leu Arg Asn Leu Val Ser Tyr Leu Lys Gln Lys Gln Ala Ala Gly Val Ile Ser Leu Pro Val Gly Gly Ser Lys Gly Arg Asp Gly Thr Gly Met Leu Tyr Ala Phe Pro Pro Cys Asp Phe Ser Gln Gln Tyr Leu GIn Ser Ala Leu Arg Thr Leu Gly Lys Leu Glu Glu Glu His Met Val Ile Val Ile Val Arg Asp Thr Ala <210> 15 <211> 153 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1879135CD1 <400> 15 Met Met Ser Gln Ser Gly His Glu Tyr Asp Pro Ile Asn Tyr Met Lys Lys Pro Leu Gly Pro Pro Pro Pro Ser Tyr Thr Cys Phe Arg Cys Gly Lys Pro Gly His Tyr Ile Lys Asn Cys Pro Thr Asn Gly Asp Lys Asn Phe Glu Ser Gly Pro Arg Ile Lys Lys Ser Thr Gly Ile Pro Arg Ser Phe Met Met Glu Val Lys Asp Pro Asn Met Lys Gly Ala Met Leu Thr Asn Thr Gly Lys Tyr Ala Ile Pro Thr Ile Asp Ala Glu Ala Tyr Ala Ile Gly Lys Lys Glu Lys Pro Pro Phe Leu Pro Glu Glu Pro Ser Ser Ser Ser Glu Glu Asp Asp Pro Ile Pro Asp Glu Leu Leu Cys Leu Ile Cys Lys Asp Ile Met Thr Asp Ala Val Val Ile Pro Cys Cys Gly Asn Ser Tyr Cys Asp Glu Cys Lys Lys Cys <210> 16 <211> 286 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2073417CD1 <400> 16 Met Ser Trp Leu Leu Phe Leu Ala His Arg Val Ala Leu Ala Ala Leu Pro Cys Arg Arg Gly Ser Arg Gly Phe Gly Met Phe Tyr Ala Val Arg Arg Gly Arg Lys Thr Gly Val Phe Leu Thr Trp Asn Glu Cys Arg Ala Gln Val Asp Arg Phe Pro Ala Ala Arg Phe Lys Lys Phe Ala Thr Glu Asp Glu Ala Trp Ala Phe Val Arg Lys Ser Ala Ser Pro Glu Val Ser Glu Gly His Glu Asn Gln His Gly Gln Glu Ser Glu Ala Lys Ala Ser Lys Arg Leu Arg Glu Pro Leu Asp Gly Asp Gly His Glu Ser Ala Glu Pro Tyr Ala Lys His Met Lys Pro Ser Met Glu Pro Ala Pro Pro Val Ser Arg Asp Thr Phe Ser Tyr Met Gly Asp Phe Val Val Val Tyr Thr Asp Gly Cys Cys Ser Ser Asn Gly Arg Arg Arg Pro Arg Ala Gly Ile Gly Val Tyr Trp Gly Pro Gly His Pro Leu Asn Val Gly Ile Arg Leu Pro Gly Arg Gln Thr Asn Gln Arg Ala Glu Ile His Ala Ala Cys Lys Ala Ile Glu Gln Ala Lys Thr Gln Asn Ile Asn Lys Leu Val Leu Tyr Thr Asp Ser Met Phe Thr Ile Asn Gly Ile Thr Asn Trp Val Gln Gly Trp Lys Lys Asn Gly Trp Lys Thr Ser Ala Gly Lys Glu Val Ile Asn Lys Glu Asp Phe Val Ala Leu Glu Arg Leu Thr Gln Gly Met Asp Ile Gln Trp Met His Val Pro GIy His Ser Gly Phe Ile Gly Asn Glu Glu Ala Asp Arg Leu Ala Arg Glu Gly Ala .Lys Gln Ser Glu Asp <210> 17 <211> 537 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2129080CD1 <400> 17 Met Leu Ala Arg Glu Thr Tyr Glu Glu Asp Arg Glu Tyr Glu Ser Gln Ala Lys Arg Leu Lys Thr Glu Glu Gly Glu Ile Asp Tyr Ser Ala Glu Glu Gly Glu Asn Arg Arg Glu Ala Thr Pro Arg Gly Gly Gly Asp Gly Gly Gly Gly Gly Arg Ser Phe Ser Gln Pro Glu Ala Gly Gly Ser His His Lys Val Ser Val Ser Pro Val Val His Val Arg Gly Leu Cys Glu Ser Val Val Glu Ala Asp Leu Val Glu Ala Leu Glu Lys Phe Gly Thr Ile Cys Tyr Val Met Met Met Pro Phe Lys Arg Gln Ala Leu Val Glu Phe Glu Asn Ile Asp Ser Ala Lys Glu Cys Val Thr Phe Ala Ala Asp Glu Pro Val Tyr Ile Ala Gly Gln Gln Ala Phe Phe Asn Tyr Ser Thr Ser Lys Arg Ile Thr Arg Pro Gly Asn Thr Asp Asp Pro Ser Gly Gly Asn Lys Val Leu Leu Leu Ser Ile Gln Asn Pro Leu Tyr Pro Ile Thr Val Asp Val Leu Tyr Thr Val Cys Asn Pro Val Gly Lys Val Gln Arg Ile Val Ile Phe Lys Arg Asn Gly Ile Gln Ala Met Val Glu Phe Glu Ser Val Leu Cys Ala Gln Lys Ala Lys Ala Ala Leu Asn Gly Ala Asp Ile Tyr Ala Gly Cys Cys Thr Leu Lys Ile Glu Tyr Ala Arg Pro Thr Arg Leu Asn Val Ile Arg Asn Asp Asn Asp Ser Trp Asp Tyr Thr Lys Pro Tyr Leu Gly Arg Arg Asp Arg Gly Lys Gly Arg Gln Arg Gln Ala Ile Leu Gly Glu His Pro Ser Ser Phe Arg His Asp Gly Tyr Gly Ser His Gly Pro Leu Leu Pro Leu Pro Ser Arg Tyr Arg Met Gly Ser Arg Asp Thr Pro Glu Leu Val Ala Tyr Pro Leu Pro Gln Ala Ser Ser Ser Tyr Met His Gly Gly Asn Pro Ser Gly Ser Val Val Met Val Ser Gly Leu His Gln Leu Lys Met Asn Cys Ser Arg Val Phe Asn Leu Phe Cys Leu Tyr Gly Asn Ile Glu Lys Val Lys Phe Met Lys Thr Ile Pro Gly Thr Ala Leu Val Glu Met Gly Asp Glu Tyr Ala Val Glu Arg Ala Val Thr His Leu Asn Asn Val Lys Leu Phe Gly Lys Arg Leu Asn Val Cys Val Ser Lys Gln His Ser Val Val Pro Ser Gln Ile Phe Glu Leu Glu Asp Gly Thr Ser Ser Tyr Lys Asp Phe Ala Met Ser Lys Asn Asn Arg Phe Thr Ser Ala Gly Gln Ala Ser Lys Asn Ile Ile Gln Pro Pro Ser Cys Val Leu His Tyr Tyr Asn Val Pro Leu Cys Val Thr Glu Glu Thr Phe Thr Lys Leu Cys Asn Asp His Glu Val Leu Thr Phe Ile Lys Tyr Lys Val Phe Asp Ala Lys Pro Ser Ala Lys Thr Leu Ser Gly Leu Leu Glu Trp Glu Cys Lys Thr Asp Ala Val Glu Ala Leu Thr Ala Leu Asn His Tyr Gln Ile Arg Val Pro Asn Gly Ser Asn Pro Tyr Thr Leu Lys Leu Cys Phe Ser Thr Ser Ser His Leu <210> 18 <211> 163 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2472867CD1 <400> 18 Met Arg Ile Glu Lys Cys Tyr Phe Cys Ser Gly Pro Ile Tyr Pro Gly His Gly Met Met Phe Val Arg Asn Asp Cys Lys Val Phe Arg Phe Cys Lys Ser Lys Cys His Lys Asn Phe Lys Lys Lys Arg Asn Pro Arg Lys Val Arg Trp Thr Lys Ala Phe Arg Lys Ala Ala Gly Lys Glu Leu Thr Val Asp Asn Ser Phe Glu Phe Glu Lys Arg Arg Asn Glu Pro Ile Lys Tyr Gln Arg Glu Leu Trp Asn Lys Thr Ile Asp Ala Met Lys Arg Val Glu Glu Ile Lys Gln Lys Arg Gln Ala Lys Phe Ile Met Asn Arg Leu Lys Lys Asn Lys Glu Leu Gln Lys Val Gln Asp Ile Lys Glu Val Lys Gln Asn Ile His Leu Ile Arg Ala Pro Leu Ala Gly Lys Gly Lys Gln Leu Glu Glu Lys Met Val Gln Gln Leu Gln Glu Asp Val Asp Met Glu Asp Ala Pro <210> 19 <211> 178 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2764755CD1 <400> 19 Met Ser Ser Phe Ser Arg Ala Pro Gln Gln Trp Ala Thr Phe Ala Arg Ile Trp Tyr Leu Leu Asp Gly Lys Met Gln Pro Pro Gly Lys Leu Ala Ala Met Ala Ser Ile Arg Leu Gln Gly Leu His Lys Pro Val Tyr His Ala Leu Ser Asp Cys Gly Asp His Val Val Ile Met Asn Thr Arg His Ile Ala Phe Ser Gly Asn Lys Trp Glu Gln Lys Val Tyr Ser Ser His Thr Gly Tyr Pro Gly Gly Phe Arg Gln Val Thr Ala Ala Gln Leu His Leu Arg Asp Pro Val Ala Ile Val Lys Leu Ala Ile Tyr Gly Met Leu Pro Lys Asn Leu His Arg Arg Thr Met Met GIu Arg Leu His Leu Phe Pro Asp Glu Tyr Ile Pro Glu Asp Ile Leu Lys Asn Leu Val Glu Glu Leu Pro Gln Pro Arg Lys Ile Pro Lys Arg Leu Asp Glu Tyr Thr Gln Glu Glu Ile Asp Ala Phe Pro Arg Leu Trp Thr Pro Pro Glu Asp Tyr Arg Leu <210> 20 <211> 140 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2875939CD1 <400> 20 Met Ala Glu Asn Arg Glu Pro Arg Gly Ala Val Glu Ala Glu Leu Asp Pro Val Glu Tyr Thr Leu Arg Lys Arg Leu Pro Ser Arg Leu Pro Arg Arg Pro Asn Asp Ile Tyr Val Asn Met Lys Thr Asp Phe Lys Ala Gln Leu Ala Arg Cys Gln Lys Leu Leu Asp Gly Gly Ala Arg Gly Gln Asn Ala Cys Ser Glu Ile Tyr Ile His Gly Leu Gly Leu Ala Ile Asn Arg Ala Ile Asn Ile Ala Leu Gln Leu Gln Ala Gly Ser Phe Gly Ser Leu Gln Val Ala Ala Asn Thr Ser Thr Val Glu Leu Val Asp Glu Leu Glu Pro Glu Thr Asp Thr Arg Glu Pro Leu Thr Arg Ile Arg Asn Asn Ser Ala Ile His Ile Arg Val Phe Arg Val Thr Pro Lys <210> 21 <211> 209 <212> PRT
