CA2338386A1 - Proteases and associated proteins - Google Patents

Abstract

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

Description

PROTEASES AND ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of proteases and associated proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative and immune disorders.
BACKGROUND OF THE INVENTION
Proteolytic processing is an essential component of normal cell growth, differentiation, remodeling, and homeostasis. The cleavage of peptide bonds within cells is necessary for the maturation of precursor proteins to their active forms, the removal of signal sequences from targeted proteins, the degradation of incorrectly folded proteins, and the controlled turnover of peptides within the cell. Proteases participate in apoptosis, inflammation, and tissue remodeling during embryonic development, wound heating, and normal growth. They are necessary components of bacterial, parasitic, and viral invasion and replication within a host. Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (See Beynon, R.J. and J.S.
Bond ( 1994) Proteolvtic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp. I-5.) The serine proteases (SPs) are a large family of proteolytic enzymes that include the digestive enzymes, trypsin and chymotrypsin; components of the complement cascade and of the blood-clotting cascade; and enzymes that control the degradation and turnover of macromolecules of the extracellular matrix. SPs are so named because of the presence of a serine residue in the active site for catalysis of protein cleavage. The active site of an SP is composed of a triad of residues including the aforementioned serine, an aspartate, and a histidine residue. SPs have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. The main sub-families are trypases which cleave after arginine or lysine; aspases which cleave after aspartate; chymases which cleave after phenylalanine or leucine; metases which cleavage after methionine; and serases which cleave after serine. Clp protease is a unique member of the serine protease family as its activity is controlled by a regulatory subunit that binds and hydrolyzes ATP. Clp protease was originally found in plant chloroplasts but is believed to be widespread in both prokaryotic and eukaryotic cells (Maurizi, M.R. et al. ( 1990) J. Biol. Chem.
265:12546-12552). SKD3, a mammalian homoiog of the bacterial Clp regulatory subunit, has recently been identified in mouse (Perier, F. et al. ( 1995) Gene 152:157-163).

Cysteine proteases are involved in diverse cellular processes ranging from the processing of precursor proteins to intracellular degradation. Mammalian cysteine proteases include lysosomal cathepsins and cytosolic calcium activated proteases, calpains. Of particular note, cysteine proteases are produced by monocytes, macrophages and other cells of the immune system S which migrate to sites of inflammation and, in their protective role, secrete various molecules to repair damaged tissue. These cells may overproduce the same molecules and cause tissue destruction in certain disorders. In autoimmune diseases such as rheumatoid arthritis, the secretion of the cysteine protease, cathepsin C, degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones. The cathepsin family of lysosomal proteases includes the cysteine proteases: cathepsins B, H, K, L, 02, and S;
and the aspartyl proteases: cathepsins D and G. Various members of this endosomal protease family are differentially expressed. Some, such as cathepsin D, have a ubiquitous tissue distribution while others, such as cathepsin L, are found only in monocytes, macrophages, and other cells of the immune system.
IS Aspartic proteases include bacterial penicillopepsin, mammalian pepsin, renin, chymosin, and certain fungal proteases. The characteristic active site residues of aspartic proteases are a pair of aspartic acid residues, for example, Asp33 and Asp213 in penicillopepsin.
Aspartic proteases are also called acid proteases because the optimum pH for their activity is between 2 and 3. In this pH range, one of the aspartate residues is ionized and the other is neutral. A
potent inhibitor of aspartic proteases is the hexapeptide pepstatin which, in the transition state, resembles normal substrates.
Carboxypeptidases A and B are the principal mammalian representatives of the metalloprotease family. Both are exopeptidases of similar structure and active site configuration.
Carboxypeptidase A, tike chymotrypsin, prefers C-terminal aromatic and aliphatic side chains of hydrophobic nature, whereas carboxypeptidase B is directed toward basic arginine and lysine residues. Active site components include zinc, which coordinates one histidine and two glutamic acid residues in the protein.
Proteasomes and ubiquitin proteases are both associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria. Proteasomes are large (2000 kDa), multisubunit complexes composed of a central catalytic core containing a variety of proteases, and terminal subunits that serve in substrate recognition and regulation of proteasome activity. The UCS mediates the elimination of abnormal proteins and regulates the half lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression. In the UCS pathway, a protein targeted for _2_ degradation is conjugated to ubiquitin, a small, heat-stable protein. The ubiquitinated protein is then recognized and degraded by a proteasome, and ubiquitin is released by ubiquitin protease for reutilization in the UCS. The UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. ( 1994) Cell 79:13-21 ). A murine proto-oncogene, Unp, encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation ofNIH 3T3 cells, and the human homolog of this gene is consistently elevated in small cell tumors and adenocarcinomas ofthe lung (Gray, D.A. (1995) Oncogene 10:2179-2183).
Many other proteolytic enzymes do not fit any of the major categories discussed above because their mechanisms of action and/or active sites have not been elucidated. These include the aminopeptidases and signal peptidases. Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Bovine leucine aminopeptidase is a zinc metalloenzyme that utilizes the sulfhydryl groups from at least three reactive cysteine residues at its active site in the binding of metal ions (Cuypers, H.T. et al.
( 1982) J. Biol. Chem.
257:7086-7091 ).
Signal peptidases are a specialized class of proteases found in all prokaryotic and eukaryotic cell types that serve in the processing of signal peptides. Signal peptides are amino-terminal sequences which direct the protein from its ribosomal assembly site to a particular cellular or extracellular location. Once the protein has been exported, removal of the signal sequence by a signal peptidase and posttranslational processing activate the protein. Signal peptidases exist as multi-subunit complexes in both yeast and mammals.
Protease inhibitors and other regulators of protease activity control the activity and effects of proteases. Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders {Murphy, G. ( 1991 ) Agents Actions Suppl. 35:69-76).
Low levels of the cystatins, low molecular weight inhibitors of the cysteine proteases, correlate with malignant progression of tumors. (Catkins, C. et al. (1995) Biol. Biochem. Hoppe Seyler 376:71-80). Also, increases in cysteine protease levels, when accompanied by reductions in inhibitor activity, are correlated with the pathology of arthritis and immunological diseases in humans.
Serpins are inhibitors of mammalian plasma serine proteases. Many serpins serve to regulate the blood clotting cascade and/or the complement cascade in mammals.
Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin. Sp32 binds the proenzyme, proacrosin, and thereby aides in packaging the enzyme into the acrosomal matrix (Baba, T. et al.
(1994) J. Biol. Chem. 269:10133-10140).

The Kunitz family of serine protease inhibitors is characterized by one or more "Kunitz domains" containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds. Members of this family include aprotinin, tissue factor pathway inhibitor (TFPI-1 and TFPI-2), inter-a-trypsin inhibitor, and bikunin (Marlor, C.W. et al. ( 1997) J. Biol. Chem. 272:12202-12208). Members of this family are potent inhibitors (in the nanomolar range) against serine proteases such as kallikrein and plasmin.
The discovery of new proteases and 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 and immune disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, proteases and associated proteins referred to collectively as "PPRG" and individually as "PPRG-1,"
"PPRG-2," "PPRG-3,"
"PPRG-4," "pPRG-S," "pPRG-6," "PPRG-7," "pPRG-8," "PPRG-9," "PPRG-10," "PPRG-1 I,"
"PPRG-12," "PPRG-13," "PPRG-14," "PPRG-I5," "PPRG-16," "PPRG-17," "PPRG-18,"
"PPRG-19," and "PPRG-20." In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO: I-20, 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:I-20 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:1-20 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucIeotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-20 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-20 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 N0:21-40, and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID N0:21-40 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the poiynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:21-40 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: I-20 and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ
ID NO: I-20 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:1-20 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 PPRG, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-20 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 PPRG, 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:I-20 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 PPRG.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods and algorithms used for identification of PPRG.
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 PPRG were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze PPRG, 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
"PPRG" refers to the amino acid sequences of substantially purified PPRG
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 PPRG, increases or prolongs the duration of the effect of PPRG. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of PPRG.
An "allelic variant" is an alternative form of the gene encoding PPRG. 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 I S changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding PPRG include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as PPRG
or a polypeptide with at least one functional characteristic of PPRG. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding PPRG, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding PPRG. 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 PPRG. 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 immunologicai activity of PPRG 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 PPRG 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 PPRG. 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 PPRG, decreases the amount or the duration of the effect of the biological or immunological activity of PPRG.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules I S which decrease the effect of PPRG.
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 PPRG polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that 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 irnmunogen 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 _g_ 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 PPRG, 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 "complementarily" refer to the natural binding of polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'." Complementarily between two single-stranded molecules may be "partial," such that only some of the nucleic acids bind, or it may be "complete," such that total complementarily exists between the single stranded molecules. The degree of complementarily between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the IS design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given poiynucleotide 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 PPRG or fragments of PPRG 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-Etmer, Norwalk CT) in the S' 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 PPRG, by northern analysis is indicative of the presence of nucleic acids encoding PPRG
in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding PPRG.
_g_ A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term ''derivative" refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
The term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity" may substitute for the word "similarity." A
partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined IS 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 MEGALIGN program (DNASTAR, Madison WI) 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 all pairs. The clusters are aligned pairwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues -l0-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 I S complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or ~tot 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.
The term "modulate" refers to a change in the activity of PPRG. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of PPRG.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein, refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In this context, "fragments" refers to those nucleic acid sequences which comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:21-40, for example, as distinct from any other sequence in the same genome. For example, a fragment of SEQ ID N0:21-40 is useful in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID N0:21-40 from related polynucleotide sequences. A fragment of SEQ ID N0:21-40 is at least about IS-20 nucleotides in length. The precise length of the fragment of SEQ ID N0:21-40 and the region of SEQ ID
N0:21-40 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. In some cases, a fragment, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded poiypeptide.
While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
The term "oligonucleotide" refers to a nucleic acid sequence of at least about nucleotides to 60 nucleotides, preferably about 1 S to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray. "Oligonucleotide" is substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
The term "sample" is used in its broadest sense. A sample suspected of containing nucleic acids encoding PPRG, or fragments thereof, or PPRG 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 PPRG polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
t 0 The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to PPRG. This definition may also include, for example, "allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains.
Species variants are polynucieotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
THE INVENTION
The invention is based on the discovery of new human proteases and associated proteins (PPRG), the polynucleotides encoding PPRG, and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative and immune disorders.
Table I lists the Incyte clones used to assemble full length nucleotide sequences encoding PPRG. 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 PPRG were identified, and column 4 shows the cDNA libraries from which these clones were isolated. Column S 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 ~ were used to assemble the consensus nucleotide sequence of each PPRG and are useful as fragments in hybridization technologies.

The columns of Table 2 show various properties of each of the polypeptides of the invention: column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites;
column 4 shows potential glycosylation sites; column S shows the amino acid residues comprising signature sequences and motifs; column 6 shows the identity of each polypeptide; and column 7 shows analytical methods used to identify each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding PPRG. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists tissue categories which express PPRG as a fraction of total tissue categories expressing PPRG. Column 3 lists diseases, disorders, or conditions associated with those tissues expressing PPRG. Column 4 lists the vectors used to subctone the cDNA library. Of particular note is the kidney-specific expression of SEQ ID
N0:29 in S out of 7 libraries (71 %). Also of note is expression of SEQ ID N0:34 in cervical tumor libraries (60%).
The columns of Table 4 show descriptions of the tissues used to construct the cDNA
libraries from which cDNA clones encoding PPRG were isolated. Column 1 references the nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
The following fragments of the nucleotide sequences encoding PPRG are useful, for example, in hybridization or amplification technologies to identify SEQ ID
N0:21-40 and to distinguish between SEQ ID N0:21-40 and related polynucleotide sequences. The useful fragments include the fragment of SEQ ID N0:21 from about nucleotide I to about nucleotide 56;
the fragment of SEQ ID N0:22 from about nucleotide 161 to about nucleotide 213; the fragment of SEQ ID N0:23 from about nucleotide 110 to about nucleotide 158; the fragment of SEQ ID
N0:24 from about nucleotide 117 to about nucleotide 174; the fragment of SEQ
ID N0:25 from about nucleotide 191 to about nucleotide 245; the fragment of SEQ 1D N0:26 from about nucleotide 204 to about nucleotide 269; the fragment of SEQ 1D N0:27 from about nucleotide 181 to about nucleotide 221; the fragments of SEQ ID N0:28 from about nucleotide 509 to about nucleotide 553, and from about nucleotide 1751 to about nucleotide 1795; the fragment of SEQ ID
N0:29 from about nucleotide 326 to about nucletide 370; the fragment of SEQ ID
N0:30 from about nucleotide 1190 to about nucleotide 1234; the fragment of SEQ ID N0:31 from about nucleotide 283 to about nucleotide 324; the fragment of SEQ ID N0:32 from about nucleotide 380 to about nucleotide 424; the fragments of SEQ ID N0:33 from about nucleotide 272 to about nucleotide 316, and from about nucleotide 1784 to about nucleotide I 831; the fragment of SEQ ID
N0:34 from about nucleotide 217 to about nucleotide 261; the fragment of SEQ
ID N0:35 from about nucleotide 2397 to about nucleotide 2441; the fragment of SEQ ID N0:36 from about nucleotide 218 to about nucleotide 262; the fragments of SEQ ID N0:37 from about nucleotide 165 to about nucleotide 209, and from about nucleotide 651 to about nucleotide 695; the fragment of SEQ ID N0:38 from about nucleotide 812 to about nucleotide 856; the fragment of SEQ ID
N0:39 from about nucleotide 541 to about nucleotide 585; and the fragment of SEQ ID N0:40 from about nucleotide 163 to about nucleotide 207. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
The invention also encompasses PPRG variants. A preferred PPRG 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 PPRG amino acid sequence, and which contains at least one functional or structural characteristic of PPRG.