w213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 3591363CD1 <400> 21 Met Ala Ala Ala Met Ala Ala Ser Ser Leu Thr Val Thr Leu Gly Arg Leu Ala Ser Ala Cys Ser His Ser Ile Leu Arg Pro Ser Gly Pro Gly Ala Ala Ser Leu Trp Ser Ala Ser Arg Arg Phe Asn Ser Gln Ser Thr Ser Tyr Leu Pro Gly Tyr Val Pro Lys Thr Ser Leu Ser Ser Pro Pro Trp Pro Glu Val Val Leu Pro Asp Pro Val Glu Glu Thr Arg His His Ala Glu Val Val Lys Lys Val Asn Glu Met Ile Val Thr Gly Gln Tyr Gly Arg Leu Phe Ala Val Val His Phe Ala Ser Arg Gln Trp Lys Val Thr Ser Glu Asp Leu Ile Leu Ile Gly Asn Glu Leu Asp Leu Ala Cys Gly Glu Arg Ile Arg Leu Glu Lys Val Leu Leu Val Gly Ala Asp Asn Phe Thr Leu Leu Gly Lys Pro Leu Leu Gly Lys Asp Leu Val Arg Val Glu Ala Thr Val Ile Glu Lys Thr Glu Ser Trp Pro Arg Ile Ile Met Arg Phe Arg Lys Arg Lys Asn Phe Lys Lys Lys Arg Ile Val Thr Thr Pro Gln Thr Val Leu Arg Ile Asn Ser Ile Glu Ile Ala Pro Cys Leu Leu <210> 22 <211> 162 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 3702292CD1 <400> 22 Met Ala Pro Lys Ala Lys Glu Ala Pro Ala His Pro Lys Ala Glu Ala Lys Ala Lys Ala Leu Lys Ala Lys Lys Ala Val Leu Lys Gly Val Arg Ser His Thr Gln Lys Gln Lys Ile Arg Met Ser Leu Thr Phe Arg Arg Pro Lys Thr Leu Arg Leu Arg Arg Gln Pro Arg Tyr Pro Arg Lys Ser Thr Pro Arg Arg Asn Lys Leu Gly His Tyr Ala Ile Ile Lys Phe Pro Leu Ala Thr Glu Ser Ala Val Lys Lys Ile Glu Glu Asn Asn Thr Leu Val Phe Thr Val Asp Val Lys Ala Asn Lys His Gln Ile Arg Gln Ala Val Lys Lys Leu Tyr Asp Ser Asp Val Ala Lys Val Thr Thr Leu Ile Cys Pro Asp Lys Glu Asn Lys Ala Tyr Val Arg Leu Ala Pro Asp Tyr Asp Ala Phe Asp Val Val Thr Lys Leu Gly Ser Pro Lys Leu Ser Pro Ala Gly <210> 23 <211> 623 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 3778908CD1 <400> 23 Met Ala Thr Glu His Val Asn Gly Asn Gly Thr Glu Glu Pro Met Asp Thr Thr Ser Ala Val Ile His Ser Glu Asn Phe Gln Thr Leu Leu Asp Ala Gly Leu Pro Gln Lys Val Ala Glu Lys Leu Asp Glu Ile Tyr Val Ala Gly Leu Val Ala His Ser Asp Leu Asp Glu Arg Ala Ile Glu Ala Leu Lys Glu Phe Asn Glu Asp Gly Ala Leu Ala Val Leu Gln Gln Phe Lys Asp Ser Asp Leu Ser His Val Gln Asn Lys Ser Ala Phe Leu Cys Gly Val Met Lys Thr Tyr Arg Gln Arg Glu Lys Gln Gly Thr Lys Val Ala Asp Ser Ser Lys Gly Pro Asp Glu Ala Lys Ile Lys Ala Leu Leu Glu Arg Thr Gly Tyr Thr Leu Asp Val Thr Thr Gly Gln Arg Lys Tyr Gly Gly Pro Pro Pro Asp Ser Val Tyr Ser Gly Gln Gln Pro Ser Val Gly Thr Glu Ile Phe Val Gly Lys Ile Pro Arg Asp Leu Phe Glu Asp Glu Leu Val Pro Leu Phe Glu Lys Ala Gly Pro Ile Trp Asp Leu Arg Leu Met Met Asp Pro Leu Thr Gly Leu Asn Arg Gly Tyr Ala Phe Val Thr Phe Cys Thr Lys Giu Ala Ala Gln Glu Ala Val Lys Leu Tyr Asn Asn His Glu Ile Arg Ser Gly Lys His Ile Gly Val Cys Ile Ser Val Ala Asn Asn Arg Leu Phe Val Gly Ser Ile Pro Lys Ser Lys Thr Lys Glu Gln Ile Leu Glu Glu Phe Ser Lys Val Thr Glu Gly Leu Thr Asp Val Ile Leu Tyr His Gln Pro Asp Asp Lys Lys Lys Asn Arg Gly Phe Cys Phe Leu Glu Tyr Glu Asp His Lys Thr Ala Ala Gln Ala Arg Arg Arg Leu Met Ser Gly Lys Val Lys Val Trp Gly Asn Val Gly Thr Val Glu Trp Ala Asp Pro Ile Glu Asp Pro Asp Pro Glu Val Met Ala Lys Val Lys Val Leu Phe Val Arg Asn Leu Ala Asn Thr Val Thr Glu Glu Ile Leu Glu Lys Ala Phe Ser Gln Phe Gly Lys Leu Glu Arg Val Lys Lys Leu Lys Asp Tyr Ala Phe Ile His Phe Asp Glu Arg Asp Gly Ala Val Lys Ala Met Glu Glu Met Asn GIy Lys Asp Leu Glu Gly Glu Asn Ile Glu Ile Val Phe Ala Lys Pro Pro Asp Gln Lys Arg Lys Glu Arg Lys Ala Gln Arg Gln Ala Ala Lys Asn Gln Met Tyr Asp Asp Tyr Tyr Tyr Tyr Gly Pro Pro His Met Pro Pro Pro Thr Arg Gly Arg Gly Arg Gly Gly Arg Gly Gly Tyr Gly Tyr Pro Pro Asp Tyr Tyr Gly Tyr Glu Asp Tyr Tyr Asp Tyr Tyr Gly Tyr Asp Tyr His Asn Tyr Arg Gly Gly Tyr Glu Asp Pro Tyr Tyr Gly Tyr Glu Asp Phe Gln Val Gly Ala Arg Gly Arg Gly Gly Arg Gly Ala Arg Gly Ala Ala Pro Ser Arg Gly Arg Gly Ala Ala Pro Pro Arg Gly Arg Ala Gly Tyr Ser Gln Arg Gly Gly Pro Gly Ser Ala Arg Gly Val Arg Gly Ala Arg Gly Gly Ala Gln Gln Gln Arg Gly Arg Gly Val Arg Gly Ala Arg Gly Gly Arg Gly Gly Asn Val Gly Gly Lys Arg Lys Ala Asp Gly Tyr Asn Gln Pro Asp Ser Lys Arg Arg Gln Thr Asn Asn Gln Asn Trp Gly Ser Gln Pro Ile Ala Gln Gln Pro Leu Gln Gly Gly Asp His Ser Gly Asn Tyr Gly Tyr Lys Ser Glu Asn Gln Glu Phe Tyr Gln Asp Thr Phe Gly Gln Gln Trp Lys <220> 24 <211> 786 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 4163642CD1 <400> 24 Met Ser Phe Ser Arg Ala Leu Leu Trp Ala Arg Leu Pro Ala Gly Arg Gln Ala Gly His Arg Ala Ala Ile Cys Ser Ala Leu Arg Pro His Phe Gly Pro Phe Pro Gly Val Leu Gly Gln Val Ser Val Leu Ala Thr Ala Ser Ser Ser Ala Ser Gly Gly Ser Lys Ile Pro Asn Thr Ser Leu Phe Val Pro Leu Thr Val Lys Pro Gln Gly Pro Ser Ala Asp Gly Asp Val Gly Ala Glu Leu Thr Arg Pro Leu Asp Lys Asn Glu Val Lys Lys Val Leu Asp Lys Phe Tyr Lys Arg Lys Glu Ile Gln Lys Leu Gly Ala Asp Tyr Gly Leu Asp Ala Arg Leu Phe His Gln Ala Phe Ile Ser Phe Arg Asn Tyr Ile Met Gln Ser His Ser Leu Asp Val Asp Ile His Ile Val Leu Asn Asp Ile Cys Phe Gly Ala Ala His Ala Asp Asp Leu Phe Pro Phe Phe Leu Arg His Ala Lys Gln Ile Phe Pro Val Leu Asp Cys Lys Asp Asp Leu Arg Lys Ile Ser Asp Leu Arg Ile Pro Pro Asn Trp Tyr Pro Asp Ala Arg Ala Met Gln Arg Lys Ile Ile Phe His Ser Gly Pro Thr Asn Ser Gly Lys Thr Tyr His Ala Ile Gln Lys Tyr Phe Ser Ala Lys Ser Gly Val Tyr Cys Gly Pro Leu Lys Leu Leu Ala His Glu Ile Phe Glu Lys Ser Asn Ala Ala Gly Val Pro Cys Asp Leu Val Thr Gly Glu Glu Arg Val Thr Val Gln Pro Asn Gly Lys Gln Ala Ser His Val Ser Cys Thr Val Glu Met Cys Ser Val Thr Thr Pro Tyr Glu Val Ala Val Ile Asp Glu Ile Gln Met Ile Arg Asp Pro Ala Arg Gly Trp Ala Trp Thr Arg Ala Leu Leu Gly Leu Cys Ala Glu Glu Val His Leu Cys Gly Glu Pro Ala Ala Ile Asp Leu Val Met Glu Leu Met Tyr Thr Thr Gly Glu Glu Val Glu Val Arg Asp Tyr Lys Arg Leu Thr Pro Ile Ser Val Leu Asp His Ala Leu Glu Ser Leu Asp Asn Leu Arg Pro Gly Asp Cys Ile Val Cys Phe Ser Lys Asn Asp Ile Tyr Ser Val Ser Arg Gln Ile Glu Ile Arg Gly Leu Glu Ser Ala Val Ile Tyr Gly Ser Leu Pro Pro Gly Thr Lys Leu Ala Gln Ala Lys Lys Phe Asn Asp Pro Asn Asp Pro Cys Lys Ile Leu Val Ala Thr Asp Ala Ile Gly Met Gly Leu Asn Leu Ser Ile Arg Arg Ile Ile Phe Tyr Ser Leu Ile Lys Pro Ser Ile Asn Glu Lys Gly Glu Arg Glu Leu Glu Pro Ile Thr Thr Ser Gln Ala Leu Gln Ile Ala Gly Arg Ala Gly Arg Phe Ser Ser Arg Phe Lys Glu Gly Glu Val Thr Thr Met Asn His Glu Asp Leu Ser Leu Leu Lys Glu Ile Leu Lys Arg Pro Val Asp Pro Ile Arg Ala Ala Gly Leu His Pro Thr Ala Glu Gln Ile Glu Met Phe Ala Tyr His Leu Pro Asp Ala Thr Leu Ser Asn Leu Ile Asp Ile Phe Val Asp Phe Ser Gln Val Asp Gly Gln Tyr Phe Val Cys Asn Met Asp Asp Phe Lys Phe Ser Ala Glu Leu Ile Gln His Ile Pro Leu Ser Leu Arg Val Arg Tyr Val Phe Cys Thr Ala Pro Ile Asn Lys Lys Gln Pro Phe Val Cys Ser Ser Leu Leu Gln Phe Ala Arg Gln Tyr Ser Arg Asn Glu Pro Leu Thr Phe Ala Trp Leu Arg Arg Tyr Ile Lys Trp Pro Leu Leu Pro Pro Lys Asn Ile Lys Asp Leu Met Asp Leu Glu Ala Val His Asp Val Leu Asp Leu Tyr Leu Trp Leu Ser Tyr Arg Phe Met Asp Met Phe Pro Asp Ala Ser Leu Ile Arg Asp Leu Gln Lys Glu Leu Asp Gly Ile Ile Gln Asp Gly Val His Asn Ile Thr Lys Leu Ile Lys Met Ser Glu Thr His Lys Leu Leu Asn Leu Glu Gly Phe Pro Ser Gly Ser Gln Ser Arg Leu Ser Gly Thr Leu Lys Ser Gln Ala Arg Arg Thr Arg Gly Thr Lys Ala Leu Gly Ser Lys Ala Thr Glu Pro Pro Ser Pro Asp Ala Gly Glu Leu Ser Leu Ala Ser Arg Leu Val Gln Gln Gly Leu Leu Thr Pro Asp Met Leu Lys Gln Leu Glu Lys Glu Trp Met Thr Gln Gln Thr Glu His Asn Lys Glu Lys Thr Glu Ser Gly Thr His Pro Lys Gly Thr Arg Arg Lys Lys Lys Glu Pro Asp Ser Asp <210> 25 <211> 260 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 4906154CD1 <400> 25 Met Thr Pro Val Gln Arg Gly Gly Pro Gly Ala Leu Val Ala Leu Gly Trp Gly Arg Arg Lys Ala Glu Asp Lys Glu Trp Met Pro Val Thr Lys Leu Gly Arg Leu Val Lys Asp Met Lys Ile Lys Ser Leu Glu Glu Ile Tyr Leu Phe Ser Leu Pro Ile Lys Glu Ser Glu Ile Ile Asp Phe Phe Leu Gly Ala Ser Leu Lys Asp Glu Val Leu Lys Ile Met Pro Val Gln Lys Gln Thr Arg Ala Gly Gln Arg Thr Arg Phe Lys Ala Phe Val Ala Ile Gly Asp Tyr Asn Gly His Val Gly Leu Gly Val Lys Cys Ser Lys Glu Val Ala Thr Ala Ile Arg Gly Ala Ile Ile Leu Ala Lys Leu Ser Ile Val Pro Val Arg Arg Gly Tyr Trp Gly Asn Lys Ile Gly Lys Pro His Thr Val Pro Cys Lys Val Thr Gly Arg Cys Gly Ser Val Leu Val Arg Leu Ile Pro Ala Pro Arg Gly Thr Gly Ile Val Ser Ala Pro Val Pro Lys Lys Leu Leu Met Met Ala Gly Ile Asp Asp Cys Tyr Thr Ser Ala Arg Gly Cys Thr Ala Thr Leu Gly Asn Phe Ala Lys Ala Thr Phe Asp Ala Ile Ser Lys Thr Tyr Ser Tyr Leu Thr Pro Asp Leu Trp Lys Glu Thr Val Phe Thr Lys Ser Pro Tyr Gln Glu Phe Thr Asp His Leu Val Lys Thr His Thr Arg Val Ser Val Gln Arg Thr Gln Ala Pro Ala Val Ala Thr Thr <210> 26 <211> 1872 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 399781CB1 <400> 26 gccctctagc tgtgtgtgtc tgaggctcgg ccgcctgagc cgcggacggt ttgctgagcc 60 cgttagtgcg cccggccgag acacgccgcc gccatgtccc gctacctgcg tccccccaac 120 acgtctctgt tcgtcaggaa cgtggccgac gacaccaggt ctgaagactt gcggcgtgaa 180 tttggtcgtt atggtcctat agttgatgtg tatgttccac ttgatttcta cactcgccgt 240 ccaagaggat ttgcttatgt tcaatttgag gatgttcgtg atgctgaaga cgctttacat 300 aatttggaca gaaagtggat ttgtggacgg cagattgaaa tacagtttgc ccagggggat 360 cgaaagacac caaatcagat gaaagccaag gaagggagga atgtgtacag ttcttcacgc 420 tatgatgatt atgacagata cagacgttct agaagccgaa gttatgaaag gaggagatca 480 agaagtcggt cttttgatta caactataga agatcgtata gtcctagaaa cagtagaccg 540 actggaagac cacggcgtag agaagccatt ccgacaatga tagaccaaac tgcagctgga 600 atacccagta cagttctgct tactacactt caagaaagat ctgaaagcgg aaaaagaacc 660 aaagaagggc agttcaagcg accaaagggt gggtggaagg tgctgcagta tgaatactgt 720 acgaatattt tgactctggt ctgaaaagat aaaagaatgt tatcgaaaac tacatggaat 780 aattgaagtc ccttcaagtt tgaaagtaag cattttagga caaataaaag gaaattcaac 840 tttgtacttg tggaaactaa tccctaaata tgaataggtt tatattgatt catgggtaac 900 aggtccataa taaattattg gaaactagga tgtctgaata tcaaggaaga cagccatagt 960 ctcttacagt gcctctgttg gtctgtctca aactgaattg ggtgggaaaa ggtatggtcc 1020 aatataaaag ttccattttt gccattattg gcaaatcttg cctttgttta ttttggtgcc 1080 agtgttttct gcttaatcat ttgctttgtt ggcatctgtg tttatttact tgtacaccac 1140 atgcagttta catctgtctt aactactcct tcccaggtaa attccaatta tatttgacat 1200 ccagctaaga gggcccatct cttctcacct ctttcctagt cagtatattc agcaaatatt 1260 tattgagccc ttactgtggg caaatcattg tactggataa ttgagaaaaa tagataattc 1320 ccttattcag taaatgtcta ctgagcacaa tctagtgaat cattacagta tggcctcatt 1380 gttttgtttg aggtgtgtta ttcataacaa tattttacac cattcgtatc aatgtaatta 1440 tagaacacaa tatacgatca aggataagta attgtgtggt tatctgccat ttaaaagtat 1500 ccagtatttg atcacattat tataaataat gaaaaaatga tttaatctgt aataaactgg 1560 tttattgtgc agtgactgta atatactaga gttataataa attgtttact ctgcctcacc 1620 aaacacatgc taggatataa cccccaaaat aagtatttaa ctttgcatta ggtataaagg 1680 agactgggtg ctataattag attattttga ggcagacaga gagctgttat cctaactgat 1740 ttagtatgtt ctgtaattga gaaaatgttc accaaattat actttttagt gatttacatg 1800 tacattttat aggggacatg ttctgtgtat agcgaataaa taacttttat agtatcaaaa 1860 aaaaaaaaaa as 1872 <210> 27 <211> 3834 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1806542CB1 <400> 27 ctgcggcggc ggcggcggcg ttaccggccc tcgcgctctt tcccttcctg gggcgccgac 60 cccgcccgct tgcttgcttg cttgcttgcc tgcctgcctg cctgcccggc gccacgcaag 120 agaaggtgcc aggggacgca gagcgactag aggcgcgcgg tcccggccag caccgtctct 180 ggcgttgtag ctgcggccgt ggcggaggac tacggcgaca aggacgaggg ccgctctccc 240 agctctctgc gtgccgcgcc gctccgctcc gctggctgac catctggagt gcaggctggg 300 aggcgggatg gagtgatagg gaagatgttt ataaattctt ctgtgggatc agagggcacg 360 cctattacaa ccagaaaact acaagtataa cagcgaggat ggatgaacag gctctattag 420 ggctaaatcc aaatgctgat tcagacttta gacaaagggc cctggcctat tttgagcagt 480 taaaaatttc cccagatgcc tggcaggtgt gtgcagaagc tctagcccag aggacataca 540 gtgatgatca tgtgaagttt ttctgctttc aagtactgga acatcaagtt aaatacaaat 600 actcagaact aaccactgtt caacaacagc taattaggga gacgctcata tcatggctgc 660 aagctcagat gctgaatccc caaccagaga agacctttat acgaaataaa gccgcccaag 720 tcttcgcctt gctttttgtt acagagtatc tcactaagtg gcccaagttt ttttttgaca 780 ttctctcagt agtggaccta aatccaaggg gagtagatct ctacctgcga atcctcatgg 840 ctattgattc agagttggtg gatcgtgatg tggtgcatac atcagaggag gctcgtagga 900 atactctcat aaaagatacc atgagggaac agtgcattcc aaatctggtg gaatcatggt 960 accaaatatt acaaaattat cagtttacta attctgaagt gacgtgtcag tgccttgaag 1020 tagttggggc ttatgtctct tggatagact tatcccttat agccaatgat aggtttataa 1080 atatgctgct aggtcatatg tcaatagaag ttctacggga agaagcatgt gactgtttat 1140 ttgaagttgt aaataaagga atggaccctg ttgataaaat gaaactagtg gaatctttgt 1200 gtcaagtatt acagtctgct gggtttttca gcattgacca ggaagaagat gttgacttcc 1260 tggccagatt ttctaagttg gtaaatggaa tgggacagtc attgatagtt agttggagta 1320 aattaattaa gaatggggat attaagaatg ctcaagaggc actacaagct attgaaacaa 1380 aagtggcact gatgttgcag ctactaattc atgaggatga tgatatttct tctaatatta 1440 ttggattttg ttacgattat cttcatattt tgaaacagct tacagtgctc tcggatcagc 1500 aaaaagctaa tgtagaggca atcatgttgg ccgttatgaa aaaattgact tacgatgaag 1560 aatataactt tgaaaatgag ggtgaagatg aagccatgtt tgtagaatat agaaaacaac 1620 tgaagttact gttggacagg cttgctcaag tttcaccaga gttactactg gcctctgttc 1680 gcagagtttt tagttctaca ctgcagaatt ggcagactac acggtttatg gaagttgaag 1740 tagcaataag attgctgtat atgttggcag aagctcttcc agtatctcat ggtgctcact 1800 tctcaggtga tgtttcaaaa gctagtgctt tgcaggatat gatgcgaact ctggtaacat 1860 caggagtcag ttcctatcag catacatctg tgacattgga gttcttcgaa actgttgtta 1920 gatatgaaaa gtttttcaca gttgaacctc agcacattcc atgtgtacta atggctttct 1980 tagatcacag aggtctgcgg cattccagtg caaaagttcg gagcaggacg gcttacctgt 2040 tttctagatt tgtcaaatct ctcaataagc aaatgaatcc t.ttcattgag gatattttga 2100 atagaataca agatttatta gagctttctc cacctgagaa t.ggccaccag tccttactga 2160 gcagcgatga tcaacttttt atttatgaga cagctggagt gctgattgtt aatagtgaat 2220 atccggcaga aaggaaacaa gccttaatga ggaatctgtt gactccacta atggagaagt 2280 ttaaaattct gttagaaaag ttgatgctgg cacaagatga agaaaggcaa gcctctctag 2340 cagactgtct taaccatgct gttggatttg caagtcgaac cagtaaagct ttcagcaaca 2400 aacagactgt gaaacaatgt ggctgttccg aagtttatct ggactgttta cagacattct 2460 tgccagccct cagttgtccc ttacaaaagg atattctcag aagtggagtc cgtactttcc 2520 ttcatcgaat gattatttgc ctggaggaag aagttcttcc gttcattcca tctgcttcag 2580 aacatatgct caaagattgt gaagcaaaag atctccagga gttcattcct cttatcaacc 2640 agattacggc caaattcaag atacaggtat ccccgttttt acaacagatg ttcatgcccc 2700 tgcttcatgc aatttttgaa gtgctgctcc ggccagcaga agaaaatgac cagtctgctg 2760 ctttagagaa gcagatgttg cggaggagtt actttgcttt cctgcaaaca gtcacaggca 2820 gtgggatgag cgaagttata gcaaatcaag gtgcagagaa tgtagaaaga gtgttggtta 2880 ctgttatcca aggagcagtt gaatatccag atccaattgc acagaaaaca tgttttatca 2940 tcctctcaaa gttggtagaa ctctggggag gtaaagatgg accagtggga tttgctgatt 3000 ttgtttataa gcacattgtc cccgcatgtt tcctagcacc tttaaaacaa acctttgacc 3060 tggcagatgc acaaacagta ttggctttat ctgagtgtgc agtgacactg aaaacaattc 3120 atctcaaacg gggcccagaa tgtgttcagt atcttcaaca agaatacctg ccctccttgc 3180 aagtagctcc agaaataatt caggagtttt gtcaagcgct tcagcagcct gatgctaaag 3240 tttttaaaaa ttacttaaag gtgttcttcc agagagcaaa gccctgagga ctggatttcc 3300 ctgtgcctac ttcatgatca tgaattccag ttaatttata aagaggcgat ttttgtgtgc 3360 cattcacact ggtctttttc acattgtttt gagcttattg cagtatatgt tttgggattt 3420 ttctgtaaaa tgggtgtaat tttcctaata caggtatgta acaacaaaag aagttgcctg 3480 catgccggtc caaattgttc tgtataaaga tgctcttaaa agacacaaga gttatcctag 3540 aaccttaatt cttttttatt tgaaatttta agtcaagtcc tttataaaga ccatagcagt 3600 ggaaaacagt gtacttttta aaaaattgct gaatataaaa tctttgaaaa ttttctttat 3660 gtgtgaagac acaaagtatg ggggaagaca gcaatcaaaa ctaacttttt gtagatagcc 3720 atttcatttc tttaaactgt ttcaacgcca atatgtattc tacaaaagag aatggtttta 3780 ggctccagtg ttatactttt ttttatatat atatataaaa ataaacttta cgtt 3834 <210> 28 <211> 2178 <212> DNA
<213> Homo sapiens <220>