The invention also encompasses polynucleotides which encode PPRG. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:21-40, which encodes PPRG.
The invention also encompasses a variant of a polynucleotide sequence encoding PPRG.
In particular, such a variant polynucleotide sequence will have at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding PPRG. A particular aspect of the invention encompasses a variant of a polynucieotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:21-40 which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID N0:21-40. Any one ofthe polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of PPRG.
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 PPRG, 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 PPRG, and all such variations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode PPRG and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring PPRG under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding PPRG 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 PPRG 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 PPRG
and PPRG 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 PPRG 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
N0:21-40 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.
152:507-5 I I.) 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 SO% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ~g/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 ~cg/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 I S 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 exonucieases 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 Biolo y, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. ( 1995) Molecular Biolo~y and BiotechnoloQV, Wiley VCH, New York NY, pp.856-853.) The nucleic acid sequences encoding PPRG 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 S 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.
I:I 11-I 19.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about SO% 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 oiigo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into S' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 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 PPRG may be cloned in recombinant DNA molecules that direct expression of PPRG, 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 PPRG.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter PPRG-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter giycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding PPRG may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. ( 1980) Nucleic Acids Symp. Ser. 7:225-232.) Alternatively, PPRG 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 431 A peptide synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of PPRG, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY.) In order to express a biologically active PPRG, the nucleotide sequences encoding PPRG
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 PPRG. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding PPRG. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding PPRG 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 translationat 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 PPRG 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, IS New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding PPRG. 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 PPRG.
For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding PPRG 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 PPRG
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 PPRG are needed, e.g. for the production of antibodies, vectors which direct high level expression of PPRG

may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of PPRG. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel, 1995, supra; Grant et ai. ( 1987) Methods Enzymol. 153:516-54; and Scorer, C.
A. et al. ( 1994) Bio/Technology 12:181-184.) Plant systems may also be used for expression of PPRG. Transcription of sequences encoding PPRG may be driven viral promoters, e.g., the 35S and 195 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 al. ( 1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. ( 1984) Science 224:838-843; and Winter, J. et al. ( 1991 ) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technolosy (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 PPRG
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 PPRG in host cells. (See, e.g., Logan, J. and T. Shenk ( 1984) Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat.
Genet. 15:345-355.) For long term production of recombinant proteins in mammalian systems, stable expression of PPRG in cell lines is preferred. For example, sequences encoding PPRG can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk or apr cells, respectively.
(See, e.g., Wigler, M. et al. ( 1977) Cell 11:223-232; Lowy, 1, et al. ( 1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418;
and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci.
U.S.A. 77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-I4.) 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. U.S.A. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), !3 glucuronidase and its substrate Q-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. Bioi. SS:I2I-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 PPRG is inserted within a marker gene sequence, transformed cells containing sequences encoding PPRG can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding PPRG
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 PPRG
and that express PPRG 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 PPRG 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 S fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PPRG 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) Serolosical Methods a Laboratory Manual, APS Press, St.
Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Current Protocols in ImmunoloEV, 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 PPRG
IS include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding PPRG, 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 PPRG 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 PPRG may be designed to contain signal sequences which direct secretion of PPRG 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, Bethesda MD) and may be chosen to ensure the S correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding PPRG 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 PPRG protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of PPRG
activity. Heterotogous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the PPRG
encoding sequence and the heteroiogous protein sequence, so that PPRG 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 PPRG 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 PPRG may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI 431A peptide synthesizer (Perkin-Elmer). Various fragments of PPRG 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 PPRG and proteases and associated proteins. In addition, the expression of PPRG is closely associated with cell proliferative conditions, including cancer, and with inflammation and the immune response. Therefore, PPRG appears to play a role in cell proliferative and immune disorders. In the treatment of cell proliferative and immune disorders associated with increased PPRG expression or activity, it is desirable to decrease the expression or activity of PPRG. In the treatment of the above conditions associated with decreased PPRG
expression or activity, it is desirable to increase the expression or activity of PPRG.
Therefore, in one embodiment, PPRG 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 PPRG. 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;
and an immune disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma.
In another embodiment, a vector capable of expressing PPRG 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 PPRG including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified PPRG 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 PPRG
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of PPRG
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of PPRG including, but not limited to, those listed above.
In a further embodiment, an antagonist of PPRG may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PPRG. Examples of such disorders include, but are not limited to, those described above. In one aspect, an antibody which specifically binds PPRG 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 PPRG.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding PPRG may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of PPRG 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 PPRG may be produced using methods which are generally known in the art. In particular, purified PPRG may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PPRG.
Antibodies to PPRG 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 PPRG or with any fragment or oiigopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitropheno(.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium~arvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to PPRG 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 PPRG 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 PPRG 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. U.S.A.
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Ceil 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. U.S.A. 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 PPRG-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. U.S.A. 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. U.S.A. 86:
3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-299.) Antibody fragments which contain specific binding sites for PPRG may also be generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between PPRG and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering PPRG 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 PPRG. Affinity is expressed as an association constant, Ka, which is defined as the molar concentration of PPRG-antibody complex t 5 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 PPRG epitopes, represents the average affinity, or avidity, of the antibodies for PPRG. The Ka determined for a preparation of monoclonal antibodies, which are monospecific for a particular PPRG epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'z L/mole are preferred for use in immunoassays in which the PPRG-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of PPRG, 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 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is preferred for use in procedures requiring precipitation of PPRG-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 PPRG, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding PPRG 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 PPRG. Thus, complementary molecules or fragments may be used to modulate PPRG 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 PPRG.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding PPRG. (See, e.g., Sambrook, supra; Ausubel, 1995, supra.) Genes encoding PPRG can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding PPRG. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding PPRG. Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +1 O 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.1. Can, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-I77.) 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 PPRG.
Specifc 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 PPRG. Such DNA sequences may be IS 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) Nat. Biotech. 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 PPRG, antibodies to PPRG, and mimetics, agonists, antagonists, or inhibitors of PPRG. 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, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remin~ton'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 get, 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 weft as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include t5 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, encapsutating, 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 acids. 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 PPRG, 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 PPRG or fragments thereof, antibodies of PPRG, and agonists, antagonists or inhibitors of PPRG, 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 EDS° {the dose therapeutically effective in 50% of the population) or LDS° (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic IS effects is the therapeutic index, which can be expressed as the LDS°/EDS° 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 fig, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

DIAGNOSTICS
In another embodiment, antibodies which specifically bind PPRG may be used for the diagnosis of disorders characterized by expression of PPRG, or in assays to monitor patients being treated with PPRG or agonists, antagonists, or inhibitors of PPRG. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for PPRG include methods which utilize the antibody and a label to detect PPRG 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 PPRG, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of PPRG
expression. Normal or standard values for PPRG expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to I S PPRG under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of PPRG 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 PPRG 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 PPRG
may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of PPRG, and to monitor regulation of PPRG
levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucieotide sequences, including genomic sequences, encoding PPRG ar closely related molecules may be used to identify nucleic acid sequences which encode PPRG.
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 PPRG, 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 PPRG encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:21-40 or from genomic sequences including promoters, enhancers, and introns of the PPRG gene.
Means for producing specific hybridization probes for DNAs encoding PPRG
include the cloning of polynucleotide sequences encoding PPRG or PPRG 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.
Polynucleotide sequences encoding PPRG may be used for the diagnosis of disorders associated with expression of PPRG. 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; and an immune disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with fymphocytotoxins, 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, poiymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Wemer syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma. The polynucleotide sequences encoding PPRG
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 PPRG expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding PPRG may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding PPRG 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 PPRG 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 PPRG, 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 PPRG, 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 PPRG 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 polynucieotide encoding PPRG, or a fragment of a poiynucleotide complementary to the S polynucleotide encoding PPRG, 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 PPRG include radiolabeiing 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 polynucieotide 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. U.S.A. 93:10614-10619; Baldeschweiler et al. (1995) PCT application W09S/251116;
Shalom D. et al. ( 1995) PCT application W095/3SSOS; Heller, R.A. et al. ( 1997) Proc. Natl. Acad.
2S Sci. U.S.A. 94:2150-2155; and Heller, M.J. et al. ( 1997) U.S. Patent No.
5,605,662.) In another embodiment of the invention, nucleic acid sequences encoding PPRG
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 PI constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 1 S:34S-3SS; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask, B.J. ( 1991 ) Trends Genet. 7:149-I S4.) 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, su ra 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 PPRG on a physical chromosomal map and a specific disorder, or a S 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 positionai cloning or other gene discovery techniques. Once the disease or syndrome has been IS crudely Localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to I 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, PPRG, 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 PPRG 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 PPRG, or fragments thereof, and washed. Bound PPRG is then detected by methods well known in the art.
Purified PPRG can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding PPRG specifically compete with a test compound for WO 00/09709 PCTlUS99/17818 binding PPRG. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with PPRG.
In additional embodiments, the nucleotide sequences which encode PPRG 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. 60/096,114 and U.S. Ser. No. 60/119,768, 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 (ysed 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 S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT piasmid (Stratagene), pSPORTI plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or SOLR from Stratagene or DHSa, DH l OB, or ElectroMAX DH I OB from Life Technologies.
II. Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector system (Stratagene) or cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep 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 PCB in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-I4). 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 (Bobbins 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-Elmer) 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 polynuc(eotide 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 S 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 Conned, 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. Struct. Biol. 6:361-365.) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID N0:21-40. 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, supra, ch. 7;
Ausubel, 1995, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQ (Incyte Pharmaceuticals).
This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defned as:
% seguence 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 IS within a 1% 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 PPRG 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 2S libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3 V. Extension of PPRG Encoding Polynucleotides The full length nucleic acid sequences of SEQ ID N0:21-27 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. For each nucleic acid sequence, one primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide. Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new unknown nucleotide sequence for the region of interest. The initial primers were designed from the cDNA using OLIGO 4.06 (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries (Life Technologies) were used to extend the sequence: If more than one extension is necessary or desired, additional sets of primers are designed to further extend the known region.
High fidelity amplification was obtained by following the instructions for the XL-PCR kit (Perkin-Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was performed using the PTC200 thermal cycier (M.J. Research) beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:
Step 1 94C for 1 min (initial denaturation) Step 2 65 C for 1 min Step 3 68C for 6 min Step 4 94C for 15 sec Step 5 65C for 1 min Step 6 68C for 7 min Step 7 Repeat steps 4-6 for an additional 15 cycles Step 8 94C for IS sec Step 9 65C for 1 min Step 10 68C for 7:15 min Step 1 i Repeat steps 8-10 for an additional 12 cycles Step 12 72C for 8 min Step 13 4C (and holding) A 5 ul to 10 ~cl aliquot of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6% to 0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were excised from the gel, purified using the QIAQUICK kit (QIAGEN), and trimmed of overhangs using Klenow enzyme to facilitate relegation and cloning.
After ethanol precipitation, the products were redissolved in 13 ~1 of legation buffer, 1/.cl T4-DNA ligase ( 15 units) and l~cl T4 polynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours, or overnight at 16°C.
Competent E. coli cells (in 40 Icl of appropriate media) were transformed with 3 ~l of legation mixture and cultured in 80 ~1 of SOC medium. (See, e.g., Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C, the E. coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra, Appendix A, p. I) containing carbenicillin (2x carb). The following day, several colonies were randomly picked from each plate and cultured in i 50 ,ul of liquid LB/2x carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ~1 of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 ~I from each sample was transferred into a PCR array.
For PCR amplification, 18 ul of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene-specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
Step 1 94C for 60 sec Step 2 94C for 20 sec Step 3 SSC for 30 sec Step 4 72 C for 90 sec Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6 72C for 180 sec Step 7 4C (and holding) Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs, and appropriate clones were selected, ligated into plasmid, and sequenced.
In like manner, the nucleotide sequence of SEQ ID N0:21-27 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for 5' extension, and an appropriate genomic library.
The full length nucleic acid sequences of SEQ ID N0:28-40 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg2+, (NHQ)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, S min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing I00 p.l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in 1 X 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 ~l to 10 ~l 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 W1), and sonicated or sheared prior to religation into pUC I 8 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.$%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. toll cells.
Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step I: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, I min; Step 4: 72°C, 2 min;
Step S: 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 ( I :2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In Iike manner, the nucleotide sequences of SEQ 1D N0:28-40 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 N0:21-40 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 uCi of [y-'2PJ 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 I 0' 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 IS 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 dodeeyl 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 ai. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides Sequences complementary to the PPRG-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring PPRG.
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 PPRG. 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 PPRG-encoding transcript.
IX. Expression of PPRG
I S Expression and purification of PPRG is achieved using bacterial or virus-based expression systems. For expression of PPRG 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 PPRG upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of PPRG in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autoeraphica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding PPRG by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E.K. et ai. ( 1994) Proc. Natl.
Acad. Sci. U.S.A.
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.) In most expression systems, PPRG 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 enryme from Schistosoma iaponicum, 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 proteolyticaily cleaved from PPRG at specifically engineered sites. FLAG, an 8-amino acid S peptide, enables immunoaffinity purification using commercially available monoclonal and polyelonal 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 PPRG obtained by these methods can be used directly in the following activity assay.
X. Demonstration of PPRG Activity Protease activity of PPRG is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantified by spectrophotometric (or fluorometric) absorption of the released chromophore.
(Beynon, R.J. and J.S. Bond ( 1994) Proteolytic Enzymes: A Practical Approach Oxford I S University Press, New York NY, pp.25-55.) Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases), animopeptidase (leucine aminopeptidase), or carboxypeptidase (Carboxypeptidase A and B, procollagen C-proteinase). Chromogens commonly used are 2-naphthylamine, 4-nitroaniline, and furylacrylic acid. Assays are performed atambient temperature and contain an aliquot of the enzyme and the appropriate substrate in a suitable buffer. Reactions are carried out in an optical cuvette and followed by measurement of the increase/decrease in absorbance of the chromogen released during hydrolysis of the peptide substrate. The change in absorbance is proportional to the enryme activity in the assay.
Regulation of protease activity (agonism or antagonism) by PPRG is measured using an appropriate protease assay as described above in the presence or absence of PPRG as an agonist or inhibitor of this activity. Protease activity is measured in the absence of PPRG (control activity) and in the presence of varying amounts of PPRG. The change in protease activity compared to the control is proportional to the amount of PPRG in the assay and is a measure of the protease regulatory activity of PPRG.
For example, for inhibitory activity of PPRG-2, the assay is carried out as described above for PPRG using a calcium activated protease, such as calpain, assayed in the absence and in the presence of various concentrations of PPRG-2. Inhibition of calpain protease activity is proportional to the activity of PPRG-2 in the assay. Similarly, for inhibitory activity of PPRG-4 and PPRG-9, assays are carried out as described above for PPRG using pancreatic trypsin assayed in the absence and in the presence of various concentrations of PPRG-4 or PPRG-9. Inhibition of pancreatic trypsin protease activity is proportional to the activity of PPRG-4 or PPRG-9 in the assay.
XI. Functional Assays PPRG function is assessed by expressing the sequences encoding PPRG at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.

5-10 ~g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome 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 cytomeiry (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 PPRG on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding PPRG 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 PPRG and other genes of interest can be analyzed by northern analysis or microarray techniques.

XII. Production of PPRG Specific Antibodies PPRG 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.
S Alternatively, the PPRG amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic cegions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.) Typically, oligopeptides 1S 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-ICLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity 1S 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 PPRG Using Specific Antibodies Naturally occurring or recombinant PPRG is substantially purified by immunoaffinity chromatography using antibodies specific for PPRG. An immunoaffinity column is constructed by covalently coupling anti-PPRG 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 PPRG are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PPRG (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/PPRG 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 PPRG is collected.
XIV. Identification of Molecules Which Interact with PPRG
PPRG, or biologically active fragments thereof, are labeled with 'ZSI Bolton-Hunter reagent. (See, e.g., Bolton, A.E. and W.M. Hunter ( 1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled PPRG, washed, and any wells with labeled PPRG complex are assayed. Data obtained using different concentrations of PPRG are used to calculate values for the number, affinity, and association of PPRG 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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a y s o y .o U rn -6a-SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
BANDMAN, Olga HILLMAN, Jennifer L.
BAUGHN, Mariah R.
AZIMZAI, Yalda GUEGLER, Karl J.
CORLEY, Neil C.
YUE, Henry TANG, Y. Tom REDDY, Roopa PATTERSON, Chandra AU-YOUNG, Janice SHI, Leo L.
LU, Dyung Aina M.
<120> PROTEASES AND ASSOCIATED PROTEINS
<130> PF-OS69 PCT
<140> To Be Assigned <141> Herewith <150> 60/096,114; 60/119,768 <151> 1998-08-10; 1999-02-11 <160> 40 <170> PERL Program <210> 1 <211> 206 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1220330 <400> 1 Met Pro Ser Arg Arg Arg Asp Ala Ile Lys Val Met Gln Arg Phe Ala Gly Leu Pro Glu Thr Gly Arg Met Asp Pro Gly Thr Val Ala Thr Met Arg Lys Pro Arg Cys Ser Leu Pro Asp Val Leu Gly Val Ala Gly Leu Val Arg Arg Arg Arg Arg Tyr Ala Leu Ser Gly Ser Val Trp Lys Lys Arg Thr Leu Thr Trp Arg Val Arg Ser Phe Pro Gln Ser Ser Gln Leu Ser Gln Glu Thr Val Arg Val Leu Met Ser Tyr Ala Leu Met Ala Trp Gly Met Glu Ser Gly Leu Thr Phe His Glu Val Asp Ser Pro Gln Gly Gln Glu Pro Asp Ile Leu Ile Asp ll0 115 120 Phe Ala Arg Ala Phe His Gln Asp Ser Tyr Pro Phe Asp Gly Leu Gly Gly Thr Leu Ala His Ala Phe Phe Pro Gly Glu His Pro Ile Ser Gly Asp Thr His Phe Asp Asp Glu Glu Thr Trp Thr Phe Gly Ser Lys Ala Ser Gln Gln Leu Glu Gln Glu Leu Ala Gly Gly Ser Pro Val Asp Glu Glu Leu Gly Phe Ser Arg Gly Trp Arg Val Asn Pro Leu Gly Pro Gly Ser Pro Glu Arg Leu Ser <210> 2 <211> 754 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1342493 <400> 2 Met Ala Phe Ala Ser Trp Trp Tyr Lys Thr His Val Ser Glu Lys Thr Ser Glu Ser Pro Ser Lys Pro Gly Glu Lys Lys Gly Ser Asp Glu Lys Lys Ala Ala Ser Leu Gly Ser Ser Gln Ser Ser Arg Thr Tyr Ala Gly Gly Thr Ala Ser Ala Thr Lys Val Ser Ala Ser Ser Gly Ala Thr Ser Lys Ser Ser Ser Met Asn Pro Thr Glu Thr Lys Ala Val Lys Thr Glu Pro Glu Lys Lys Ser Gln Ser Thr Lys Leu Ser Val Val His Glu Lys Lys Ser Gln Glu Gly Lys Pro Lys Glu His Thr Glu Pro Lys Ser Leu Pro Lys Gln Ala Ser Asp Thr Gly Ser Asn Asp Ala His Asn Lys Lys Ala Val Ser Arg Ser Ala Glu Gln Gln Pro Ser Glu Lys Ser Thr Glu Pro Lys Thr Lys Pro Gln Asp Met Ile Ser Ala Gly Gly Glu Ser Val Ala Gly Ile Thr Ala Ile Ser Gly Lys Pro Gly Asp Lys Lys Lys Glu Lys Lys Ser Leu Thr Pro Ala Val Pro Val Glu Ser Lys Pro Asp Lys Pro Ser Gly Lys Ser Gly Met Asp Ala Ala Leu Asp Asp Leu Ile Asp Thr Leu Gly Gly Pro Glu Glu Thr Glu Glu Glu Asn Thr Thr Tyr Thr Gly Pro Glu Val Ser Asp Pro Met Ser Ser Thr Tyr Ile Glu Glu Leu Giy Lys Arg Glu Val Thr Ile Pro Pro Lys Tyr Arg Glu Leu Leu Ala Lys Lys Glu Gly Ile Thr Gly Pro Pro Ala Asp Ser Ser Lys Pro Ile Gly Pro Asp Asp Ala Ile Asp Ala Leu Ser Ser Asp Phe Thr Cys Gly Ser Pro Thr Ala Ala Gly Lys Lys Thr Glu Lys Glu Glu Ser Thr Glu Val Leu Lys Ala Gln Ser Ala Gly Thr Val Arg Ser Ala Ala Pro Pro Gln Glu Lys Lys Arg Lys Val Glu Lys Asp Thr Met Ser Asp Gin Ala Leu Glu Ala Leu Ser Ala Ser Leu Gly Thr Arg Gln Ala Glu Pro GIu Leu Asp Leu Arg Ser Ile Lys Glu Val Asp Glu Ala Lys Ala Lys Glu Glu Lys Leu Glu Lys Cys Gly Glu Asp Asp Glu Thr Ile Pro Ser Glu Tyr Arg Leu Lys Pro Ala Thr Asp Lys Asp Gly Lys Pro Leu Leu Pro Glu Pro Glu Glu Lys Pro Lys Pro Arg Ser Glu Ser Glu Leu Ile Asp Glu Leu Ser Glu Asp Phe Asp Arg Ser Glu Cys Lys Glu Lys Pro Ser Lys Pro Thr Glu Lys Thr Glu Glu Ser Lys Ala Ala Ala Pro Ala Pro Val Ser Glu Ala Val Cys Arg Thr Ser Met Cys Ser Ile Gln Ser Ala Pro Pro Glu Pro Ala Thr Leu Lys Gly Thr Val Pro Asp Asp Ala Val Glu Ala Leu Ala Asp Ser Leu Gly Lys Lys Glu Ala Asp Pro Glu Asp Gly Lys Pro Val Met Asp Lys Val Lys Giu Lys Ala Lys Glu Glu Asp Arg Glu Lys Leu Gly Glu Lys Glu Glu Thr Ile Pro Pro Asp Tyr Arg Leu Glu Glu Val Lys Asp Lys Asp Gly Lys Pro Leu Leu Pro Lys Glu Ser Lys Glu Gln Leu Pro Pro Met Ser Glu Asp Phe Leu Leu Asp