<221> misc_feature <223> Incyte Identification No.: 2263514CB1 <400> 28 cagctcccta agcggttgtc accgctggag acggttggga gaaccgttgt ggcgagcgct 60 acacgaggca aacgacttct cccttctttg aactggaccc cgcgagcacc agagtcggcg 120 taactatcgc ctgacaggca tttaaatcaa acggtattga gatggattgg gttatgaaac 180 ataatggtcc aaatgacgct agtgatggga cagtacgact tcgtggacta ccatttggtt 240 gcagcaaaga ggaaatagtt cgagttcttt caaggtatat tgagatcttc agaagtagca 300 ggagtgaaat caaaggattt tatgatccac caagaagatt gctgggacag cgaccgggac 360 catatgatag accaatagga ggaagagggg gttattatgg agctgggcgt ggaagttatg 420 gaggttttga tgactatggt ggctataata attacggcta tgggaatgat ggctttgatg 480 acagaatgag agatggaaga ggtatgggag gacatggcta tggtggagct ggtgatgcaa 540 gttcaggttt tcatggtggt catttcgtac atatgagagg gttgcctttt cgtgcaactg 600 aaaatgccat tgctaatttc ttctcaccac taaatccaat acgagttcat attgatattg 660 gagctgatgg cagagccaca ggagaagcag atgtagagtt tgtgacacat gaagatgcag 720 tagctgccat gtctaaagat aaaaataaca tgcaacatcg atatattgaa ctcttcttga 780 attctactcc tggaggcggc tctggcatgg gaggttctgg aatgggaggc tacggaagag 840 atggaatgga taatcaggga ggctatggat cagttggaag aatgggaatg gggaacaatt 900 acagtggagg atatggtact cctgatggtt tgggtggtta tggccgtggt ggtggaggca 960 gtggaggtta ctatgggcaa ggcggcatga gtggaggtgg atggcgtggg atgtactgaa 1020 agcaaaaaca ccaacataca agtcttgaca acagcatctg gtctactaga ctttcttaca 1080 gatttaattt cttttgtatt ttaagaactt tataatgact gaaggaatgt gttttcaaaa 1140 tattatttgg taaagcaaca gattgtgatg ggaaaatgtt ttctgtaggt ttatttgttg 1200 catactttga cttaaaaata aatttttata ttcaaaccac tgatgttgat actttttata 1260 tactagttac tcctaaagat gtgctgcctt cataagattt gggttgatgt attttactat 1320 tagttctaca agaagtagtg tggtgtaatt ttagaggata atggttcacc tctgcgtaaa 1380 ctgcaagtct taagcagaca tctggaatag agcttgacaa ataattagtg taactttttt 1440 ctttagttcc tcctggacaa cactgtaaat ataaagccta aagatgaagt ggcttcagga 1500 gtataaattc agctaattat ttctatatta ttatttttca aatgtcattt atcaggcata 1560 gctctgaaac attgatgatc taagaggtat tgatttctga atattcataa ttgtgttacc 1620 tgggtatgag agtgttggaa gctgaattct agccctagat tttggagtaa aaccccttca 1680 gcacttgacc gaaataccaa aaatgtctcc aaaaaattga tagttgcagg ttatcgcaag 1740 atgtcttaga gtagggttaa ggttctcagt gacacaagaa ttcagtatta agtacatagg 1800 tatttactat ggagtataat tctcacaatt gtattttcag ttttctgccc aatagagttt 1860 aaataactgt ataaatgatg actttaaaaa aatgtaagca acaagtccat gtcatagtca 1920 ataaaaacaa tcctgcagtt gggttttgta tctgatccct gcttggagtt ttagtttaaa 1980 gaatctatat gtagcaagga aaaggtgctt tttaatttta atccctttga tcaatatggc 2040 ttttttccaa attggctaat ggatcaaaat gaaacctgtt gatgtgaatt cagttattga 2100 acttgttact tgtttttgcc agaaatgtta ttaataaatg tcaatgtggg agataaaaaa 2160 aaaaaaaaaa aaactggg 2178 <210> 29 <211> 1503 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2738270CB1 <400> 29 acgacctttt ggccaggtta gggagggggc gacgctgaga tgggggcggc ggcggcggaa 60 gcggatcgca ctctctttgt gggcaacctt gaaacgaaag tgaccgagga gctccttttc 120 gagcttttcc accaggctgg gccagtaata aaggtgaaaa ttccaaaaga taaggatggt 180 aaaccaaagc agtttgcgtt tgtgaatttc aaacatgaag tgtctgttcc ttatgcaatg 240 aatctactta atggaatcaa actttatgga aggcctatca aaattcaatt tagatcagga 300 agtagtcatg ccccacaaga tgtcagtttg tcatatcccc aacatcatgt tggaaattca 360 agccctacct ccacatctcc tagcagcagg tacgaaagga ctatggataa catgacttca 420 tcagcacaga taattcagag atctttctct tctccagaaa attttcagag acaagcagtg 480 atgaacagtg ctttgagaca aatgtcatat ggtggaaaat ttggttcttc acctctggat 540 caatcaggat tttcaccatc agttcaatca cacagtcata gtttcaatca gtcttcaagc 600 tcccagtggc gccaaggtac accatcatca cagcgtaaag tcagaatgaa ttcttatccc 660 tacctagcag atagacatta tagccgggaa cagcgttaca ctgatcatgg gtctgaccat 720 cattacagag gaaagagaga tgatttcttc tatgaagaca ggaatcatga tgactggagc 780 catgactatg ataacagaag agacagtagt agagatggaa aatggcgctc atctcgacac 840 taacacatgt taaaaggaca ttgtttttat agggtcattt taggcccttt gactaagttg 900 atatggaaat attttgttga aaaactgtac agagcagctt tacaagttgt cacattttct 960 ttataaattt ttttaaagct acagtttaat acaaaatgaa ttgcggtttt attacattaa 1020 taacctttca cctcagggtt ttatgaagag gaaagggttt tatgcaaaag aaagtgctac 1080 aattcctaat cattttagac actttaggag ggggtgaagt tgtatgataa agcagatatt 1140 ttaattattt gttatctttt tgtattgcaa gaaatttctt gctagtgaat caagaaaaca 1200 tccagattga cagtctaaaa tggctactgg tattttagtt aattcaaaaa tgaaactttt 1260 cagtgattca ctttactaac attctatttg agaaggctta ttggtaaagt ttggggataa 1320 aggcattgct taacttctta tataatttag gtataaattc tgtgacatgc tcttgagctt 1380 taccctagtt gaacatacat gtgtagattt acacatactg tttcattcta aaaattttag 1440 aattgttcat taaaacccca tttgaggtat aaggtcactc aggaaggtta aaatatctcc 1500 acc 1503 <210> 30 <211> 2548 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2824412CB1 <400> 30 tctgggagtc cgaccaggca tgcctcctca gcctcagggg cctgcaccct tacgtcgtcc 60 tgactcatct gatgaccgtt atgtaatgac aaaacatgcc accatttatc caactgaaga 120 ggagttacag gcagttcaga aaattgtttc tattactgaa cgtgctttaa aactcgtttc 180 agacagtttg tctgaacatg agaagaacaa gaacaaagag ggagatgata agaaagaggg 240 aggtaaagac agagctttga aaggagtttt gcgagtggga gtattggcaa aaggattact 300 tctccgagga gatagaaatg tcaaccttgt tttgctgtgc tcagagaaac cttcaaagac 360 attattaagc cgtattgcag aaaacctacc caaacagctt gctgttataa gccctgagaa 420 gtatgacata aaatgtgctg tatctgaagc ggcaataatt ttgaattcat gtgtggaacc 480 caaaatgcaa gtcactatca cactgacatc tccaattatt cgagaagaga acatgaggga 540 aggagatgta acctcgggta tggtgaaaga cccaccggac gtcttggaca ggcaaaaatg 600 ccttgacgct ctggctgctc tacgccacgc taagtggttc caggctagag ctaatggtct 660 gcagtcctgt gtgattatca tacgcattct tcgagacctc tgtcagcgag ttccaacttg 720 gtctgatttt ccaagctggg ctatggagtt actagtagag aaagcaatca gcagtgcttc 780 tagccctcag agccctgggg atgcactgag aagagttttt gaatgcattt cttcagggat 840 tattcttaaa ggtagtcctg gacttctgga tccttgtgaa aaggatccct ttgatacctt 900 ggcaacaatg actgaccagc agcgtgaaga catcacatcc agtgcacagt ttgcattgag 960 actccttgca ttccgccaga tacacaaagt tctaggcatg gatccattac cgcaaatgag 1020 ccaacgtttt aacatccaca acaacaggaa acgaagaaga gatagtgatg gagttgatgg 1080 atttgaagct gaggggaaaa aagacaaaaa agattatgat aacttttaaa aagtgtctgt 1140 aaatcttcag tgttaaaaaa acagatgccc atttgttggc tgtttttcat tcataataat 1200 gtctacattg aaaaatttat caagaattta aaggatttca tggaagaacc aagtttttct 1260 atgatattaa aaaatgtaca gtgttaggta ttatttgaat ggaaagacac ccaaaaaaaa 1320 aaatgtgctc cgactagggg gaaaacagta gttccgattt tttcccatta tttttatttt 1380 attttctggt tgccctagct tcccccccta tttttgtgtc ttttattaac tagtgcattg 1440 tcttattaaa tcttcactgt atttaatgca ggatgtgtgc ttcagttgct ctgtgtattt 1500 tgatatttta atttagaggt tttgtttgct ttttgacact agttgtaagt tactttgtta 1560 tagatggtat cctttacccc ttcttaatat tttacagcag tacgtttttt tgtaacgtga 1620 gactgcagag tttgtttttc tatatgtgaa ggattacaac acaaaaagtt atcctgccat 1680 tcgagtgctc agaactgaat gtttctgcag atcttgtggc atttgtctct agtgtgatat 1740 ataaaggtgt aattaagaca gagttctgtt aatctaatca agtttgctgt tagttgtgca 1800 ttagcagtat aaaagctaat atatactata tggtcttgca acagttttaa agcctctgca 1860 taattgataa taaaaatgca tgacattctt gtttttaata gacttttaaa atcataattt 1920 taggtttaac acgtagatct ttgtacagtt gactttttga catagcaagg ccaaaaataa 1980 ctttctgaat atttttttct tgtgtataag tggaaagggc atttttcaca tataagtggg 2040 ctaaccaata ttttcaaaag aacttcatca ttgtacaact aacaacagta actagccctt 2100 aattatggtg acagttcctt attggtgtgt gtgagattac tctagcaact attacagtat 2160 aacacagatg atcttctcca cacaccccat cacccagata atttacagtt ctgttaacag 2220 tgaggttgat aaagtattac tgataaaaaa ttatctaagg aaaaaaacag aaaattattt 2280 ggtgtggcca tcttacctgc ttatgtctcc tacacaaagc taaatattct agcagtgatg 2340 taatgaaaaa ttacatctta ctgttgatat atgtatgctc tggtacacag atgtcatttt 2400 gttgtcacag cactacagtg aaatacacaa aaaatgaaat tcatataatg acttaaatgt 2460 attatatgtt agaattgaca acataaacta cttttgcttt gaaatgatgt atgcttcagt 2520 aaaatcatat tcaaatttaa aaaaaaaa 2548 <210> 31 <211> B11 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 002690CB1 <400> 31 cgcatgcgtt ccctgaaatt gccgccaccg gctctacctt ccagtttcca gttccggcct 60 ccaaggggcg ggcagaagtt ggaaacatgc ggctgtcggt cgctgcagcg atctcccatg 120 gccgcgtatt tcgccgtatg ggcctcggtc ccgagtcccg catccatctg ttgcggaact 180 tgctcacagg gctggtgcgg cacgaacgca tcgaggcacc atgggcgcgt gtggacgaaa 240 tgaggggcta cgcggagaag ctcatcgact atgggaagct gggagacact aacgaacgag 300 ccatgcgcat ggctgacttc tggctcacag agaaggattt gatcccaaag ctgtttcaag 360 tactggcccc tcggtacaaa gatcaaactg ggggctacac aagaatgctg cagatcccaa 420 atcggagttt ggatcgggcc aagatggcag tgatcgagta taaagggaat tgcctcccac 480 ccctgcctct gcctcgcaga gacagccacc ttacactcct aaaccagctg ctgcagggtt 540 tgcggcagga cctcaggcaa agccaggaag caagcaacca cagctcccac acagctcaaa 600 caccagggat ttaactggat ctgaagagtc tgcagccctt aatcagtacc catgatcaca 660 ggcctttgga gcacttttac tctctgagaa gaactggagc tagagatgta aaatggacag 720 tcttgatggg gttgagaacc ttctggggag ccagatgacc ctctctttgc acaatagata 780 aaagtcttta tatgaatata aaaaaaaaaa a 811 <210> 32 <211> 1457 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 041108CB1 <400> 32 tacggaagct gggtcttctt gctgtgaggt cgcgttcccc agtgttacgg agggtccttg 60 aggcaggagt gaaaattggg tctgggggtt agtcctgggg tggaggtctg ggcacgccgg 120 gtcggacccc ctccatcttc ggttttgcac accccgcttt ccagcgcgga gtcgcggcgg 180 gtagggcggc gtcgcgtgcg tgacgtcatc cagcggcgcc tcgcgaggct ccagtggcct 240 tgacctcccg cggcgtggga ggctgcgcgg cgatgctgca gttcgtccgg gccggggcgc 300 gggcctggct tcggcctacc ggcagccagg gcctgagttc cctggcggaa gaggcagcgc 360 gtgcgaccga gaacccggag caggtggcga gcgagggtct cccggagccc gtgctgcgca 420 aagtcgagct cccggtaccc actcatcgac gcccagtgca ggcctgggtc gagtccttgc 480 ggggcttcga gcaggagcgc gtgggcctgg ccgacctgca ccccgatgtt ttcgccaccg 540 cgcccaggct ggacatactg caccaggttg ctatgtggca gaagaacttc aagagaatta 600 gctatgccaa gaccaagacg agagccgagg