Ala Leu Ser Glu Asp Phe Ser Gly Pro Gln Asn Ala Ser Ser Leu Lys Phe Glu Asp Ala Lys Leu Ala Ala Ala Ile Ser Glu Val Val Ser Gln Thr Pro Ala Ser Thr Thr Gln Ala Gly Ala Pro Pro Arg Asp Thr Ser Gln Ser Asp Lys Asp Leu Asp Asp Aia Leu Asp Lys Leu Ser Asp Ser Leu Gly Gln Arg Gln Pro Asp Pro Asp Glu Asn Lys Pro Met Glu Asp Lys Val Lys Glu Lys Ala Lys Ala Glu His Arg Asp Lys Leu Gly Glu Arg Asp Asp Thr Ile Pro Pro Glu Tyr Arg His Leu Leu Asp Asp Asn Gly Gln Asp Lys Pro Val Lys Pro Pro Thr Lys Lys Ser Glu Asp Ser Lys Lys Pro Ala Asp Asp Gln Asp Pro Ile Asp Ala Leu Ser Gly Asp Leu Asp Ser Cys Pro Ser Thr Thr Glu Thr Ser Gln Asn Thr Ala Lys Asp Lys Cys Lys Lys Ala Ala Ser Ser Ser Lys Ala Pro Lys Asn Gly Gly Lys Ala Lys Asp Ser Ala Lys Thr Thr Glu Glu Thr Ser Lys Pro Lys Asp Asp <210> 3 <211> 308 <222> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1698270 <400> 3 Met Gly Glu Ile Lys Val Ser Pro Asp Tyr Asn Trp Phe Arg Gly Thr Val Pro Leu Lys Lys Ile Ile Val Asp Asp Asp Asp Ser Lys Ile Trp Ser Leu Tyr Asp Ala Gly Pro Arg Ser Ile Arg Cys Pro Leu Ile Phe Leu Pro Pro Val Ser Gly Thr Ala Asp Val Phe Phe Arg Gln Ile Leu Ala Leu Thr Gly Trp Gly Tyr Arg Val Ile Ala Leu Gln Tyr Pro Val Tyr Trp Asp His Leu Glu Phe Cys Asp Gly Phe Arg Lys Leu Leu Asp His Leu Gln Leu Asp Lys Val His Leu Phe Gly Ala Ser Leu Gly Gly Phe Leu Ala Gln Lys Phe Ala Glu Tyr Thr His Lys Ser Pro Arg Val His Ser Leu ile Leu Cys Asn Ser Phe Ser Asp Thr Ser Ile Phe Asn Gln Thr Trp Thr Ala Asn Ser Phe Trp Leu Met Pro Ala Phe Met Leu Lys Lys Ile Val Leu Gly Asn Phe Ser Ser Gly Pro Val Asp Pro Met Met Ala Asp Ala Ile Asp Phe Met Val Asp Arg Leu Glu Ser Leu Gly Gln Ser Glu Leu Ala Ser Arg Leu Thr Leu Asn Cys Gln Asn Ser Tyr Val Glu Pro His Lys Ile Arg Asp Ile Pro Val Thr Ile Met Asp Val Phe Asp Gln Ser Ala Leu Ser Thr Glu Ala Lys Glu Glu Met Tyr Lys Leu Tyr Pro Asn Ala Arg Arg Ala His Leu Lys Thr Gly Gly Asn Phe Pro Tyr Leu Cys Arg Ser Ala Glu Val Asn Leu Tyr Val Gln Ile His Leu Leu Gln Phe His Gly Thr Lys Tyr Ala Ala Ile Asp Pro Ser Met Val Ser Ala Glu Glu Leu Glu Val Gln Lys Gly Ser Leu Gly Ile Ser Gln Glu Glu Gln <210> 4 <211> 164 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2012492 <400> 4 Met Lys Thr Gln Asp Gly Gly Ile His Ser Glu Gly Ala Ala Ala Glu His Ser Lys Phe Gly Asn His Gln Lys Gly Trp Pro Leu Phe Asn Met Gly Ser Ser Gly Leu Leu Ser Leu Leu Val Leu Phe Val Leu Leu Ala Asn Val Gln Gly Pro Gly Leu Thr Asp Trp Leu Phe Pro Arg Arg Cys Pro Lys Ile Arg Glu Glu Cys Glu Phe Gln Glu Arg Asp Val Cys Thr Lys Asp Arg Gln Cys Gln Asp Asn Lys Lys Cys Cys Val Phe Ser Cys Gly Lys Lys Cys Leu Asp Leu Lys Gln Asp Val Cys Glu Met Pro Lys Glu Thr Gly Pro Cys Leu Ala Tyr Phe Leu His Trp Trp Tyr Asp Lys Lys Asp Asn Thr Cys Ser Met Phe Val Tyr Gly Gly Cys Gln Gly Asn Asn Asn Asn Phe Gln Ser Lys Ala Asn Cys Leu Asn Thr Cys Lys Asn Lys Arg Phe Pro <2i0> 5 <211> 565 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2309875 <400> 5 Met Pro Gln Ala Ser Glu His Arg Leu Gly Arg Thr Arg Glu Pro Pro Val Asn Ile Gln Pro Arg Val Gly Ser Lys Leu Pro Phe Ala Pro Arg Ala Arg Ser Lys Glu Arg Arg Asn Pro Ala Ser Gly Pro Asn Pro Met Leu Arg Pro Leu Pro Pro Arg Pro Gly Leu Pro Asp Glu Arg Leu Lys Lys Leu Glu Leu Gly Arg Gly Arg Thr Ser Gly Pro Arg Pro Arg Gly Pro Leu Arg Ala Asp His Gly Val Pro Leu Pro Gly Ser Pro Pro Pro Thr Val Ala Leu Pro Leu Pro Ser Arg Thr Asn Leu Ala Arg Ser Lys Ser Val Ser Ser Gly Asp Leu Arg Pro Met Gly Ile Ala Leu Gly Gly His Arg Gly Thr Gly Glu Leu Gly Ala Ala Leu Ser Arg Leu Ala Leu Arg Pro Glu Pro Pro Thr Leu Arg Arg Ser Thr Ser Leu Arg Arg Leu Gly Gly Phe Pro Gly Pro Pro Thr Leu Phe Ser Ile Arg Thr Glu Pro Pro Ala Ser His Gly Ser Phe His Met Ile Ser Ala Arg Ser Ser Glu Pro Phe Tyr Ser Asp Asp Lys Met Ala His His Thr Leu Leu Leu Gly Ser Gly His Val Gly Leu Arg Asn Leu Gly Asn Thr Cys Phe Leu Asn Ala Val Leu Gln Cys Leu Ser Ser Thr Arg Pro Leu Arg Asp Phe Cys Leu Arg Arg Asp Phe Arg Gln Glu Val Pro Gly Gly Gly Arg Ala GIn Glu Leu Thr Glu Ala Phe Ala Asp Val Ile Gly Ala Leu Trp His Pro Asp Ser Cys Glu Ala Val Asn Pro Thr Arg Phe Arg Ala Val Phe Gln Lys Tyr Val Pro Ser Phe Ser Gly Tyr Ser Gln Gln Asp Ala Gln Glu Phe Leu Lys Leu Leu Met Glu Arg Leu His Leu Glu Ile Asn Arg Arg Gly Arg Arg Ala Pro Pro Ile Leu Ala Asn Gly Pro VaI Pro Ser Pro Pro Arg Arg Gly Gly Ala Leu Leu Glu Glu Pro Glu Leu Ser Asp Asp Asp Arg Ala Asn Leu Met Trp Lys Arg Tyr Leu Glu Arg Glu Asp Ser Lys Ile Val Asp Leu Phe Val Gly Gln Leu Lys Ser Cys Leu Lys Cys Gln Ala Cys Gly Tyr Arg Ser Thr Thr Phe Glu Val Phe Cys Asp Leu Ser Leu Pro Ile Pro Lys Lys Gly Phe Ala Gly Gly Lys Val Ser Leu Arg Asp Cys Phe Asn Leu Phe Thr Lys Glu Glu Glu Leu Glu Ser Glu Asn Ala Pro Val Cys Asp Arg Cys Arg Gln Lys Thr Arg Ser Thr Lys Lys Leu WO 00/09709 PCT/US99/1?818 Thr Val Gln Arg Phe Pro Arg Ile Leu Val Leu His Leu Asn Arg Phe Ser Ala Ser Arg Gly Ser Ile Lys Lys Ser Ser Val Gly Val Asp Phe Pro Leu Gln Arg Leu Ser Leu Gly Asp Phe Ala Ser Asp Lys Ala Gly Ser Pro Val Tyr Gln Leu Tyr Ala Leu Cys Asn His Ser Gly Ser Val His Tyr Gly His Tyr Thr Ala Leu Cys Arg Cys Gln Thr Gly Trp His Val Tyr Asn Asp Ser Arg Val Ser Pro Val Ser Glu Asn Gln Val Ala Ser Ser Glu Gly Tyr Val Leu Phe Tyr Gln Leu Met Gln Glu Pro Pro Arg Cys Leu <210> 6 <211> 421 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2479394 <400> 6 Met Arg Trp Ile Leu Phe Ile GIy Ala Leu Ile Gly Ser Ser Ile Cys Gly Gln Glu Lys Phe Phe Gly Asp Gln Val Leu Arg Ile Asn Val Arg Asn Gly Asp Glu Ile Ser Lys Leu Ser Gln Leu Val Asn Ser Asn Asn Leu Lys Leu Asn Phe Trp Lys Ser Pro Ser Ser Phe Asn Arg Pro Val Asp Val Leu Val Pro Ser Val Ser Leu Gln Ala Phe Lys Ser Phe Leu Arg Ser Gln Gly Leu Glu Tyr Ala Val Thr Ile Glu Asp Leu Gln Ala Leu Leu Asp Asn Glu Asp Asp Glu Met Gln His Asn Glu Gly Gln Glu Arg Ser Ser Asn Asn Phe Asn Tyr Gly Ala Tyr His Ser Leu Glu Ala Ile Tyr His Glu Met Asp Asn Ile AIa Ala Asp Phe Pro Asp Leu Ala Arg Arg Val Lys Ile Gly His Ser Phe Glu Asn Arg Pro Met Tyr Val Leu Lys Phe Ser Thr GIy Lys Gly Val Arg Arg Pro Ala Val Trp Leu Asn Ala Gly Ile His Ser Arg Glu Trp Ile Ser Gln Ala Thr Ala Ile Trp Thr Ala Arg Lys Ile Val Ser Asp Tyr Gln Arg Asp Pro Ala Ile Thr Ser Ile Leu Glu Lys Met Asp Ile Phe Leu Leu Pro Val Ala Asn Pro Asp Gly Tyr Val Tyr Thr Gln Thr Gln Asn Arg Leu Trp Arg Lys Thr Arg Ser Arg Asn Pro Gly Ser Ser Cys Ile Gly Ala Asp Pro Asn Arg Asn Trp Asn Ala Ser Phe Ala Gly Lys Gly Ala Ser Asp Asn Pro Cys Ser Glu Val Tyr His Gly Pro His Ala Asn Ser Glu Val Glu Val Lys Ser Val Val Asp Phe Ile Gln Lys His Gly Asn Phe Lys GIy Phe Ile Asp Leu His Ser Tyr Ser Gln Leu Leu Met Tyr Pro Tyr Gly Tyr Ser Val Lys Lys Ala Pro Asp Ala Glu Glu Leu Asp Lys Val Ala Arg Leu Ala Ala Lys Ala Leu Ala Ser Val Ser Gly Thr Glu Tyr Gln Val Gly Pro Thr Cys Thr Thr Val Tyr Pro Ala Ser Gly Ser Ser Ile Asp Trp Ala Tyr Asp Asn Gly Ile Lys Phe Ala Phe Thr Phe Glu Leu Arg Asp Thr Gly Thr Tyr Gly Phe Leu Leu Pro Ala Asn Gln Ile Ile Pro Thr Ala Glu Glu Thr Trp Leu Gly Leu Lys Thr Ile Met Glu His Val Arg Asp Asn Leu Tyr <210> 7 <211> 666 <2I2> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2613215 <400> 7 Met Ala Ala Ser Arg Lys Pro Pro Arg Val Arg Val Asn His Gln Asp Phe Gln Leu Arg Asn Leu Arg Ile Ile Glu Pro Asn Glu Val Thr His Ser Gly Asp Thr Gly Val Glu Thr Asp Gly Arg Met Pro Pro Lys Val Thr Ser Glu Leu Leu Arg Gln Leu Arg Gln Ala Met Arg Asn Ser Glu Tyr Val Thr Glu Pro Ile Gln Ala Tyr Ile Ile Pro Ser Gly Asp Ala His Gln Ser Glu Tyr Ile Ala Pro Cys Asp Cys Arg Arg Ala Phe Val Ser Gly Phe Asp Gly Ser Ala Gly Thr Ala Ile Ile Thr Glu Glu His Ala Ala Met Trp Thr Asp Gly Arg Tyr Phe Leu Gln Ala Ala Lys Gln Met Asp Ser Asn Trp Thr Leu Met Lys Met Gly Leu Lys Asp Thr Pro Thr Gln Glu Asp Trp Leu Val Ser Val Leu Pro Glu Gly Ser Arg Val Gly Val Asp Pro Leu Ile Ile Pro Thr Asp Tyr Trp Lys Lys Met Ala Lys Val Leu Arg Ser Ala Gly His His Leu Ile Pro Val Lys Glu Asn Leu Val Asp Lys Ile Trp Thr Asp Arg Pro Glu Arg Pro Cys Lys Pro Leu Leu Thr Leu Gly Leu Asp Tyr Thr Gly Ile Ser Trp Lys Asp Lys Val Ala Asp Leu Arg Leu Lys Met Ala Glu Arg Asn Val Met Trp Phe Val Val Thr Ala Leu Asp Glu Ile Ala Trp Leu Phe Asn Leu Arg Gly Ser Asp Val Glu His Asn Pro Val Phe Phe Ser Tyr Ala Ile Ile Gly Leu Glu Thr Ile Met Leu Phe Ile Asp Gly Asp Arg Ile Asp Ala Pro Ser Val Lys Glu His Leu Leu Leu Asp Leu Gly Leu Glu Ala Glu Tyr Arg Ile Gln Val His Pro Tyr Lys Ser Ile Leu Ser Glu Leu Lys Ala Leu Cys Ala Asp Leu Ser Pro Arg Glu Lys Val Trp Val Ser Asp Lys Ala Ser Tyr Ala Val Ser Glu Thr Ile Pro Lys Asp His Arg Cys Cys Met Pro Tyr Thr Pro Ile Cys Ile Ala Lys Ala Val Lys Asn Ser Ala Glu Ser Glu Gly Met Arg Arg Ala His Ile Lys Asp Ala Val Ala Leu Cys Glu Leu Phe Asn Trp Leu Glu Lys Glu Val Pro Lys Gly Gly Val Thr Glu Ile Ser Ala Ala Asp Lys Ala Glu Glu Phe Arg Arg Gln Gln Ala Asp Phe Val Asp Leu Ser Phe Pro Thr Ile Ser Ser Thr Gly Pro Asn Gly Ala Ile Ile His Tyr Ala Pro Val Pro Glu Thr Asn Arg Thr Leu Ser Leu Asp Glu Val Tyr Leu Ile Asp Ser Gly Ala Gln Tyr Lys Asp Gly Thr Thr Asp Val Thr Arg Thr Met His Phe Gly Thr Pro Thr Ala Tyr Glu Lys Glu Cys Phe Thr Tyr Val Leu Lys Gly His Ile Ala Val Ser Ala Ala Val Phe Pro Thr Gly Thr Lys Gly His Leu Leu Asp Ser Phe Ala Arg Ser Ala Leu Trp Asp Ser Gly Leu Asp Tyr Leu His Gly Thr Gly His Gly Vai Gly Ser Phe Leu Asn Val His Glu Gly Pro Cys Gly Ile Ser Tyr Lys Thr Phe Ser Asp Glu Pro Leu Glu Ala Gly Met Ile Val Thr Asp Glu Pro Gly Tyr Tyr Glu Asp Gly Ala Phe Gly Ile Arg Ile Glu Asn Val Val Leu Val Val Pro Val Lys Thr Lys Tyr Asn Phe Asn Asn Arg Gly Ser Leu Thr Phe Glu Pro Leu Thr Leu Val Pro Ile Gln Thr Lys Met Ile Asp Val Asp Ser Leu Thr Asp Lys Glu Cys Asp Trp Leu Asn Asn Tyr His Leu Thr Cys Arg Asp Val Ile Gly Lys Glu Leu G1n Lys Gln Gly Arg Gln Glu Ala Leu Glu Trp Leu Ile Arg Glu Thr Gln Pro Ile Ser Lys Gln His <210> 8 <211> 952 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 001528 <400> 8 Met Ala Glu Gly Gly Ala Ala Asp Leu Asp Thr Gln Arg Ser Asp Ile Ala Thr Leu Leu Lys Thr Ser Leu Arg Lys Gly Asp Thr Trp Tyr Leu Val Asp Ser Arg Trp Phe Lys Gln Trp Lys Lys Tyr Val Gly Phe Asp Ser Trp Asp Lys Tyr Gln Met Gly Asp Gln Asn Val Tyr Pro Gly Pro Ile Asp Asn Ser Gly Leu Leu Lys Asp Gly Asp Ala Gln Ser Leu Lys Glu His Leu Ile Asp Glu Leu Asp Tyr Ile Leu Leu Pro Thr Glu Gly Trp Asn Lys Leu Val Ser Trp Tyr Thr Leu Met Glu Gly Gln Glu Pro Ile Ala Arg Lys Val Val Glu Gln Gly Met Phe Val Lys Arg Cys Lys Val Glu Val Tyr Leu Thr Glu Leu Lys Leu Cys Glu Asn Gly Asn Met Asn Asn Val Val Thr Arg Arg Phe Ser Lys Ala Asp Thr Ile Asp Thr Ile Glu Lys Glu Ile Arg Lys Ile Phe Ser Ile Pro Asp Glu Lys Glu Thr Arg Leu Trp Asn Lys Tyr Met Ser Asn Thr Phe Glu Pro Leu Asn Lys Pro Asp Ser Thr Ile Gln Asp Ala Gly Leu Tyr Gln Gly Gln Val Leu Val Ile Glu Gln Lys Asn Glu Asp Gly Thr Arg Pro Arg Gly Pro Ser Thr Pro Asn Val Lys Asn Ser Asn Tyr Cys Leu Pro Ser Tyr Thr Ala Tyr Lys Asn Tyr Asp Tyr Ser Glu Pro Gly Arg Asn Asn Glu Gln Pro Gly Leu Cys Gly Leu Ser Asn Leu Gly Asn Thr Cys Phe Met Asn Ser Ala Ile Gln Cys Leu Ser Asn Thr Pro Pro Leu Thr Glu Tyr Phe Leu Asn Asp Lys Tyr Gln Glu Glu Leu Asn Phe Asp Asn Pro Leu Gly Met Arg Gly GIu Ile Ala Lys Ser Tyr Ala Glu Leu Ile Lys Gln Met Trp Ser Gly Lys Phe Ser Tyr Val Thr Pro Arg Ala Phe Lys Thr Gln Val Gly Arg Phe Ala Pro Gln Phe Ser Gly Tyr Gln Gln Gln Asp Cys Gln Glu Leu Leu Ala Phe Leu Leu Asp Gly Leu His Glu Asp Leu Asn Arg Ile Arg Lys Lys Pro Tyr Ile Gln Leu Lys Asp Ala Asp Gly Arg Pro Asp Lys Val Val Ala Glu Glu Ala Trp Glu Asn His Leu Lys Arg Asn Asp Ser Ile Ile Val Asp Ile Phe His Gly Leu Phe Lys Ser Thr Leu Val Cys Pro Glu Cys Ala Lys Ile Ser Val Thr Phe Asp Pro Phe Cys Tyr Leu Thr Leu Pro Leu Pro Met Lys Lys Glu Arg Thr Leu Glu Val Tyr Leu Val Arg Met Asp Pro Leu Thr Lys Pro Met Gln Tyr Lys Val Val Val Pro Lys Ile Gly Asn Ile Leu Asp Leu Cys Thr Ala Leu Ser Ala Leu Ser Gly Ile Pro Ala Asp Lys Met Ile Val Thr Asp Ile Tyr Asn His Arg Phe His Arg Ile Phe Ala Met Asp Glu Asn Leu Ser Ser Ile Met Glu Arg Asp Asp Ile Tyr Val Phe Glu Ile Asn Ile Asn Arg Thr Glu Asp Thr Glu His Val Ile Ile Pro Val Cys Leu Arg Glu Lys Phe Arg His Ser Ser Tyr Thr His His Thr Gly Ser Ser Leu Phe Gly Gln Pro Phe Leu Met Ala Val Pro Arg Asn Asn Thr Glu Asp Lys Leu Tyr Asn Leu Leu Leu Leu Arg Met Cys Arg Tyr Val Lys Ile Ser Thr Glu Thr Glu Glu Thr Glu Gly Ser Leu His Cys Cys Lys Asp Gln Asn Ile Asn Gly Asn Gly Pro Asn Gly Ile His Glu Glu Gly Ser Pro Ser Glu Met Glu Thr Asp Glu Pro Asp Asp Glu Ser Ser Gln Asp Gln Glu Leu Pro Ser Glu Asn Glu Asn Ser Gln Ser Glu Asp Ser Val Gly Gly Asp Asn Asp Ser Glu Asn Gly Leu Cys Thr Glu Asp Thr Cys Lys Gly Gln Leu Thr Gly His Lys Lys Arg Leu Phe Thr Phe Gln Phe Asn Asn Leu Gly Asn Thr Asp Ile Asn Tyr Ile Lys Asp Asp Thr Arg His Ile Arg Phe Asp Asp Arg Gln Leu Arg Leu Asp Glu Arg Ser Phe Leu Ala Leu Asp Trp Asp Pro Asp Leu Lys Lys Arg Tyr Phe Asp Glu Asn Ala Ala Glu Asp Phe Glu Lys His Glu Ser Val Glu Tyr Lys Pro Pro Lys Lys Pro Phe Val Lys Leu Lys Asp Cys Ile Glu Leu Phe Thr Thr Lys Glu Lys Leu Gly Ala Glu Asp Pro Trp Tyr Cys Pro Asn Cys Lys Glu His Gln Gln Ala Thr Lys Lys Leu Asp Leu Trp Ser Leu Pro Pro Val Leu Val Val His Leu Lys Arg Phe Ser Tyr Ser Arg Tyr Met Arg Asp Lys Leu Asp Thr Leu Val Asp Phe Pro Ile Asn Asp Leu Asp Met Ser Glu Phe Leu Ile Asn Pro Asn Ala Gly Pro Cys Arg Tyr Asn Leu Ile Ala Val Ser Asn His Tyr Gly Gly Met Gly Gly Gly His Tyr Thr Ala Phe Ala Lys Asn Lys Asp Asp Gly Lys Trp Tyr Tyr Phe Asp Asp Ser Ser Val Ser Thr Ala Ser Glu Asp Gln Ile Val Ser Lys Ala Ala Tyr Val Leu Phe Tyr Gln Arg Gln Asp Thr Phe Ser Gly Thr Gly Phe Phe Pro Leu Asp Arg Glu Thr Lys Gly Ala Ser Ala Ala Thr Gly Ile Pro Leu Glu Ser Asp Glu Asp Ser Asn Asp Asn Asp Asn Asp Ile Glu Asn Glu Asn Cys Met His Thr Asn <210> 9 <211> 166 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 998626 <400> 9 Met Leu His Pro Glu Thr Ser Pro Gly Arg Gly His Leu Leu Ala Val Leu Leu Ala Leu Leu Gly Thr Ala Trp Ala Glu Val Trp Pro Ser Leu His Cys Cys Lys Asp Gln Asn Ile Asn Pro Gln Leu Gln Glu Gln Ala Pro Met Ala Gly Ala Leu Asn Arg Lys Glu Ser Phe Leu Leu Leu Ser Leu His Asn Arg Leu Arg Ser Trp Val Gln Pro Pro Ala Ala Asp Met Arg Arg Leu Asp Trp Ser Asp Ser Leu Ala Gln Leu Ala Gln Ala Arg Ala Ala Leu Cys Gly Ile Pro Thr Pro Ser Leu Ala Ser Gly Leu Trp Arg Thr Leu Gln Val Gly Trp Asn Met Gln Leu Leu Pro Ala Gly Leu Ala Ser Phe Val Glu Val Val Ser Leu Trp Phe Ala Glu Gly Gln Arg Tyr Ser His Ala Ala Gly Glu Cys Ala Arg Asn Ala Thr Cys Thr His Tyr Thr Gln Leu Val Trp Ala Thr Ser Ser Gln Leu Gly Cys Gly Arg His <210> 10 <211> 543 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1393301 <400> 10 Met Arg Lys Pro Ala Ala Gly Phe Leu Pro Ser Leu Leu Lys VaI

Leu Leu Leu Pro Leu Ala Pro Ala Ala Ala Gln Asp Ser Thr Gln Ala Ser Thr Pro Gly Ser Pro Leu Ser Pro Thr Glu Tyr Glu Arg Phe Phe Ala Leu Leu Thr Pro Thr Trp Lys Ala Glu Thr Thr Cys Arg Leu Arg Ala Thr His Gly Cys Arg Asn Pro Thr Leu Val Gln Leu Asp Gln Tyr Glu Asn His Gly Leu Val Pro Asp Gly Ala Val Cys Ser Asn Leu Pro Tyr Ala Ser Trp Phe Glu Ser Phe Cys Gln Phe Thr His Tyr Arg Cys Ser Asn His Val Tyr Tyr Ala Lys Arg VaI Leu Cys Ser Gln Pro Val Ser Ile Leu Ser Pro Asn Thr Leu Lys Glu Ile Glu Ala Ser Ala Glu Val Ser Pro Thr Thr Met Thr Ser Pro Ile Ser Pro His Phe Thr Val Thr Glu Arg Gln Thr Phe Gln Pro Trp Pro Glu Arg Leu Ser Asn Asn Val Glu Glu Leu Leu Gln Ser Ser Leu Ser Leu Gly Gly Gln Glu Gln Ala Pro Glu His Lys Gln Glu Gln Gly Val Glu His Arg Gln Glu Pro Thr Gln Glu His Lys Gln Glu Glu Gly Gln Lys Gln Glu Glu Gln Glu Glu Glu Gln Glu Glu Glu Gly Lys Gln Glu Glu Gly Gln Gly Thr Lys Glu Gly Arg Glu Ala Val Ser Gln Leu Gln Thr Asp Ser Glu Pro Lys Phe His Ser Glu Ser Leu Ser Ser Asn Pro Ser Ser Phe Ala Pro Arg Val Arg Glu Val Glu Ser Thr Pro Met Ile Met Glu Asn Ile Gln Glu Leu Ile Arg Ser Ala Gln Glu Ile Asp Glu Met Asn Glu Ile Tyr Asp Glu Asn Ser Tyr Trp Arg Asn Gln Asn Pro Gly Ser Leu Leu Gln Leu Pro His Thr Glu Ala Leu Leu Val Leu Cys Tyr Ser Ile Val Glu Asn Thr Cys Ile Ile Thr Pro Thr Ala Lys Ala Trp Lys Tyr Met Glu Glu Glu Ile Leu Gly Phe Gly Lys Ser Val Cys Asp Ser Leu Gly Arg Arg His Met Ser Thr Cys Ala Leu Cys Asp Phe Cys Ser Leu Lys Leu Glu Gln Cys His Ser Glu Ala Ser Leu Gln Arg Gln Gln Cys Asp Thr Ser His Lys Thr Pro Phe Val Ser Pro Leu Leu Ala Ser Gln Ser Leu Ser Ile Gly Asn Gln Val Gly Ser Pro Glu Ser Gly Arg Phe Tyr Gly Leu Asp Leu Tyr Gly Gly Leu His Met Asp Phe Trp Cys Ala Arg Leu Ala Thr Lys Gly Cys Glu Asp Val Arg Val Ser Gly Trp Leu Gln Thr Glu Phe Leu Ser Phe Gln Asp Gly Asp Phe Pro Thr Lys Ile Cys Asp Thr Asp Tyr Ile Gln Tyr Pro Asn Tyr Cys Ser Phe Lys Ser Gln Gln Cys Leu Met Arg Asn Arg Asn Arg Lys Val Ser Arg Met Arg Cys Leu Gln Asn Glu Thr Tyr Ser Ala Leu Ser Pro Gly Lys Ser Glu Asp Val Val Leu Arg Trp Ser Gln Glu Phe Ser Thr Leu Thr Leu Gly Gln Phe Gly <210> lI
<211> 83 <212> PRT
<213> Homo sapiens <220>