tgcggggcgg tggccggaag ccttggccgc 660 agaaaggcac tgggcgggcc cggcatggca gcatccgctc tccgctctgg cgaggaggag 720 gtgttgccca tggcccccgg ggccccacaa gttactacta catgctgccc atgaaggtgc 780 gggcgctggg tctcaaagtg gcactgaccg tcaagctggc ccaggacgac ctgcacatca 840 tggactccct agagctgccc accggagacc cacagtacct gacagagctg gcgcactacc 900 gccgctgggg ggactccgta ctcctcgtgg acttaacaca cgaggagatg ccacagagca 960 tcgtggaggc cacctctagg cttaagacct tcaacttgat cccggctgtt ggcctaaatg 1020 tgcacagcat gctcaagcac cagacgctgg tcctgacgct gcccaccgtc gccttcctgg 1080 aggacaagct gctctggcag gactcacgtt acagacccct ctaccccttc agcctgccct 1140 acagcgactt cccccgaccc ctaccccacg ctacccaggg cccagcggcc accccgtacc 1200 actgttgatg tgaagcacct cttctgagcc aggccgagcc cctggccgac ttgggagcct 1260 caggcccacg cccacccttc gaggaaggtg tcacctggac cccttcattc cacggaggaa 1320 gctgaggcca cagggagcgg ccatcgccat tgggaagggg cgactccacg gagagcccag 1380 acgggcttct gcatccattc cctctttttg tttttaaaat aaattgtatt tttgaatcaa 1440 ggaggaaaaa aaaaaaa 1457 <210> 33 <211> 1357 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 869138CB1 <400> 33 ctggtggcgt tcaagatgtc gaccaagaat ttccgagtca gtgacgggga ctggatttgc 60 cctgacaaaa aatgtggaaa tgtaaacttt gctagaagaa ccagctgtaa tcgatgtggt 120 cgggagaaaa caactgaggc caagatgatg aaagctgggg gcactgaaat aggaaagaca,180 cttgcagaaa agagccgagg cctatttagt gctaatgact ggcaatgtaa aacttgcagc 240 aatgtgaatt gggccagaag atcagagtgt aatatgtgta atactccaaa gtatgctaaa 300 ttagaagaaa gaacaggata tggtggtggt tttaatgaaa gagaaaatgt tgaatatata 360 gaaagagaag aatctgatgg tgaatatgat gagtttggac gtaaaaagaa aaaatacaga 420 gggaaagcag ttggtcctgc atctatatta aaggaagttg aagataaaga atcagaggga 480 gaagaagagg atgaggatga agatctttct aaatataagt tagatgagga tgaggatgaa 540 gatgacgctg atctctcaaa atataatctt gatgccagtg aagaagaaga tagtaataaa 600 aagaaatcta atagacgaag tcgctcaaag tctcgatctt cacattcacg atcttcatca 660 cgctcatcct ccccctcaag ttcaaggtct aggtccaggt cccgttcaag aagttcttcc 720 agttcgcagt caagatctcg ttccagttcc agagaacgtt cgagatctcg tgggtcgaaa 780 tcaagatcca gctccaggtc ccacaggggc tcttcttccc cacgaaaaag atcttattca 840 agttcatcat cttctcctga gaggaacaga aagagaagtc gttctagatc ttcttcatct 900 ggtgatcgca aaaaaagacg aacaagatca cggtcacccg aaagacgcca caggtcatca 960 tctggatcat cccattctgg ttcccgttca agttcaaaaa agaaataatg tattaaaatt 1020 tacatcttaa aaaaatccag tacagtgcat gaagcatatt tttaaagaag ttggtgtctt 1080 acttggtcag aagtgctaaa tctgctagta gaggtgcatg cctttcattg cttttcaaaa 1140 caatacagct gtgtttattt gtgaagttaa aagtaaatag cattttaagc cataatgtcc 1200 caaaatagat gttctgtcat tcattattta caaccatttg cttcatttaa aaccatttca 1260 gctataacaa agtactttgc ttcctaattt aaacccattt ttgtcatttc caaatacatc 1320 ctgtccattg gctaagacag gattacctag gcttgct 1357 <210> 34 <211> 1326 <212> DNA
<213> Homo Sapiens <221> unsure <222> 1313 <223> a or t or g or c, unknown, or other <220>
<221> misc_feature <223> Incyte Identification No.: 934406CB1 <400> 34 ttttcttcgg ggactatcct tgtctgatca ggcgggaaag acggtgccgc ccgacaatgc 60 gcggaggtag gagggggaag tggaggcggg agtgaagtct cgcgagaaga gtcggttgcc 120 gtagcagagc cctctagctg tgtgtgtctg aggctcggcc gcctgagccg cggacggttt 180 gctgagcccg ttagtgcgcc cggccgagac acgccgccgc catgtcccgc tacctgcgtc 240 cccccaacac gtctctgttc gtcaggaacg tggccgacga caccaggtct gaagacttgc 300 ggcgtgaatt tggtcgttat ggtcctatag ttgatgtgta tgttccactt gatttctaca 360 ctcgccgtcc aagaggattt gcttatgttc aatttgagga tgttcgtgat gctgaagacg 420 ctttacataa tttggacaga aagtggattt gtggacggca gattgaaata cagtttgccc 480 agggggatcg aaagacacca aatcagatga aagccaagga agggaggaat gtgtacagtt 540 cttcacgcta tgatgattat gacagataca gacgttctag aagccgaagt tatgaaagga 600 ggagatcaag aagtcggtct tttgattaca actatagaag atcgtatagt cctagaaaca 660 gtagaccgac tggaagacca cggcgtagca gaagccattc cgacaatgat agaccaaact 720 gcagctggaa tacccagtac agttctgctt actacacttc aagaaagatc tgaaagcgga 780 aaaagaacca aagaagggca gttcaagcga ccaaagggtg ggtggaaggt gctgcagtat 840 gaatactgta cgaatatttt gactctggtc tgaaaagata aaagaatgtt atcgaaaact 900 acatggaata attgaagtcc cttcaagttt gaaagtaagc attttaggac aaataaaagg 960 aaattcaact ttgtacttgt ggaaactaat ccctaaatat gaataggttt atattgattc 1020 atgggtaaca ggtccataat aaattattgg aaactaggat gtctgaatat caaggaagac 1080 agccatagtc tcttacagtg cctctgttgg tctgtctcaa actgaattgg gtgggaaaag 1140 gtatggtcca atataaaagt tccatttttg ccattattgg gcaaatcttg cctttgttta 1200 ttttggtgcc agtgttttct gcttaatcat ttgctttgtt ggcatctgtg tttatttact 1250 tgtacaccac atgcagttta catctgtctt aactactcct tcccaggtaa ttnccattat 1320 attgac 1326 <210> 35 <211> 3301 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1315083CB1 <400> 35 gaagggaagt aacgtcagcc tgagaactga gtagctgtac tgtgtggcgc cttattctag 60 gcacttgttg ggcagaatgt cacacctgcc gatgaaactc ctgcgtaaga agatcgagaa 120 gcggaacctc aaattgcggc agcggaacct aaagtttcag ggggcctcaa atctgaccct 180 atcggaaact caaaatggag atgtatctga agaaacaatg ggaagtagaa aggttaaaaa 240 atcaaaacaa aagcccatga atgtgggctt atcagaaact caaaatggag gcatgtctca 300 agaagcagtg ggaaatataa aagttacaaa gtctccccag aaatccactg tattaaccaa 360 tggagaagca gcaatgcagt cttccaattc agaatcaaaa aagaaaaaga agaaaaagag 420 aaaaatggtg aatgatgctg agcctgatac gaaaaaagca aaaactgaaa acaaagggaa 480 atctgaagaa gaaagtgccg agactactaa agaaacagaa aataatgtgg agaagccaga 540 taatgatgaa gatgagagtg aggtgcccag tctgcccctg ggactgacag gagcttttga 600 ggatacttcg tttgcttctc tatgtaatct tgtcaatgaa aacactctga aggcaataaa 660 agaaatgggt tttacaaaca tgactgaaat tcagcataaa agtatcagac cacttctgga 720 aggcagggat cttctagcag ctgcaaaaac aggcagtggt aaaaccctgg cttttctcat 780 ccctgcagtt gaactcattg ttaagttaag gttcatgccc aggaatggaa caggagtcct 840 tattctctca cctactagag aactagccat gcaaaccttt ggtgttctta aggagctgat 900 gactcaccac gtgcatacct atggcttgat aatgggtggc agtaacagat ctgctgaagc 960 acagaaactt ggtaatggga tcaacatcat tgtggccaca ccaggccgtc tgctggacca 1020 tatgcagaat accccaggat ttatgtataa aaacctgcag tgtctggtta ttgatgaagc 1080 tgatcgtatc ttggatgtgg ggtttgaaga ggaattaaag caaattatta aacttttgcc 1140 aacacgtaga cagactatgc tcttttctgc cacccaaact cgaaaagttg aagacctggc 1200 aaggatttct ctgaaaaagg agccattgta tgttggcgtt gatgatgata aagcgaatgc 1260 aacagtggat ggtcttgaac agggatatgt tgtttgtcct tctgaaaaga gattccttct 1320 gctctttaca ttccttaaga agaaccgaaa gaagaagctt atggtcttct tttcatcttg 1380 tatgtctgtg aaataccact atgagttgct gaactacatt gatttgcccg tcttggccat 1440 tcatggaaag caaaagcaaa ataagcgtac aaccacattc ttccagttct gcaatgcaga 1500 ttcgggaaca ctattgtgta cggatgtggc agcgagagga ctagacattc ctgaagtcga 1560 ctggattgtt cagtatgacc ctccggatga ccctaaggaa tatattcatc gtgtgggtag 1620 aacagccaga ggcctaaatg ggagagggca tgccttgctc attttgcgcc cagaagaatt 1680 gggttttctt cgttacttga aacaatccaa ggttccatta agtgaatttg acttttcctg 1740 gtctaaaatt tctgacattc agtctcagct tgagaaattg attgaaaaga attactttct 1800 tcataagtca gcccaggaag catataagtc atacatacga gcctatgatt cccattctct 1860 gaaacagatc tttaatgtta ataacctaaa tttgcctcag gttgctctgt catttggttt 1920 caaggtgcct cccttcgttg atctgaacgt caacagtaat gaaggcaagc agaaaaagcg 1980 aggaggtggt ggtggatttg gctaccagaa aaccaagaaa gttgagaaat ccaaaatctt 2040 taaacacatt agcaagaaat catctgacag caggcagttc tctcactgaa cacatgcctt 2100 cctttcatct tgaataactt tgtcctaaaa tgaatttttt ttccccttga tttaacagga 2160 tttttgtaga ctttagaatt tggacttacc taacaagagt ataaattgac ttgggttgca 2220 agcactgagc actgttactt ctatcacgtc tctcttttat ttctgggata taaaacaggc 2280 tttaagtttc ttggttgccc aagggcagag caaggaatat ctggtgtttc ttgtgatgat 2340 aatattttaa ttttaaatat ccctccctca tacaagtgta tgttaccatt ttaatataat 2400 tctttttgta cctttccttc ttgttttgtg aagatttttg tggcatggat tgctgtgctc 2460 actgctgtaa aaggtgacct agtgtactgg gcagctggtg gcggtgcaga aaagagtctc 2520 aggttatttt ttgtttttag ttatttcttg gaccttgaca gtatctaatg actcctcctg 2580 aaaatgctgc agtataaaag agcaaagagc tttgggaaat acctaagaag caccttaaga 2640 ttagggtggc attgctttta tagattcttg attttaaagc aacaggcctt tctcaggtgt 2700 tgcatttttt ggagcaaaaa ctatgggttg taatttgaat aaagtgtcac taagcagtta 2760 taacgtttga tggctggggg gtaggaagag gatggaattg agatgtttga gcctcattta 2820 catcaataga ggtgtaatgt actgcatttc ttcatttggt aacataacaa agactttcat 2880 acaaagaacg atgatgctcc tcattaagat ttgtttaatt caaggtggtt tggatttggt 2940 aagcctttgc actctgtaga gtacttagaa gacaagggca acttacttgg agttagagcc 3000 aagctgtcag acggtgccca gcacacatta atgttagctt ctttctgaga aaaaaatacc 3060 tcttccaggc cctgaaacaa aaaatacatt tgctgtgaag attgaaaatg aacaaagtta 3120 gaaaaaaaaa cagcaaaatc agtgatttag tcagatgagt ttttcgttgt aggagcactt 3180 gatttctagt gtgttttgta cagtatataa ctacaagata gtacattttg tagcagttca 3240 aagccaaagt tgctagcatc attttgctgt tgtgccagtt aatcatagga tcccattaag 3300 g 3301 <210> 36 <211> 1703 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1444908CB1 <400> 36 gcttcccctc cgtcatccat cgctcggcgg tcttctcttc tccatgggtc tgctctgcgc 60 gttccatcga gtttctcggc catcgcgcgc ctgcgccatt gggctgtcag tcagaggcgg 120 cgtggagatc gctgggagcg gttgcggcgt gcggggagct gagttatagc tgtgacttct 180 gccctgccag gccgcacaca agctggctga cccggtttgt aaaaatggaa tttcaagcag 240 tagtgatggc agtaggtgga ggatctcgga tgacagacct aacttccagc attcccaaac 300 ctctgcttcc agttgggaac aaacctttaa tttggtaccc attgaacctg cttgagcgtg 360 ttggatttga agaagtcatt gtggttacaa ccagggatgt tcaaaaggct ctatgtgcag 420 aattcaagat gaaaatgaag ccagatattg tgtgtattcc tgatgatgct gacatgggaa 480 ctgcagattc tttgcgctac atatatccaa aacttaagac agatgtgctg gtgctgagct 540 gtgatctgat aacagacgtt gccttacatg aggttgtgga cctgtttaga gcttatgatg 600 catcacttgc tatgttgatg agaaaaggcc aagatagcat agaacctgtt cccggtcaaa 660 aggggaaaaa aaaagcagtg gagcagcgtg acttcattgg agtggacagc acaggaaaga 720 ggctgctctt catggctaat gaagcagact tggatgaaga gctggtcatt aagggatcca 780 tcctacagaa gcatcctaga atacgtttcc acacgggtct tgtggatgcc cacctctact 