<221> misc feature <223> Incyte Clone No: 1444055 <400> 11 Met Ile Gly Trp Asp Ser Leu Arg Leu IIe Leu Gly Asn Thr Asp Asn Val Ser Arg Arg Asp Ser Thr Arg Gly Ser Ile Phe Ile Thr Gln Leu Ile Ala Cys Phe Gln Arg Tyr Ser Trp Arg Cys His Leu Glu Glu Val Phe Trp Lys Val Gln Gln Ala~ Phe Glu Ser Pro Glu Ala Thr Val Gln Met Pro Thr Ile Glu Arg Val Ser Met Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn <210> 12 <211> 648 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1650177 <400> 12 Met Leu Gly Ser Leu Val Leu Arg Arg Lys Ala Leu Ala Pro Arg Leu Leu Leu Arg Leu Leu Arg Ser Pro Thr Leu Arg Gly His Gly Gly Ala Ser Gly Arg Asn Val Thr Thr Gly Ser Leu Gly Glu Pro Gln Trp Leu Arg Val Ala Thr Gly Gly Arg Pro Gly Thr Ser Pro Ala Leu Phe Ser Gly Arg Gly Ala Ala Thr Gly Gly Arg Gln Gly Gly Arg Phe Asp Thr Lys Cys Leu Ala Ala Ala Thr Trp Gly Arg Leu Pro Gly Pro Glu Glu Thr Leu Pro Gly Gln Asp Ser Trp Asn Gly Val Pro Ser Arg Ala Gly Leu Gly Met Cys Ala Leu AIa Ala Ala Leu Val Val His Cys Tyr Ser Lys Ser Pro Ser Asn Lys Asp Ala Ala Leu Leu Glu Ala Ala Arg Ala Asn Asn Met Gln Glu Val Ser Ser Val Val Gln Val Leu Leu Ala Ala Gly Ala Asp Pro Asn Leu Gly Asp Asp Phe Ser Ser Val Phe Lys Thr Ala Lys Glu Gln Gly Ile His Ser Leu Glu Val Leu Ile Thr Arg Glu Asp Asp Phe Asn Asn Arg Leu Asn Asn Arg Ala Ser Phe Lys Gly Cys Thr Ala Leu His Tyr Ala Val Leu Ala Asp Asp Tyr Arg Thr Val Lys Glu Leu Leu Asp Gly Gly Ala Asn Pro Leu Gln Arg Asn Glu Met Gly His Thr Pro Leu Asp Tyr Ala Arg Glu Gly Glu Val Met Lys Leu Leu Arg Thr Ser Glu Ala Lys Tyr Gln Glu Lys Gln Arg Lys Arg Glu Ala Glu Glu Arg Arg Arg Phe Pro Leu Glu Gln Arg Leu Lys Glu His Ile Ile Gly Gln Glu Ser Ala Ile Ala Thr Val Gly Ala Ala Ile Arg Arg Lys Glu Asn Gly Trp Tyr Asp Glu Glu His Pro Leu Val Phe Leu Phe Leu Gly Ser Ser Gly Ile Gly Lys Thr Glu Leu Ala Lys Gln Thr Ala Lys Tyr Met His Lys Asp Ala Lys Lys Gly Phe Ile Arg Leu Asp Met Ser Glu Phe Gln Glu Arg His Glu Val Ala Lys Phe Ile Gly Ser Pro Pro Gly Tyr Val Gly His Glu Glu Gly Gly Gln Leu Thr Lys Lys Leu Lys Gln Cys Pro Asn Ala Val Val Leu Phe Asp Glu Val Asp Lys Ala His Pro Asp Val Leu Thr Ile Met Leu Gln Leu Phe Asp Glu Gly Arg Leu Thr Asp Gly Lys Gly Lys Thr Ile Asp Cys Lys Asp Ala Ile Phe Ile Met Thr Ser Asn Val Ala Ser Asp Glu Ile Ala Gln His Ala Leu Gln Leu Arg Gln Glu Ala Leu Glu Met Ser Arg Asn Arg Ile Ala Glu Asn Leu Gly Asp Val Gln Ile Ser Asp Lys Ile Thr Ile Ser Lys Asn Phe Lys Glu Asn Val Ile Arg Pro Ile Leu Lys Ala His Phe Arg Arg Asp Glu Phe Leu Gly Arg Ile Asn Glu Ile Val Tyr Phe Leu Pro Phe Cys His Ser Glu Leu Ile Gln Leu Val Asn Lys Glu Leu Asn Phe Trp Ala Lys Arg Ala Lys Gln Arg His Asn Ile Thr Leu Leu Trp Asp Arg Glu Val Ala Asp Val Leu Val Asp Gly Tyr Asn Val His Tyr Gly Ala Arg Ser Ile Lys His Glu Val Glu Arg Arg Val Val Asn Gln Leu Ala Ala Ala Tyr Glu Gln Asp Leu Leu Pro Gly Gly Cys Thr Leu Arg Ile Thr Val Glu Asp Ser Asp Lys Gln Leu Leu Lys Ser Pro Glu Leu Pro Ser Pro Gln Ala Glu Lys Arg Leu Pro Lys Leu Arg Leu Glu Ile Ile Asp Lys Asp Ser Lys Thr Arg Arg Leu Asp Ile Arg Ala Pro Leu His Pro Glu Lys Val Cys Asn Thr Ile <210> 13 <211> 672 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1902576 <400> 13 Met Arg Ala Gly Arg Gly Ala Thr Pro Ala Arg Glu Leu Phe Arg Asp Ala Ala Phe Pro Ala Ala Asp Ser Ser Leu Phe Cys Asp Leu Ser Thr Pro Leu Ala Gln Phe Arg Glu Asp Ile Thr Trp Arg Arg Pro Gln Glu Ile Cys Ala Thr Pro Arg Leu Phe Pro Asp Asp Pro Arg Glu Gly Gln Val Lys Gln Gly Leu Leu Gly Asp Cys Trp Phe Leu Cys Ala Cys Ala Ala Leu Gln Lys Ser Arg His Leu Leu Asp Gln Val Ile Pro Pro Gly Gln Pro Ser Trp Ala Asp Gln Glu Tyr Arg Gly Ser Phe Thr Cys Arg Ile Trp Gln Phe Gly Arg Trp Val Glu Val Thr Thr Asp Asp Arg Leu Pro Cys Leu Ala Gly Arg Leu Cys Phe Ser Arg Cys Gln Arg Glu Asp Val Phe Trp Leu Pro Leu Leu Glu Lys Val Tyr Ala Lys Val His Gly Ser Tyr Glu His Leu Trp Ala Gly Gln Val Ala Asp Ala Leu Val Asp Leu Thr Gly Gly Leu Ala Glu Arg Trp Asn Leu Lys Gly Val Ala Gly Ser Gly Gly Gln Gln Asp Arg Pro Gly Arg Trp Glu His Arg Thr Cys Arg Gln Leu Leu His Leu Lys Asp Gln Cys Leu Ile Ser Cys Cys Val Leu Ser Pro Arg Ala Gly Ala Arg Glu Leu Gly Glu Phe His Ala Phe Ile Val Ser Asp Leu Arg Glu Leu Gln Gly Gln Ala Gly Gln Cys Ile Leu Leu Leu Arg Ile Gln Asn Pro Trp Gly Arg Arg Cys Trp Gln Gly Leu Trp Arg Glu Gly Gly Glu Gly Trp Ser Gln Val Asp Ala Ala Val Ala Ser Glu Leu Leu Ser Gln Leu Gln Glu Gly Glu Phe Trp Val Glu Glu Glu Glu Phe Leu Arg Glu Phe Asp Glu Leu Thr Val Gly Tyr Pro Val Thr Glu Ala Gly His Leu Gln Ser Leu Tyr Thr Glu Arg Leu Leu Cys His Thr Arg Ala Leu Pro Gly Ala Trp Val Lys Gly Gln Ser Ala Gly Gly Cys Arg Asn Asn Ser Gly Phe Pro Ser Asn Pro Lys Phe Trp Leu Arg Val Ser Glu Pro Ser Glu Val Tyr Ile Ala Val Leu Gln Arg Ser Arg Leu His Ala Ala Asp Trp Ala Gly Arg Ala Arg Ala Leu Val Gly Asp Ser His Thr Ser Trp Ser Pro Ala Ser Ile Pro Gly Lys His Tyr Gln Ala Val Gly Leu His Leu Trp Lys Val Glu Lys Arg Arg Val Asn Leu -Pro Arg Val Leu Ser Met Pro Pro Val Ala Gly Thr Ala Cys His Ala Tyr Asp Arg Glu Val His Leu Arg Cys Glu Leu Ser Pro Gly Tyr Tyr Leu Ala VaI Pro Ser Thr Phe Leu Lys Asp Ala Pro Gly Glu Phe Leu Leu Arg Val Phe Ser Thr Gly Arg Val Ser Leu Ser Ala Ile Arg Ala Val Ala Lys Asn Thr Ala Pro Gly Ala Ala Leu Pro Ala Gly Glu Trp Gly Thr Val Gln Leu Arg Gly Ser Trp Arg Val Gly Gln Thr Ala Gly Gly Ser Arg Asn Phe Ala Ser Tyr Pro Thr Asn Pro Cys Phe Pro Phe Ser Val Pro Glu Gly Pro Gly Pro Arg Cys Val Arg Ile Thr Leu His Gln His Cys Arg Pro Ser Asp Thr Glu Phe His Pro Ile Gly Phe His Ile Phe Gln Val Pro Glu Gly Gly Arg Ser Gln Asp Ala Pro Pro Leu Leu Leu Gln Glu Pro Leu Leu Ser Cys Val Pro His Arg Tyr Ala Gln Glu Val Ser Arg Leu Cys Leu Leu Pro Ala Gly Thr Tyr Lys Val Val Pro Ser Thr Tyr Leu Pro Asp Thr Glu Gly Ala Phe Thr Val Thr Ile Ala Thr Arg Ile Asp Arg Pro Ser Ile His Ser Gln Glu Met Leu Gly Gln Phe Leu Gln Glu Val Ser Val Met Ala Val Met Lys Thr <210> 14 <211> 80 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2024210 <400> 14 Met Lys Leu Ser Gly Met Phe Leu Leu Leu Ser Leu Ala Leu Phe Cys Phe Leu Thr Gly Val Phe Ser Gln Gly Gly Gln Val Asp Cys Gly Glu Phe Gln Asp Pro Lys Val Tyr Cys Thr Arg Glu Ser Asn Pro His Cys Gly Ser Asp Gly Gln Thr Tyr Gly Asn Lys Cys Ala Phe Cys Lys Ala Ile Val Lys Ser Gly Gly Lys Ile Ser Leu Lys His Pro Gly Lys Cys <210> 15 <211> 795 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2523109 <400> 15 Met Ala Val Leu Leu Leu Leu Leu Arg Ala Leu Arg Arg Gly Pro Gly Pro Gly Pro Arg Pro Leu Trp Gly Pro Gly Pro Ala Trp Ser Pro Gly Phe Pro Ala Arg Pro Gly Arg Gly Arg Pro Tyr Met Ala Ser Arg Pro Pro Gly Asp Leu Ala Glu Ala Gly Gly Arg Ala Leu Gln Ser Leu Gln Leu Arg Leu Leu Thr Pro Thr Phe Glu Gly Ile Asn Gly Leu Leu Leu Lys Gln His Leu Val Gln Asn Pro Val Arg Leu Trp Gln Leu Leu Gly Gly Thr Phe Tyr Phe Asn Thr Ser Arg Leu Lys G1n Lys Asn Lys Glu Lys Asp Lys Ser Lys Gly Lys Ala Pro Glu Glu Asp Glu Glu Glu Arg Arg Arg Arg Glu Arg Asp Asp Gln Met Tyr Arg Glu Arg Leu Arg Thr Leu Leu Val Ile Ala Val Val Met Ser Leu Leu Asn Ala Leu Ser Thr Ser Gly Gly Ser Ile Ser Trp Asn Asp Phe Val His Glu Met Leu Ala Lys Gly Glu Val Gln Arg Val Gln Val Val Pro Glu Ser Asp Val Val Glu Val Tyr Leu His Pro Gly Ala Val Val Phe Gly Arg Pro Arg Leu Ala Leu Met Tyr Arg Met Gln Val Ala Asn Ile Asp Lys Phe Glu Glu Lys Leu Arg Ala Ala Glu Asp Glu Leu Asn Ile Glu Ala Lys Asp Arg Ile Pro Val Ser Tyr Lys Arg Thr Gly Phe Phe Gly Asn Ala Leu Tyr Ser Val Gly Met Thr Ala Val Gly Leu Ala Ile Leu Trp Tyr Val Phe Arg Leu Ala Gly Met Thr Gly Arg Glu Gly Gly Phe Ser Ala Phe Asn Gln Leu Lys Met Ala Arg Phe Thr Ile Val Asp Gly Lys Met Gly Lys Gly Val Ser Phe Lys Asp Val Ala Gly Met His Glu Ala Lys Leu Glu Val Arg Glu Phe Val Asp Tyr Leu Lys Ser Pro Lys Arg Phe Leu Gln Leu Gly Ala Lys Val Pro Lys Gly Ala Leu Leu Leu Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala Lys Ala Val Ala Thr Glu Ala Gln Val Pro Phe Leu Ala Met Ala Gly Pro Glu Phe Val Glu Val Ile Gly Gly Leu Gly Ala Ala Arg Val Arg Ser Leu Phe Lys Glu Ala Arg Ala Arg Ala Pro Cys Ile Val Tyr Ile Asp Glu Ile Asp Ala Val Gly Lys Lys Arg Ser Thr Thr Met Ser Gly Phe Ser Asn Thr Glu Glu Glu Gln Thr Leu Asn Gln Leu Leu Val Glu Met Asp Gly Met Gly Thr Thr Asp His Val Ile Val Leu Ala Ser Thr Asn Arg Ala Asp Ile Leu Asp Gly Ala Leu Met Arg Pro Gly Arg Leu Asp Arg His Val Phe Ile Asp Leu Pro Thr Leu Gln Glu Arg Arg Glu Ile Phe Glu Gln His Leu Lys Ser Leu Lys Leu Thr Gln Ser Ser Thr Phe Tyr Ser Gln Arg Leu Ala Glu Leu Thr Pro Gly Phe Ser Gly Ala Asp Ile Ala Asn Ile Cys Asn Glu Ala Ala Leu His Ala Ala Arg Glu Gly His Thr Ser Val His Thr Leu Asn Phe Glu Tyr Ala Val Glu Arg Val Leu Ala Gly Thr Ala Lys Lys Ser Lys Ile Leu Ser Lys Glu Glu Gln Lys Val Val Ala Phe His Glu Ser Gly His Ala Leu