840 gtttgaaaaa atacatcgtg gatttcctaa tggaaaatgg gtcaataact tctatccgga 900 gtgaactgat tccatattta gtgagaaaac agttttcctc agcttcctca caacagggac 960 aagaagaaaa agaggaggat ctaaagaaaa aggagctgaa gtccttagat atctacagtt 1020 ttataaaaga agccaataca ctgaacctgg ctccctatga tgcctgctgg aatgcctgtc 1080 gaggagacag gtgggaagac ttgtccagat cacaggtgcg ctgctatgtc cacatcatga 1140 aagaggggct ctgctctcga gtgagcacac tgggactcta catggaagca aacagacagg 1200 tgcccaaatt gctgtctgct ctctgtccag aagaaccacc agtccattcg tcagcccaga 1260 ttgtcagcaa acacctggtt ggagttgaca gcctcattgg gccagagaca cagattggag 1320 agaagtcatc cattaagcgc tcagtcattg gctcatcctg tctcataaaa gatagagtga 1380 ctattaccaa ttgccttctc atgaactcag tcactgtgga ggaaggaagc aatatccaag 1440 gcagtgtcat ctgcaacaat gctgtgatcg agaagggtgc agacatcaag gactgcttga 1500 ttggaagtgg ccagaggatt gaagccaaag ctaaacgagt gaatgaggtg atcgtgggga 1560 atgaccagct catggagatc tgagttctga gcaagtcaga ctccttcctt ttggcctcca 1620 aagccacaga tgttggccgg cccacctgtt taactctgta tttatttccc aataaagaag 1680 ggcttccaaa ggcaaaaaaa aaa 1703 <210> 37 <211> 2536 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1557481CB1 <400> 37 gacttccggg gcggcggttg catcagattc taggaagtgt ctgtagccgc agctgcgggt 60 ccgggattcc cagccatggc agattcctcc gggcagcagg gcaaaggccg gcgtgtgcag 120 ccccagtggt cccctcctgc tgggacccag ccatgcagac tccaccttta caacagcctc 180 accaggaaca aggaagtgtt catacctcaa gatgggaaaa aggtgacgtg gtattgctgt 240 gggccaaccg tctatgacgc atctcacatg gggcacgcca ggtcctacat ctcttttgat 300 atcttgagaa gagtgttgaa ggattacttc aaatttgatg tcttttattg catgaacatt 360 acggatattg atgacaagat catcaagagg gcccggcaga accacctgtt cgagcagtat 420 cgggagaaga ggcctgaagc ggcacagctc ttggaggatg ttcaggccgc cctgaagcca 480 ttttcagtaa aattaaatga gaccacggat cccgataaaa agcagatgct cgaacggatt 540 cagcacgcag tgcagcttgc cacagagcca cttgagaaag ctgtgcagtc cagactcacg 600 ggagaggaag tcaacagctg tgtggaggtg ttgctggaag aagccaagga tttgctctct 660 gactggctgg attctacact tggctgtgat gtcactgaca attccatctt ctccaagctg 720 cccaagttct gggaggggga cttccacaga gacatggaag ctctgaatgt tctccctcca 780 gatgtcttaa cccgggttag tgagtatgtg ccagaaattg tgaactttgt ccagaagatt 840 gtggacaacg gttacggcta tgtctccaat gggtctgtct actttgatac agcgaagttt 900 gcttctagcg agaagcactc ctatgggaag ctggtgcctg aggccgttgg agatcagaaa 960 gcccttcaag aaggggaagg tgacctgagc atctctgcag accgcctgag tgagaagcgc 1020 tctcccaacg actttgcctt atggaaggcc tctaagcccg gagaaccgtc ctggccgtgc 1080 ccttggggaa agggtcgtcc gggctggcat atcgagtgct r_ggccatggc aggcaccctc 1140 ctaggggctt cgatggacat tcacggaggt gggttcgacc tccggttccc ccaccatgac 1200 aatgagctgg cacagtcgga ggcctacttt gaaaacgact gctgggtcag gtacttcctg 1260 cacacaggcc acctgaccat tgcaggctgc aaaatgtcaa agtcactaaa aaacttcatc 1320 accattaaag atgccttgaa aaagcactca gcacggcagt tgcggctggc cttcctcatg 1380 cactcgtgga aggacaccct ggactactcc agcaacacca tggagtcagc gcttcaatat 1440 gagaagttct tgaatgagtt tttcttaaat gtgaaagata tccttcgcgc tcctgttgac 1500 atcactggtc agtttgagaa gtggggagaa gaagaagcag aactgaataa gaacttttat 1560 gacaagaaga cagcaattca caaagccctc tgtgacaatg ttgacacccg caccgtcatg 1620 gaagagatgc gggccttggt cagtcagtgc aacctctata tggcagcccg gaaagccgtg 1680 aggaagaggc ccaaccaggc tctgctggag aacatcgccc tgtacctcac ccatatgctg 1740 aagatctttg gggccgtaga agaggacagc tccctgggat tcccggtcgg agggcctgga 1800 accagcctca gtctcgaggc cacagtcatg ccctaccttc aggtgttatc agaattccga 1860 gaaggagtgc ggaagattgc ccgagagcaa aaagtccctg agattctgca gctcagcgat 1920 gccctgcggg acaacatcct gcccgagctt ggggtgcggt ttgaagacca cgaaggactg 1980 cccacagtgg tgaaactggt agacagaaac accttattaa aagagagaga agaaaagaga 2040 cgggttgaag aggagaagag gaagaagaaa gaggaggcgg cccggaggaa acaggaacaa 2100 gaagcagcaa agctggccaa gatgaagatt ccccccagtg agatgttctt gtcagaaacc 2160 gacaaatact ccaagtttga tgaaaatggt ctgcccacac atgacatgga gggcaaagag 2220 ctcagcaaag ggcaagccaa gaagctgaag aagctcttcg aggctcagga gaagctctac 2280 aaggaatatc tgcagatggc ccagaatgga agcttccagt gagggggcac aggactgact 2340 ttttaaacca ttgtggacta gtggctgctg tctgcctcag tgacaatgtc ccagcgctcc 2400 tatcatgttt acagtcaccc ttgggtccta aattaagagt tgtgttcatg taggttcgtg 2460 tcgtcgttgg ctctgagaca ttgataataa atttttctca acagtgaaaa aaaaaaaaaa 2520 gaaaaaaaaa aaaaaa 2536 <210> 38 <211> 1350 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1747456CB1 <400> 38 cggaggagcc cgggcggggg ggaggaggag ggggaggagg gagcggagat ctcggggctc 60 ggagccggcc gccgctccgc tccgatcgct gtggggcttg gttttttggg ggtggggggg 120 cgggggggct cagatatgga ggcaaatggg agccaaggca cctcgggcag cgccaacgac 180 tcccagcacg accccggtaa aatgtttatc ggtggactga gctggcagac ctcaccagat 240 agccttagag actattttag caaatttgga gaaattagag aatgtatggt catgagagat 300 cccactacga aacgctccag aggcttcggt ttcgtcacgt tcgcagaccc agcaagtgta 360 gataaagtat taggtcagcc ccaccatgag ttagattcca agacgattga ccccaaagtt 420 gcatttcctc gtcgagcgca acccaagatg gtcacaagaa caaagaaaat atttgtaggc 480 gggttatctg cgaacacagt agtggaagat gtaaagcaat atttcgagca gtttggcaag 540 gtggaagatg caatgctgat gtttgataaa actaccaaca ggcacagagg gtttggcttt 600 gtcacttttg agaatgaaga tgttgtggag aaagtctgtg agattcattt ccatgaaatc 660 aataataaaa tggtagaatg taagaaagct cagccgaaag aagtcatgtt cccacctggg 720 acaagaggcc gggcccgggg actgccttac accatggacg cgttcatgct tggcatgggg 780 atgctgggat atcccaactt cgtggcgacc tatggccgtg gctaccccgg atttgctcca 840 agctatggct atcagttccc aggcttccca gcagcggctt atggaccagt ggcagcagcg 900 gcggtggcgg cagcaagagg atcaggctcc aacccggcgc ggcccggagg cttcccgggg 960 gccaacagcc caggacctgt cgccgatctc tacggccctg ccagccagga ctccggagtg 1020 gggaattaca taagtgcggc cagcccacag ccgggctcgg gcttcggcca cggcatagct 1080 ggacctttga ttgcaacggc ctttacaaat ggataccatt gagcaggtgc tttcgttgcc 1140 atctcactct gagagcatac ctggatgtcc aggcaagact gggcgaagtt tctgagtggc 1200 cctttgttta ggtgatgtcc tcagacctgg acccccacca gcctcactcc ccatcccaac 1260 cagagatggc tcacttcgga tcgagggttg actacatctc atcatctcac gaatctgctg 1320 taatataaga caacagcttt taaatgtgta 1350 <210> 39 <211> 2190 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1748626CB1 <400> 39 cctgcgggag ccccgtgccc gtcacgccgc cgcagccttc gccctggatg ccgctgctgc 60 cgccgccgtg ggactgtccc gggagcgggc cctggactac tacgggctgt acgacgaccg 120 tgggcgcccc tatggctacc cagctgtgtg tgaggaggac ctgatgcccg aggatgacca 180 gcgggccacg cgcaacctct tcattggtaa cctggaccac agcgtatctg aggtggagct 240 gcgaagggcc ttcgagaaat atggcatcat cgaggaggtg gtcatcaaga ggcctgcccg 300 tggccagggc ggtgcctatg ccttcctcaa gttccagaac ctggacatgg cccatagggc 360 taaggtggcc atgtcgggcc gagtgattgg tcgcaacccc attaagatag gctatggcaa 420 ggccaacccc accactcgtc tctgggtggg tggcctggga cctaacacgt cactggcggc 480 tctggcccga gagtttgacc gctttgggag cattcggacc attgatcacg tcaaaggaga 540 tagctttgcc tatattcagt acgagagctt ggacgcagcc caggccgcct gtgctaaaat 600 gaggggtttt cccttgggtg gaccagaccg caggctccgc gtggattttg ccaaagcaga 660 ggagactcgg tacccccagc agtaccagcc ctcgccactc cctgtgcatt atgagctgct 720 cacagatgga tacacccggc accgcaacct ggacgccgac ctggtgcggg acaggacgcc 780 cccacacctt ctgtactcag accgagaccg gacttttttg gaaggggact ggaccagccc 840 cagtaaaagc tctgaccgcc gaaacagcct tgagggctac agtcgctcag tgcgcagccg 900 gagtggtgag cgttgggggg cagatggaga ccgtggtttg cccaagccct gggaagagag 960 gcggaaacgg agaagccttt ccagtgaccg tgggaggaca acccattcac catatgagga 1020 acggagtagg accaagggca gtgggcagca gtcagagcgg ggctccgacc gcacccctga 1080 gcgcagccgc aaggagaacc actccagtga agggaccaag gagtccagca gcaactccct 1140 cagcaacagc agacatgggg ctgaggaacg gggccaccac caccaccacc acgaggctgc 1200 agactcttcc cacgggaaga aggcaagaga cagcgagcgc aatcaccgga ccacagaggc 1260 cgagcccaag cctctggaag agccaaaaca cgagaccaaa aagctgaaga atctttcaga 1320 gtacgctcag acactacagc tgggttggaa tgggcttctg gtgttgaaaa acagctgctt 1380 ccccacgtct atgcatatcc tagaggggga ccagggggtg atcagcagtc tcctcaaaga 1440 ccacacttct gggagcaagc tgacccagct gaagatcgcc cagcgccttc gactggacca 1500 gcccaagctt gacgaggtca cacgacgcat caagcagggg agccccaacg gctatgcggt 1560 cctcttagcc acccaggcaa cccccagtgg gcttggcact gaggggatgc ccacagtaga 1620 gcccggtctg cagaggcggc ttctcaggaa cctggtctcc tacttgaaac agaagcaggc 1680 cgcaggggtg atcagcttgc cagtgggggg gtccaagggc agagacggca caggcatgct 1740 ctacgccttc ccaccctgcg acttttccca gcagtacctc cagtcagcac taaggacatt 1800 gggcaagcta gaagaagaac acatggtgat agtcatcgtc agagacactg cctagcccaa 1860 gcctgtcttt cccagcgtca tgtttgtgtc acaaaagcag ttattttaaa atctgatccc 1920 ctctctaccc taccactttg gtttgaatta tctcctgggt tattttggtt catttgggtg 1980 gggatcaaag tcctgtccac caccaaaact aagttcttag attttggggg attttttttt 2040 ttaaacgatg agaagggaat ccggttatgt tgatttctag tgtacaagat actgtctgct 2100 gtggttctgt atttttttat tttttgacca actgtatgga aagttgtcag taaaaccttt 2160 gacagaggat ggatttttaa aaaaaaaaaa 2190 <210> 40 <211> 680 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 1879135CB1 <400> 40 gtctttggct gtgtggtctt aaatgtgttt ctaatgtgtg tgtcaaataa ttacctgtta 60 aacagactgc caatctggct gaagccaatg cttctgaaga agataaaatt aaagcaatga 120 tgtcgcaatc tggccatgaa tacgacccaa tcaattacat gaagaaacct ctaggtccac 180 cacctccatc ttacacgtgt ttccgttgtg gtaaacctgg acattatatt aagaattgcc 240 caacaaatgg ggataaaaac tttgaatctg gtcctaggat taaaaagagc actggaattc 300 ccagaagttt catgatggaa gtgaaagatc ctaatatgaa aggtgcaatg cttaccaaca 360 ctggaaaata tgcaatacca actatagatg cagaagcata tgcaattggg aagaaagaga 420 aacctccctt cttaccagag gagccatctt cttcctcaga agaagatgat cctatcccag 480 atgaattgtt gtgtctcatc tgcaaggata ttatgactga tgctgttgtg attccctgct 540 gtggaaacag ttactgtgat gaatgtaaga agtgctgaat cttggaagat gtatatttta 600 gaatatttgt atttacttgg aatggctctt cccaacctca tatgttttaa taataaaata 660 aataatgttg aaaaaaaaaa 680 <210> 41 <211> 1150 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2073417CB1 <400> 41 gacggaagtg cggtgttgag cgccggcggc tcgcgcccac gctgggccgg gagtcgaaat 60 gcttcccggt gccgggagtg agcgatgagc tggcttctgt tcctggccca cagagtcgcc 120 ttggccgcct tgccctgccg ccgcggctct cgcgggttcg ggatgttcta tgccgtgagg 180 aggggccgca agaccggggt ctttctgacc tggaatgagt gcagagcaca ggtggaccgg 240 tttcctgctg ccagatttaa gaagtttgcc acagaggatg aggcctgggc ctttgtcagg 300 aaatctgcaa gcccggaagt ttcagaaggg catgaaaatc aacatggaca agaatcggag 360 gcgaaagcca gcaagcgact ccgtgagcca ctggatggag atggacatga aagcgcagag 420 ccgtatgcaa agcacatgaa gccgagcatg gagccggcgc ctccagttag cagagacacg 480 ttttcctaca tgggagactt cgtcgtcgtc tacactgatg gctgctgctc cagtaatggg 540 cgtagaaggc cgcgagcagg aatcggcgtt tactgggggc caggccatcc tttaaatgta 600 ggcattagac ttcctgggcg gcagacaaac caaagagcgg aaattcatgc agcctgcaaa 660 gccattgaac aagcaaagac tcaaaacatc aataaactgg ttctgtatac agacagtatg 720 tttacgataa atggtataac taactgggtt caaggttgga agaaaaatgg gtggaagaca 780 agtgcaggga aagaggtgat caacaaagag gactttgtgg cactggagag gcttacccag 840 gggatggaca ttcagtggat gcatgttcct ggtcattcgg gatttatagg caatgaagaa 900 gctgacagat tagccagaga aggagctaaa caatcggaag actgagccat gtgactttag 960 tccttgggag aacttgagcc agcggctgtc ttgctgcctg tacttactgg tgtggaaaat 1020 agcctgcagg taggaccatt gcagtgatgg gcagatgcgt ctttcacacg gaatcaggca 1080 cagtggcctt ctgtgacatg tgtttataaa aaatggttaa gtatataata aattgaacat 1140 ctttgagatt 1150 <210> 42 <211> 2545 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2129080CB1 <400> 42 ggcagggagc ggagacggag gaggaggagg gagaggctga atgttggctc gggagacgta 60 cgaggaggac cgggagtacg agagccaggc caagcgtctc aagaccgagg agggggagat 120 cgactactcg gccgaggaag gcgagaaccg ccgggaagcg acgccccggg gcgggggcga 180 tggcggcggc ggcggccgga gcttctctca gccggaggca ggtggaagtc atcataaagt 240 ttctgtttca cccgtcgtcc atgttcgagg actctgtgaa tctgtggtgg aagcagacct 300 cgtggaagcg ctggaaaaat ttgggacaat atgctatgtg atgatgatgc catttaaacg 360 acaggctcta gtggaatttg aaaacataga tagtgccaaa gaatgtgtga catttgctgc 420 agatgaaccc gtgtacattg ctggtcaaca ggcttttttc aactattcta caagcaaaag 480 gatcactcgg ccaggaaata ctgatgatcc atcaggaggc aacaaagttc ttctgctctc 540 aattcagaat ccgctttatc caattacagt ggatgtttta tatactgtat gcaaccctgt 600 tggcaaagtg caacgtattg ttatattcaa gagaaatggg atacaagcaa tggttgagtt 660 tgaatcagtc ctttgtgccc agaaagctaa agcagcactc aatggagctg atatatatgc 720 tggatgttgc acactaaaaa ttgaatatgc acggccaact cgtctaaatg ttattaggaa 780 tgacaatgac agttgggact acactaaacc atatttggga agacgagata gaggaaaggg 840 tcgccagaga caagccattt tgggagaaca cccttcttcg tttagacatg atggctatgg 900 atcccatggt ccattattgc ctttaccaag tcgttacaga atgggctctc gagatacacc 960 tgaacttgtt gcttatccat taccacaggc ttcttcctct tacatgcatg gaggaaatcc 1020 ctctggttca gttgtaatgg ttagtggatt acatcaacta aaaatgaatt gttcaagagt 1080 cttcaacctg ttctgcttat atggaaatat tgagaaggta aaatttatga agaccattcc 1140 tggtacagca ctggtagaaa tgggtgatga gtatgctgta gaaagagctg tcacacacct 1200 taataatgtc aaattatttg ggaaaagact taatgtttgc gtgtctaaac aacattcagt 1260 tgttccaagt caaatatttg agctggagga tggtaccagc agctacaaag attttgcaat 1320 gagcaaaaat aatcgcttta caagtgctgg ccaagcatct aagaatataa tccagccacc 1380 ctcctgtgtt ttgcattatt ataatgttcc attgtgtgtc acagaagaga ccttcacaaa 1440 gttgtgtaat gaccatgaag ttcttacatt catcaaatat aaagtgtttg atgcaaaacc 1500 ttcagccaaa acactttctg ggctattaga atgggagtgc aaaactgatg cagtagaagc 1560 ccttacggca ctgaatcact atcagataag agtgccgaat ggttccaatc cctatacatt 1620 gaagctttgc ttttctacat catcccattt ataagaagag aagagcatgt tagaatttat 1680 gttcaccttt attacaattt caaagctaca cttcattaaa aaaaaatcta aaatggttga 1740 tctcatgttg ccttgcttac tttaagatcc tgttctgtaa taaacatatt ttgcettgag 1800 taaatttgtt gtaagcttaa atattgaatt gttttcattt taagatagaa tatcataatg 1860 tagactatct acagcttcat tgtagattat acagatatat gatttctaac cttattactg 1920 gaatttttct tccacagtaa aaatatattt gcattcttaa tgctaattat ctgcaagtat 1980 tttttcattg tgtaagagat taatgcaggt gaaagtattg cattttaata tagaattcct 2040 attatatgtt tagatgttta agtatgttgc agttactcat attaaacata acttgtatat 2100 ttattatttt aatgaagttt gagaataacg ttacatatgt tgaattttaa gtactacaga 2160 tttaactgat tttatatttc tgaaaggcta acagacatgg atacacgtgt acagtatgca 2220 ttcaaactta tttaaattgg tgtatttttt tttaagtcac tgtccatttg tattgacatg 2280 cctctgtttc tagtccagtt tggagatttt ataaagttat aacaatgagt taatgtgttc 2340 attttcattt gttgcatgtg acttaaatac agctagtatt tggcattgag attttaatag 2400 aggttataat taacagttcc tcttatagat aataatctgg gatccatggg tgggcttcag 2460 agaatctgtg taccccctaa aattgtatgt agaattttgg atatttacat ttttattttt 2520 taactcttgg atccatcagt aatat 2545 <210> 43 <211> 907 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2472867CBI
<400> 43 caagcttggc gtttgtttgg tggggtcaca cgcgggttca acatgcgtat cgaaaagtgt 60 tatttctgtt cggggcccat ctatcctgga cacggcatga tgttcgtccg caacgattgc 120 aaggtgttca gattttgcaa atctaaatgt cataaaaact ttaaaaagaa gcgcaatcct 180 cgcaaagtta ggtggaccaa agcattccgg aaagcagctg gtaaagagct tacagtggat 240 aattcatttg aatttgaaaa acgtagaaat gaacctatca aataccagcg agagctatgg 300 aataaaacta ttgatgcgat gaagagagtt gaagaaatca aacagaagcg ccaagctaaa 360 tttataatga acagattgaa gaaaaataaa gagctacaga aagttcagga tatcaaagaa 420 gtcaagcaaa acatccatct tatccgagcc cctcttgcag gcaaagggaa acagttggaa 480 gagaaaatgg tacagcagtt acaagaggat gtggacatgg aagatgctcc ttaaaaatct 540 ctgtaaccat ttcttttatg tacatttgaa aatgcccttt ggatacttgg aactgctaaa 600 ttattttatt ttttacataa ggtcacttaa atgaaaagcg attaaaagac atctttcctg 660 cattgccatc tacataatat cagatattac ggatgttaga ttgcatctca gtgttaaatc 720 tttactgata gatgtactta agtaaatcat gaaaattcta cttgtaacta tagaagtgaa 780 ttgtggacgt aaaatggttg tgctatttgg ataatggcac taggcagcat ttgtatagta 840 actaatggca aaaattcatg gctagtgatg tataaaataa aatattcttt gcagtaaaaa 900 aaaaaaa 907 <210> 44 <211> 1104 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2764755CB1 <400> 44 cttcgtttag gtcggctgga aattatgtcc tccgtcggtt ttccgcagtt tttccaccaa 60 gcgagatatt tttgggagtt attccctaaa taactgcatt atatgctcct ttcatgacga 120 aattgctgcc gtggagaaga ctggaggaaa ctcgaggaag agggagaagc cgacaagtgc 180 tcgacgggct aggaactgtc ctgcttgggt gttagcgttt cccgccgggc cagtaaggct 240 gagtgacccg gcgtggctac taggagaagg acgtacggtc ctgctagtag aggaatatgt 300 cgagtttctc tagggcgccc cagcaatggg ccacttttgc tagaatatgg tatctcttag 360 atgggaaaat gcagccacct ggcaaacttg ctgctatggc atctataaga cttcagggat 420 tacataaacc tgtgtaccat gcactgagtg actgtgggga tcatgttgtt ataatgaaca 480 caagacacat tgcattttct ggaaacaaat gggaacaaaa agtatactct tcgcatactg 540 gctacccagg tggatttaga caagtaacag ctgctcagct tcacctgagg gatccagtgg 600 caattgtaaa actagctatt tatggcatgc tgccaaaaaa ccttcacaga agaacaatga 660 tggaaaggtt gcatcttttt ccagatgagt atattccaga agatattctt aagaatttag 720 tagaggagct tcctcaacca cgaaaaatac ctaaacgtct agatgagtac acacaagaag 780 aaatagacgc cttcccaaga ttgtggactc cacctgaaga ttatcggcta taagagaata 840 agaattgcag aaaataacag tgaagtgatt gaaactttct tctgatgagt ttctctaacc 900 tacaggatgg agtaaaacaa ctgctacagt tcagcacctg ttttatgtgc cgaatcactg 960 tggggaaagg tcaggaaggt gtagtccttc aataggaaat tgtaattaaa atataatttt 1020 atagaaccat ttttatgtaa tctgatttga atgttatagt tgataataat aaaatcactt 1080 acttggttga ctaaaaaaaa aaaa 1104 <210> 45 <211> 910 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 2875939CB1 <400> 45 cccacgcgtc cgcgattcat agctcgcagg gtacgggcgc gcgtgcgcac tccgcagccc 60 gttcaggacc ccggcgcggg cagggcgccc acgagctggc tggctgcttg cacccacatc 120 cttctttctc tgggacctgg ggtcgcggtt acttgggctg gccggcgaac ccttgagtgg 180 cctggcgggg agcgggcctc gcgcgcctgg agggccctgt ggaacgaaga gaggcacaca 240 gcatggcaga aaaccgagag ccccgcggtg ctgtggaggc tgaactggat ccagtggaat 300 acacccttag gaaaaggctt cccagccgcc tgccccggag acccaatgac atttatgtca 360 acatgaagac ggactttaag gcccagctgg cccgctgcca gaagctgctg gacggagggg 420 cccggggtca gaacgcgtgc tctgagatct acattcacgg cttgggcctg gccatcaacc 480 gcgccatcaa catcgcgctg cagctgcagg cgggcagctt cgggtccttg caggtggctg 540 ccaatacctc caccgtggag cttgttgatg agctggagcc agagaccgac acacgggagc 600 cactgactcg gatccgcaac aactcagcca tccacatccg agtcttcagg gtcacaccca 660 agtaattgaa aagacactcc tccacttatc ccctccgtga tatggctctt cgcatgctga 720 gtactggacc tcggaccaga gccatgtaag aaaaggcctg ttccctggaa gcccaaagga 780 ctctgcattg agggtggggg taattgtctc ttggtgggcc cagttagtgg gccttcctga 840 gtgtgtgtat gcggtctgta actattgcca tataaataaa aaatcctgtt gcactagtaa 900 aaaaaaaaaa 910 <210> 46 <211> 733 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 3591363CB1 <400> 46 gttcgcgtcg tttccgtttc cggccgaggc tgcgggaaga tggcggcggc catggcagca 60 tcttccctga cggtcacctt agggcggctg gcgtccgcgt gcagccacag catcctgaga 120 ccttcggggc ccggagcagc ctccctttgg tctgcttctc gaaggttcaa ttcacagagc 180 acttcatatc taccaggata tgttcctaaa acatccctga gttcaccacc ttggccagaa 240 gttgttctgc cagacccagt tgaggagacc agacaccatg cagaggtcgt gaagaaggtg 300 aatgagatga tcgtcacggg gcagtatggc aggctctttg ccgtggtgca ctttgccagc 360 cgccagtgga aggtgacctc tgaagacctg atcttaattg gaaatgaact agaccttgcg 420 tgtggagaga gaattcgact ggagaaggtc ctgctggttg gggcagacaa cttcacgctg 480 cttggcaagc cactcctcgg aaaggatctt gttcgagtag aagccacagt cattgaaaag 540 acagaatcat ggccaagaat cattatgaga ttcaggaaaa ggaaaaactt caagaagaaa 600 agaatcgtca cgaccccgca gactgtcctc cggataaaca gcattgagat tgctccgtgt 660 ttgttgtgat taccgagtta atacttacaa aaggataaaa ataaactcct gcttcccaag 720 gaaaaaaaaa aaa 733 <210> 47 <211> 918 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 3702292CB1 <400> 47 cgcatgcggc cgagtgcggg actggggctc ctggctgtgg gtgtggtacc gaggcttcag 60 cgggtgccgc ccgcctagag ggagtggagc ggtgagcacg tcaggggtgg ggggcgcagg 120 tcaagctttc accagttttt aattctttga tggggtaaat ttgagcaatt ttctcgactt 180 gtcgacattc gttattaact gagcaggaat caggagagga acccggtcct ctccacacag 240 cccagcagag agcctacgac