Val Gly Trp Met Leu Glu His Thr Glu Ala Val Met Lys Val Ser Ile Thr Pro Arg Thr Asn Ala Ala Leu Gly Phe Ala Gln Met Leu Pro Arg Asp Gln His Leu Phe Thr Lys Glu Gln Leu Phe Glu Arg Met Cys Met Ala Leu Gly Gly Arg Ala Ser Glu Ala Leu Ser Phe Asn Glu Val Thr Ser Gly Ala Gln Asp Asp Leu Arg Lys Val Thr Arg Ile Ala Tyr Ser Met Val Lys Gln Phe Gly Met Ala Pro Gly Ile Gly Pro Ile Ser Phe Pro Glu Ala Gln Glu Gly Leu Met Gly Ile Gly Arg Arg Pro WO 00!09709 PCT/US99/17818 Phe Ser Gln Gly Leu Gln Gln Met Met Asp His Glu Ala Arg Leu Leu Val Ala Lys Ala Tyr Arg His Thr Glu Lys Val Leu Gln Asp Asn Leu Asp Lys Leu Gln Ala Leu Ala Asn Ala Leu Leu Glu Lys Glu Val Ile Asn Tyr Glu Asp Ile Glu Ala Leu Ile Gly Pro Pro Pro His Gly Pro Lys Lys Met Ile Ala Pro Gln Arg Trp Ile Asp Ala Gln Arg Glu Lys Gln Asp Leu Gly Glu Glu Glu Thr Glu Glu Thr Gln Gln Pro Pro Leu Gly Gly Glu Glu Pro Thr Trp Pro Lys <210> 16 <211> 193 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2588566 <400> 16 Met Pro Asp Ser Asp Arg His Leu Ser Ser His Phe Asn Leu Arg Met Lys Gly Ser Pro Ser Glu His Gly Ser Gln Gln Ser Ile Phe Asn Arg Tyr Ala Gln Gln Arg Leu Asp Ile Asp Ala Thr Gln Leu Gln Gly Leu Leu Asn Gln Glu Leu Leu Thr Gly Pro Pro Gly Asp Met Phe Ser Leu Asp Glu Cys Arg Ser Leu Val Ala Leu Met Glu Leu Lys Val Asn Gly Arg Leu Asp Gln Glu Glu Phe Ala Arg Leu Trp Lys Arg Leu Val His Tyr Gln His Val Phe Gln Lys Val Gln Thr Ser Pro Gly Val Leu Leu Ser Ser Asp Leu Trp Lys Ala Ile Glu Asn Thr Asp Phe Leu Arg Gly Ile Phe Ile Ser Arg Glu Leu Leu His Leu Val Thr Leu Arg Tyr Ser Asp Ser Val Gly Arg Val Ser Phe Pro Ser Leu Val Cys Phe Leu Met Arg Leu Glu Ala Met Ala Lys Thr Phe Arg Asn Leu Ser Lys Asp Gly Lys Gly Leu Tyr Leu Thr Glu Met Glu Trp Met Ser Leu Val Met Tyr Asn <210> 17 <211> 663 <212> P12T
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2740570 <400> 17 Met Asp Leu Leu His Glu Glu Leu Lys Glu Gln Val Met Glu Val Glu Glu Asp Pro Gln Thr Ile Thr Thr Glu Glu Thr Met Glu Glu Asp Lys Ser Gln Ser Asp Val Asp Phe Gln Ser Cys Glu Ser Cys Ser Asn Ser Asp Arg Ala Glu Asn Glu Asn Gly Ser Arg Cys Phe Ser Glu Asp Asn Asn Glu Thr Thr Met Leu Ile Gln Asp Asp Glu Asn Asn Ser Glu Met Ser Lys Asp Trp Gln Lys Glu Lys Met Cys Asn Lys Ile Asn Lys Val Asn Ser Glu Gly Glu Phe Asp Lys Asp Arg Asp Ser Ile Ser Glu Thr Val Asp Leu Asn Asn G1n Glu Thr Val Lys Val Gln Ile His Ser Arg Ala Ser Glu Tyr Ile Thr Asp Val His Ser Asn Asp Leu Ser Thr Pro Gln Ile Leu Pro Ser Asn Glu Gly Val Asn Pro Arg Leu Ser Ala Ser Pro Pro Lys Ser Gly Asn Leu Trp Pro Gly Leu Ala Pro Pro His Lys Lys Ala Gln Ser Ala Ser Pro Lys Arg Lys Lys Gln His Lys Lys Tyr Arg Ser Val Ile Ser Asp Ile Phe Asp Gly Thr Ile Ile Ser Ser Val Gln Cys Leu Thr Cys Asp Arg Val Ser Val Thr Leu Glu Thr Phe Gln Asp Leu Ser Leu Pro Ile Pro Gly Lys Glu Asp Leu Ala Lys Leu His Ser Ser Ser His Pro Thr Ser Ile Val Lys Ala Gly Ser Cys Gly Glu Ala Tyr Ala Pro Gln Gly Trp Ile Ala Phe Phe Met Glu Tyr Val Lys Arg Phe Val Val Ser Cys Val Pro Ser Trp Phe Trp Gly Pro Val Val Thr Leu Gin Asp Cys Leu Ala Ala Phe Phe Ala Arg Asp Glu Leu Lys Gly Asp Asn Met Tyr Ser Cys Glu Lys Cys Lys Lys Leu Arg Asn Gly Val Lys Phe Cys Lys Val Gln Asn Phe Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu Leu Met Phe Ser Thr Lys Ile Ser Thr His Val Ser Phe Pro Leu Glu Gly Leu Asp Leu Gln Pro Phe Leu Ala Lys Asp Ser Pro Ala Gln Ile Val Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Ser Ser Gly His Tyr Ile Ala Tyr Cys Arg Asn Asn Leu Asn Asn Leu Trp Tyr Glu Phe Asp Asp Gln Ser Val Thr Glu Val Ser Glu Ser Thr Val Gln Asn Ala Glu Ala Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu Ala Gln Lys Glu Arg Arg Arg Ile Ser Asn Leu Leu Asn Ile Met Glu Pro Ser Leu Leu Gln Phe Tyr Ile Ser Arg Gln Trp Leu Asn Lys Phe Lys Thr Phe Ala Glu Pro Gly Pro Ile Ser Asn Asn Asp Phe Leu Cys Ile His Gly Gly Val Pro Pro Arg Lys Ala Gly Tyr Ile Glu Asp Leu Val Leu Met Leu Pro Gln Asn Ile Trp Asp Asn Leu Tyr Ser Arg Tyr Gly Gly Gly Pro Ala Val Asn His Leu Tyr Ile Cys His Thr Cys Gln Ile Glu Ala Glu Lys Ile Glu Lys Arg Arg Lys Thr Glu Leu Glu Ile Phe Ile Arg Leu Asn Arg Ala Phe Gln Lys Glu Asp Ser Pro Ala Thr Phe Tyr Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ser Phe Val Lys Gly Lys Asp Gly Asp Pro Pro Gly Pro Ile Asp Asn Thr Lys Ile Ala Val Thr Lys Cys Gly Asn Val Met Leu Arg Gln Gly AIa Asp Ser Gly Gln Ile Ser Glu Glu Thr Trp Asn Phe Leu Gln Ser Ile Tyr Gly Gly Gly Pro Glu Val Ile Leu Arg Pro Pro Val Val His Val Asp Pro Asp Ile Leu Gln Ala Glu Glu Lys Ile Glu VaI Glu Thr Arg Ser Leu <210> 18 <211> 362 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2820384 <400> 18 Met Tyr Ser Cys Glu Arg Cys Lys Lys Leu Arg Asn Gly Val Lys Tyr Cys Lys Val Leu Arg Leu Pro Glu Ile Leu Cys Ile His Leu Lys Arg Phe Arg His Glu Val Met Tyr Ser Phe Lys Ile Asn Ser His Val Ser Phe Pro Leu Glu Gly Leu Asp Leu Arg Pro Phe Leu Ala Lys Glu Cys Thr Ser Gln Ile Thr Thr Tyr Asp Leu Leu Ser Val Ile Cys His His Gly Thr Ala Gly Ser Gly His Tyr Ile Ala Tyr Cys Gln Asn Val Ile Asn Gly Gln Trp Tyr Glu Phe Asp Asp Gln Tyr Val Thr Glu Val His Glu Thr Val Val Gln Asn Ala Glu 110 lI5 120 Gly Tyr Val Leu Phe Tyr Arg Lys Ser Ser Glu Glu Ala Met Arg Glu Arg Gln Gln Val Val Ser Leu Ala Ala Met Arg Glu Pro Ser Leu Leu Arg Phe Tyr Val Ser Arg Glu Trp Leu Asn Lys Phe Asn Thr Phe Ala Glu Pro Gly Pro Ile Thr Asn Gln Thr Phe Leu Cys Ser His Gly Gly Ile Pro Pro His Lys Tyr His Tyr Ile Asp Asp Leu Val Val Ile Leu Pro Gln Asn Val Trp Glu His Leu Tyr Asn Arg Phe Gly Gly Gly Pro Ala Val Asn His Leu Tyr Val Cys Ser Ile Cys Gln Val Glu Ile Glu Ala Leu Ala Lys Arg Arg Arg Ile Glu IIe Asp Thr Phe Ile Lys Leu Asn Lys Ala Phe Gln Ala Glu Glu Ser Pro Gly Val Ile Tyr Cys Ile Ser Met Gln Trp Phe Arg Glu Trp Glu Ala Phe Val Lys Gly Lys Asp Asn Glu Pro Pro Gly Pro Ile Asp Asn Ser Arg Ile Ala Gln Val Lys Gly Ser Gly His Val Gln Leu Lys Gln Gly Ala Asp Tyr Gly Gln Ile Ser Glu Glu Thr Trp Thr Tyr Leu Asn Ser Leu Tyr Gly Gly Gly Pro Glu Ile Ala Ile Arg Gln Ser Val Ala Gln Arg Trp Ala Gln Arg Thr Cys Thr Gly Ser Arg Arg Ser Lys Pro Arg Arg Gly Pro Cys Asp Leu Leu Gly <210> 19 <211> 210 <212> PRT
<213> Homo Sapiens <220>
<22I> misc_feature <223> Incyte Clone No: 2990692 <400> 19 Met Val Ser Leu Leu Pro Gly Glu Pro Pro Gln Lys Ile Pro Arg Gly Val Tyr Gly Pro Leu Pro Glu Gly Arg Val Gly Leu Ile Leu Gly Arg Ser Ser Leu Asn Leu Lys Gly Val Gln Ile His Thr GIy Val IIe Tyr Ser Asp Tyr Lys Gly Gly Ile Gln Leu Val Ile Ser Ser Thr Val Pro Trp Ser Ala Asn Pro Gly Asp Arg Ile Ala Gln Leu Leu Leu Leu Pro Tyr Val Lys Ile Gly Glu Asn Lys Thr Glu Arg Thr Gly Gly Phe Gly Ser Thr Asn Pro Ala Gly Lys Ala Thr Tyr Trp Ala Asn Gln Val Ser Glu Asp Arg Pro Val Cys Thr Val Thr Ile Pro Gly Lys Glu Phe Glu Gly Leu Val Asp Thr Gln Ala Asp Val Ser Ile Ile Gly Ile Gly Thr Ala Ser Glu Val Tyr Gln Ser Ala Met Ile Leu His Cys Leu Gly Ser Asp Asn Gln Glu Ser Thr Val Gln Pro Met Ile Thr Ser Ile Pro Ile Asn Leu Trp Gly Arg Asp Leu Leu Gln Gln Trp His Ala Glu Ile Thr Ile Pro Ala Ser Leu Tyr Ser Pro Arg Asn Gln Lys Ile Met Thr Lys Met Gly <210> 20 <211> 283 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 4590384 <400> 20 Met Gly Leu Gly Leu Arg Gly Trp Gly Arg Pro Leu Leu Thr Val Ala Thr Ala Leu Met Leu Pro Val Lys Pro Pro Ala Gly Ser Trp Gly Ala Gln Ile Ile Gly Gly His Glu Val Thr Pro His Ser Arg Pro Tyr Met Ala Ser Val Arg Phe Gly Gly Gln His His Cys Gly Gly Phe Leu Leu Arg Ala Arg Trp Val Val Ser Ala Ala His Cys Phe Ser His Arg Asp Leu Arg Thr Gly Leu Val Val Leu Gly Ala His VaI Leu Ser Thr Ala Glu Pro Thr Gln Gln Val Phe Gly Ile Asp Ala Leu Thr Thr His Pro Asp Tyr His Pro Met Thr His Ala Asn Asp Ile Cys Leu Leu Arg Leu Asn Gly Ser Ala Val Leu Gly Pro Ala Val Gly Leu Leu Arg Leu Pro Gly Arg Arg Ala Arg Pro Pro Thr Ala Gly Thr Arg Cys Arg Val Ala Gly Trp Gly Phe Val Ser Asp Phe Glu Glu Leu Pro Pro Gly Leu Met Glu Ala Lys Val Arg Val Leu Asp Pro Asp Val Cys Asn Ser Ser Trp Lys Gly His Leu Thr Leu Thr Met Leu Cys Thr Arg Ser Gly Asp Ser His Arg Arg Gly Phe Cys Ser Ala Asp Ser Gly Gly Pro Leu Val Cys Arg Asn Arg Ala His Gly Leu Val Ser Phe Ser Gly Leu Trp Cys Gly Asp Pro Lys Thr Pro Asp Val Tyr Thr Gln Val Ser Ala Phe Val Ala Trp Ile Trp Asp Val Val Arg Arg Ser Ser Pro Gln Pro Gly Pro Leu Pro Gly Thr Thr Arg Pro Pro Gly Glu Ala Ala <210> 21 <211> 896 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 660 c223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1220330 <400> 21 atgccatcgc gccggcgcga tgccatcaaa gtcatgcaga ggttcgcggg gctgccggag 60 accggccgca tggacccagg gacagtggcc accatgcgta agccccgctg ctccctgcct 120 gacgtgctgg gcgtggcggg gctggtcagg cggcgtcgtc ggtacgctct gagcggcagc 180 gtgtggaaga agcgaaccct gacatggagg gtacgttcct tcccccagag ctcccagctg 240 agccaggaga ccgtgcgggt cctcatgagc tatgccctga tggcctgggg catggagtca 300 ggcctcacat ttcatgaggt ggattccccc cagggccagg agcccgacat cctcatcgac 360 tttgcccgcg ccttccacca ggacagctac cccttcgacg ggttgggggg caccctagcc 420 catgccttct tccctgggga gcaccccatc tccggggaca ctcactttga cgatgaggag 480 acctggactt ttgggtcaaa ggcctctcag cagctggagc aggagctggc aggcggctca 540 ccggttgatg aggagctggg cttcagccgg ggctggcgtg tgaatcctct gggtcctggc 600 agtcctgagc gcctgagctg aatacagagg gaagaggctg ggagcaaggc cgggtgctgn 660 ggcccgcagg cctgtgttct gagagtgcct gctacgagga gctctgtggt tccccaagga 720 gatgggaggg aagacctggg ggttgggggg ttggtacagg gggtaggggc agaaggaagg 780 gggcaagaag gcttgttgaa ccaaggggaa gaattggggg gaaggggggg aattggaatg 840 gaccttcaaa gggggttggt taaagggggt taattgggcc ttttaaggga aggggg 896 c210> 22 <211> 4906 <212> DNA