tagatttgca tctttacgtc ctgcgcggag gctgctacac 300 acatgcagaa gtcatgctgg tggcctggac agtgaaggga gagaagtgga tttgggagac 360 atttaggaga tggcaccaaa agcgaaggaa gctcctgctc atcctaaagc cgaagccaaa 420 gcgaaggctt taaaggccaa gaaggcagtg ttgaaaggtg tccgcagcca cacgcaaaaa 480 cagaagatcc gcatgtcact caccttcagg cggcccaaga cactgcgact ccggaggcag 540 cccagatatc ctcggaagag cacccccagg agaaacaagc ttggccacta tgctatcatc 600 aagtttccgc tggccactga gtcggccgtg aagaagatag aagaaaacaa cacgcttgtg 660 ttcactgtgg atgttaaagc caacaagcac cagatcagac aggctgtgaa gaagctctat 720 gacagtgatg tggccaaggt caccaccctg atttgtcctg ataaggagaa caaggcatat 780 gttcgacttg ctcctgatta tgatgctttc gatgttgtaa caaaattggg atcacctaaa 840 ctgagtccag ctggctaact ctaaatatat gtgtatcttt tcagcataaa aaaataatgt 900 ttttcataaa aaaaaaaa 91g <210> 48 <211> 2680 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 3778908CB1 <400> 48 agtggaactg gatcgggttt gctgccagcg gcgtgagctt cggccgccat tttacaacag 60 ctccactcgc gccggacaca gggagcagcg agcacgcgtt tcccgcaacc cgataccatc 120 ggacaggatt tctccgcctc agcccaacgg ggagatctct ggaaacatgg ctacagaaca 180 tgttaatgga aatggtactg aagagcccat ggatactact tctgcagtta tccattcaga 240 aaattttcag acattgcttg atgctggttt accacagaaa gttgctgaaa aactagatga 300 aatttacgtt gcagggctag ttgcacatag tgatttagat gaaagagcta ttgaagcttt 360 aaaagaattc aatgaagacg gtgcattggc agttcttcaa cagtttaaag acagtgatct 420 ctctcatgtt cagaacaaaa gtgccttttt atgtggagtc atgaagactt acaggcagag 480 agaaaaacaa gggaccaaag tagcagattc tagtaaagga ccagatgagg caaaaattaa 540 ggcactcttg gaaagaacag gctacacact tgatgtgacc actggacaga ggaagtatgg 600 aggaccacct ccagattccg tttattcagg tcagcagcct tctgttggca ctgagatatt 660 tgtgggaaag atcccaagag atctatttga ggatgaactt gttccattat ttgagaaagc 720 tggacctata tgggatcttc gtctaatgat ggatccactc actggtctca atagaggtta 780 tgcgtttgtc actttttgta caaaagaagc agctcaggag gctgttaaac tgtataataa 840 tcatgaaatt cgttctggaa aacatattgg tgtctgcatc tcagttgcca acaataggct 900 ttttgtgggc tctattccta agagtaaaac caaggaacag attcttgaag aatttagcaa 960 agtaacagag ggtcttacag acgtcatttt ataccaccaa ccggatgaca agaaaaaaaa 1020 cagaggcttt tgctttcttg aatatgaaga tcacaaaaca gctgcccagg caaggcgtag 1080 gttaatgagt ggtaaagtca aggtctgggg gaatgttgga actgttgaat gggctgatcc 1140 tatagaagat cctgatcctg aggttatggc aaaggtaaaa gtgctgtttg tacgcaacct 1200 tgccaatact gtaacagaag agattttaga aaaggcattt agtcagtttg ggaaactgga 1260 acgagtgaag aagttaaaag attatgcgtt cattcatttt gatgagcgag atggtgctgt 1320 caaggctatg gaagaaatga atggcaaaga cttggaggga gaaaatattg aaattgtttt 1380 tgccaagcca ccagatcaga aaaggaaaga aagaaaagct cagaggcaag cagcaaaaaa 1440 tcaaatgtat gacgattact actattatgg tccacctcat atgccccctc caacaagagg 1500 tcgagggcgt ggaggtagag gtggttatgg atatcctcca gattattatg gatatgaaga 1560 ttattatgat tattatggtt atgattacca taactatcgt ggtggatatg aagatccata 1620 ctatggttat gaagattttc aagttggagc tagaggaagg ggtggtagag gagcaagggg 1680 tgctgctcca tccagaggtc gtggggctgc tcctccccgc ggtagagccg gttattcaca 1740 gagaggaggt cctggatcag caagaggcgt tcgaggtgcg agaggaggtg cccaacaaca 1800 aagaggccgc ggggtacgtg gtgcgagggg tggccgcggt ggaaatgtag gaggaaagcg 1860 caaagctgat gggtacaacc agccagattc caagcggcgc c:agaccaata atcagaactg 1920 gggctcccaa cccattgctc agcaaccgct ccaaggtggt gatcattctg gtaactatgg 1980 ttacaaatct gaaaaccagg agttttatca ggatactttt gggcaacagt ggaagtagaa 2040 acagtagggc ctctgtaaaa ttggagactg ataggttgat cagaaactca ccctaaatct 2100 gaacgggtgc cgctataatt tgtgacatct ggcaagattt ccctttatgt atatatttta 2160 acaatccgct tggacacgaa caaagccaca cttctaactg cttctggcga actgatttta 2220 tttttaattt ttttcaataa agatattctt agatactgaa agaaatagtt aatgagtttg 2280 catttgtgct tgagaaaatt tggctcaagt ccatttggct gtagtgtcaa cgatgtttcc 2340 agtagtgttt agatttggtg tcttcaaagg tagttgatta aaaccaagtg tgtctttaat 2400 atcttgtatc agaataactt tgtatgttac caacttaaat tgctagaata aggtaaattg 2460 atacacaact gctattttta atttagaact ttgacctaat ttgggttttc aaaaccattt 2520 tggctacttg tattctttat gctgttgttt atttcaataa aaaattcaca cctaaatgta 2580 tacttactaa aattgtgttt acaattcgtt tttcacaaaa tttcctgcaa atttggttca 2640 aattgtatag catgtcaagg ccaattaaag ggttttgtga 2680 <210> 49 <211> 2568 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 4163642CB1 <400> 49 ccaggctgcg ccagacagtg tagaacctgc ggcctcgatg tccttctccc gtgccctatt 60 gtgggctcgg ctcccggcgg ggcgccaggc tggccaccgg gcagccatct gctctgccct 120 tcgtccccac tttgggccct ttcccggggt tctggggcaa gtttctgtcc ttgccaccgc 180 ctcctcctct gcctccggtg gctccaaaat accaaacacg tccttgttcg tgcccctgac 240 tgtgaaacct cagggcccca gcgccgacgg cgacgtcggg gccgagctaa cccggcctct 300 ggacaagaat gaagtaaaga aggtcttaga caaattttac aagaggaaag aaattcagaa 360 actgggtgct gattatggac ttgatgctcg tctcttccac caagctttca taagctttag 420 aaattatatt atgcagtctc attccctgga tgtggacatt cacattgttt tgaatgatat 480 ttgcttcggt gcagctcatg cggatgattt attcccattt ttcttgagac atgccaaaca 540 aatatttcct gtgttggact gtaaggatga tctacgtaaa atcagtgact taagaatacc 600 acctaactgg tacccagatg ctagagccat gcagcggaag ataatatttc attcaggccc 660 cacaaacagt ggaaagactt atcacgcaat ccagaaatac ttctcagcaa agtctggagt 720 gtattgtggc cctctaaaat tactggcaca tgagatcttc gaaaagagta atgctgctgg 780 tgtgccatgt gacttggtga caggtgaaga gcgtgtgaca gttcagccaa atgggaaaca 840 ggcttcacat gtttcttgta cagttgagat gtgcagtgtt acaactcctt atgaagtggc 900 tgtaattgat gaaattcaaa tgattagaga tccagccaga ggatgggcct ggaccagagc 960 acttctagga ctgtgtgctg aagaggttca tttgtgtgga gaacctgctg ctattgacct 1020 ggtgatggag cttatgtaca caacggggga ggaagtggag gttcgagact ataagaggct 1080 tacccccatt tctgtgctgg accatgcact agaatcttta gataaccttc ggcctgggga 1140 ctgcattgtc tgttttagca agaatgatat ttattctgtg agtcggcaga ttgaaattcg 1200 gggattagaa tcagctgtta tatatggcag tctcccacct gggaccaaac ttgctcaagc 1260 aaaaaagttt aatgatccca atgacccatg caaaatcttg gttgctacag atgcaattgg 1320 catgggactt aatttgagca taaggagaat tattttttac tcccttataa agcccagtat 1380 caatgaaaag ggagagagag aactagaacc aatcacaacc tctcaagccc tgcagattgc 1440 tggcagagct ggcagattca gctcacggtt taaagaagga gaggttacaa caatgaatca 1500 tgaagatctc agtttattaa aggaaatttt gaagaggcct gtggatccta taagggcagc 1560 tggtcttcat ccaactgctg agcagattga aatgtttgcc taccatctcc ctgatgcaac 1620 actgtccaat ctcattgata tttttgtaga cttttcacaa gttgatgggc agtattttgt 1680 ctgcaatatg gatgatttta aattttctgc agagttgatc cagcatattc cactaagtct 1740 gcgagtgagg tatgttttct gcacagctcc tatcaacaag aagcagcctt ttgtgtgttc 1800 ttcactgtta cagtttgcca ggcagtatag caggaatgag cccctgacct ttgcatggtt 1860 acgccgatac atcaaatggc ctttacttcc acctaagaat attaaagacc tcatggatct 1920 tgaagctgtc cacgatgtct tggatcttta cttgtggcta agctaccgat ttatggatat 1980 gtttccagat gccagcctta ttcgagatct ccagaaagaa ctagatggta ttatccaaga 2040 tggtgtgcac aatatcacta aattgattaa aatgtctgag acgcataagc tgttgaattt 2100 ggagggcttt ccatcaggga gccagtcacg attgtcagga accttaaaga gccaagctag 2160 aaggacacgc ggcaccaaag ctctagggag taaagctact gagccaccca gccccgatgc 2220 aggagagctg tcccttgctt ccagattggt gcagcaagga ctcctcactc cagacatgct 2280 gaaacagcta gaaaaagagt ggatgacaca acaaactgaa cacaacaaag aaaaaacaga 2340 gtctgggact catccaaaag ggacgagaag aaagaagaag gaacctgatt cggactagtt 2400 ttctgttcct gttttttttt tttatttaat tttgcaaata~aaaatttatt ttgaatcctt 2460 tttcctcata tgcatttact ccctcctcta gtattgtggc tatctggtac tgggggattt 2520 ttggtgtgtg tgtgtgtttg tgtgtgtgtt tttttgtttg t.tttttcc 2568 <210> 50 <211> 847 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Identification No.: 4906154CB1 <400> 50 caaatggcgg atgacgccgg tgcagcgggg gggcccgggg gccctggtgg ccctgggatg 60 gggccggagg aaggccgagg ataaggagtg gatgcccgtc accaagttgg gccgcttggt 120 caaggacatg aagatcaagt ccctggagga gatctatctc ttctccctgc ccattaagga 180 atcagagatc attgatttct tcctgggggc ctctctcaag gatgaggttt tgaagattat 240 gccagtgcag aagcagaccc gtgccggcca gcgcaccagg ttcaaggcat ttgttgctat 300 cggggactac aatggccacg tcggtctggg tgttaagtgc tccaaggagg tggccaccgc 360 catccgtggg gccatcatcc tggccaagct ctccatcgtc cccgtgcgca gaggctactg 420 ggggaacaag atcggcaagc cccacactgt cccttgcaag gtgacaggcc gctgcggctc 480 tgtgctggta cgcctcatcc ctgcacccag gggcactggc atcgtctccg cacctgtgcc 540 taagaagctg ctcatgatgg ctggtatcga tgactgctac acctcagccc ggggctgcac 600 tgccaccctg ggcaacttcg ccaaggccac ctttgatgcc atttctaaga cctacagcta 660 cctgaccccc gacctctgga aggagactgt attcaccaag tctccctatc aggagttcac 720 tgaccacctc gtcaagaccc acaccagagt ctccgtgcag cggactcagg ctccagctgt 780 ggctacaaca tagggttttt atacaagaaa aataaagtga attaagcgtg ttaaaaaaaa 840 aaaaaaa 847

Claims (20)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-25 and fragments thereof.

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

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

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

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

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

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

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

9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:26-50 and fragments thereof.

10. An isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity to the polynucleotide of claim 9.

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

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

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

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

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

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

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

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

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

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