c213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1342493 <400> 22 ttgggatgtg cggagtcagt gccagcccgg tcccggccaa gcggagtgtg agcccggcgc 60 ctccaacgca acaccccgcg ccctcgccgg ctcccccgcc gtgcggatcg gagccagccg 120 gttgttgcca tggcattcgc cagctggtgg tacaagacgc atgtcagtga aaaaaccagt 180 gaatcgcctt ccaaaccagg agaaaagaaa ggatcagatg agaaaaaagc agcaagcctc 240 ggcagcagtc aatcctccag aacctatgct ggtggaacag cctcggccac caaggtgtca 300 gcttcctctg gtgcaaccag caagtcttcc agtatgaatc ccacagaaac caaggctgta 360 aaaacagaac ctgagaagaa gtcacagtca accaagctgt ctgtggttca tgagaaaaaa 420 tcccaagaag gaaagccaaa agaacacaca gagccaaaaa gcctacccaa gcaggcatca 480 gatacaggaa gtaacgatgc tcacaataaa aaagcagttt ccagatcagc tgaacagcag 540 ccatcagaga aatcaacaga accaaagact aaaccacaag acatgatttc tgctggtgga 600 gagagtgttg ctggtatcac tgcaatatct ggcaagccgg gtgacaagaa aaaagaaaag 660 aaatcattaa ccccagctgt gccagttgaa tctaaaccgg ataaaccatc gggaaagtca 720 ggcatggatg ctgctttgga tgacttaata gatactttag gaggacctga agaaactgaa 780 gaagaaaata caacgtatac tggaccagaa gtttcagatc caatgagttc cacctacata 840 gaggaattgg gtaaaagaga agtcacaatt cctccaaaat atagggaact attggctaaa 900 aaggaaggga tcacagggcc tcctgcagac tcttcgaaac ccatagggcc agatgatgct 960 atagacgcct tgtcatctga cttcacctgt gggtcgccta cagctgctgg aaagaaaact 1020 gaaaaagagg aatctacaga agttttaaaa gctcagtcag cagggacagt cagaagtgct 1080 gctccacccc aagagaagaa aagaaaggtg gagaaggata caatgagtga tcaagcactc 1140 gaggctctgt cggcttcact gggcacccgg caagcagaac ctgagctcga cctccgctca 1200 attaaggaag tcgatgaggc aaaagctaaa gaagaaaaac tagagaagtg tggtgaggat 1260 gatgaaacaa tcccatctga gtacagatta aaaccagcca cggataaaga tggaaaacca 1320 ctattgccag agcctgaaga aaaacccaag cctcggagtg aatcagaact cattgatgaa 1380 ctttcagaag attttgaccg gtctgaatgt aaagagaaac catctaagcc aactgaaaag 1440 acagaagaat ctaaggccgc tgctccagct cctgtgtcgg aggctgtgtg tcggacctcc 1500 atgtgtagta tacagtcagc accccctgag ccggctacct tgaagggcac agtgccagat 1560 gatgctgtag aagccttggc tgatagcctg gggaaaaagg aagcagatcc agaagatgga 1620 aaacctgtga tggataaagt caaggagaag gccaaagaag aagaccgtga aaagcttggt 1680 gaaaaagaag aaacaattcc tcctgattat agattagaag aggtcaagga taaagatgga 1740 aagccactcc tgccaaaaga gtctaaggaa cagcttccac ccatgagtga agacttcctt 1800 ctggatgctt tgtctgagga cttctctggt ccacaaaatg cttcatctct taaatttgaa 1860 gatgctaaac ttgctgctgc catctctgaa gtggtttccc aaaccccagc ttcaacgacc 1920 caagctggag ccccaccccg tgatacctcg cagagtgaca aagacctcga tgatgccttg 1980 gataaactct ctgacagtct aggacaaagg cagcctgacc cagatgagaa caaaccaatg 2040 gaagataaag taaaggaaaa agctaaagct gaacatagag acaagcttgg agaaagagat 2100 gacactatcc cacctgaata cagacatctc ctggatgata atggacagga caaaccagtg 2160 aagccaccta caaagaaatc agaggattca aagaaacctg cagatgacca agaccccatt 2220 gatgctctct caggagatct ggacagctgt ccctccacta cagaaacctc acagaacaca 2280 gcaaaggata agtgcaagaa ggctgcttcc agctccaaag cacctaagaa tggaggtaaa 2340 gcgaaggatt cagcaaagac aacagaggaa acttccaagc caaaagatga ctaaagaaat 2400 acaagttaag gtatctggta tctgcatgta aaatcttcag ctggtggatg gtgacttttg 2460 aagaacaaaa ggctttggca acagaaaaca attgttctgg gtgatttcta gaatggtttt 2520 tgttgagtct ctgaacatcc taaatattgg tttgttattc ttttccagaa agaaaatgaa 2580 tttgactggt tcacctgtgt actgagtatt gataaacttt gaattttttt aattgccttc 2640 aattgggaga gaaagcttta tatttgtaag aaatatattt gataaagttt cttaaagcaa 2700 caccaaaaaa acaaaagaaa agctaagtga atttttgcac attctacaca cagtgcctgt 2760 aaatctcatt tgtattttca gtttgccctt aatttttttt gttagtgttt agaaaacaat 2820 gttttaaaca ttcttcagtg ttctgatttc ttattacccc ctttcctctt gggcttttga 2880 actgtatttg atgttgcttt gggataatgt ttataagtca aacataagat attgtacatt 2940 gggcacatat ctcctcttgg gctgctaata ataaattaat aacaggtaac ctggacaaac 3000 caggaagcac caaacccctt ttcagtttga actcttcttt gccaggtgtg aggacttctg 3060 catcttacag tcagcacaga acacactgag acttgaatca agtcagcaac agagcaaaat 3120 z~i4z aaaggttaga taagtccttg tgtagcaaat ttcgagcata agaaataaaa tctaattaat 3180 tcttagggta ctcatctgac ttgaactctg ttggtttact gtgttagtaa actgtgcttt 3240 ctattatcta tacataaaac ctgagcagca actgtgtctt tagagctatt gccacattag 3300 cctttgcact gtatagcgtc tggctttatg gaacttaagt ttaccaaata taaaaagaaa 3360 cttctgcttt taaaaaaatt atatatatat atattaaatt tgaaacctgc atttctccca 3420 cagcaatgta agaagtaggc tctgatgtcc taccactttg aatggttttc taatatctta 3480 atgaatagtt cctgaacatt gcactgatat catcgattag aattttgata tttaatttca 3540 tctttatttc ctggtagaga atgcaggaaa agatgtcagg tacataacat aaaacagatt 3600 gggaatttat tgtttccaaa gggcatggcc ttccttagca tcagtttgaa gcttttgtta 3660 tgacttagct gacttgtggc agcggggcaa gcaaaaacaa taacactgct tataaatggc 3720 accacatctt gttaacctcc cccccaaata ctctctgaaa gtcatgcaca tacctatggg 3780 attttacaca ccaccagctt aaaatgctat gtctctatcc atcagaaata gtcattattc 3840 tatttttaag gcagcaacaa gaaaagaaaa aacacttttc ctgagggatt tctaaccatg 3900 tatctaatcc tcccattttg ggcagtatag gtgtttgctt ttttgttttc tttttttaag 3960 aaaaaccttg aaacctttga cactgacaga tgtgtttgca aggatacggc tgcagtatta 4020 ctaatttcca tgtgtatctg gaagtatttt taaatggcat accaaaatcc agaagtttaa 4080 agatgcctat aaaagtaaac aacatttatt taaaaagaac tctgaatatg ccttcttttt 4140 taattagaaa tatcttcgag acttgggtgt ttgttaataa ctaataactg gagtaagcta 4200 caggatctaa agcagccctt tttacagtct agttaggaga gagaaaataa ttgcaaatat 4260 ccacttagag gcaaagaaca attttttatt atcaaaaagg tttctgcaca ttgttgtggc 4320 aatattgtat ctgtttagaa aatgggcttt tccaaaagca aacaaagata ggttcctcag 4380 gtgaccaaaa ctgaaaatca atatttccat gtttcattaa tcaaggcata aaatacaatt 4440 aaagcaaaat attttacatt aaaatcttgg ttgtgtattt ttttaaaaga agggaaatag 4500 tttagtttgg ggtggaaatt accagtgatt tctattttta ttaaaccatt cactacaaca 4560 aataagtata aaaattccaa ttccactttt atacctattt atttgttgta gtgaatggtt 4620 taataaatgg cagatttatg tccagaagtc actctatttt gtcgtgtatt agaggaacac 4680 attttgacat ttttcgtatc aatcatcaat catattccct tatcttgaag tttttgcctt 4740 cttatattca aaaagttcag tttgaattct cctttgccag gtgtgaggat ttccgcacct 4800 tagagtcagc gcaaaacacg ctgcaacttg aatcaatcaa gtcagcaaca gagcacaccg 4860 gacgcgtggg cggacgcgtg ggcggacgcg tgggcggacg cgtggg 4906 <210> 23 <211> 1641 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1698270 <400> 23 gccgcctcca cggcccgcgc tcgtactgga gcgaagagcg gcctcctgaa ggaggggaag 60 ggacgtgggg gcggccacgg caggattaac ctccatttca gctaatcatg ggagagatta 120 aagtctctcc tgattataac tggtttagag gtacagttcc ccttaaaaag attattgtgg 180 atgatgatga cagtaagata tggtcgctct atgacgcggg cccccgaagt atcaggtgtc 240 ctctcatatt cctgccccct gtcagtggaa ctgcagatgt ctttttccgg cagattttgg 300 ctctgactgg atggggttac cgggttatcg ctttgcagta tccagtttat tgggaccatc 360 tcgagttctg tgatggattc agaaaacttt tagaccattt acaattggat aaagttcatc 420 tttttggcgc ttctttggga ggctttttgg cccagaaatt tgctgaatac actcacaaat 480 ctcctagagt ccattcccta atcctctgca attccttcag tgacacctct atcttcaacc 540 aaacttggac tgcaaacagc ttttggctga tgcctgcatt tatgctcaaa aaaatagttc 600 ttggaaattt ttcatctggc ccggtggacc ctatgatggc tgatgccatt gatttcatgg 660 tagacaggct agaaagtttg ggtcagagtg aactggcttc aagacttacc ttgaattgtc 720 aaaattctta tgtggaacct cataaaattc gggacatacc tgtaactatt atggatgtgt 780 ttgatcagag tgcgctttca actgaagcta aagaagaaat gtacaagctg tatcctaatg 840 cccgaagagc tcatctgaaa acaggaggca atttcccata cctgtgcaga agtgcagagg 900 tcaatcttta tgtacagata catttgctgc aattccatgg aaccaaatac gcggccattg 960 acccatcaat ggtcagtgcc gaggagcttg aggtgcagaa aggcagcctt ggcatcagcc 1020 aggaggagca gtagtgtgtc tctcgctgtc aatgatgagt tgacccggtg tgttcttgta 1080 tagtcagtgg catcagcacc cgtcagccgg ccttttcctt caggttcgtc aggctcaccg 1140 gttctcactg tgtctgggaa gtaggactga tggtcatctt catgacaggc ggcatctcca 1200 ctaagcctgt gtaactgttc cctctttggt tttcttagct tttgaatttg aagaagtact 1260 tttgaagact cccattttaa gaaccgtgca gattttgcta ccaaaagtct tcaccactgt 1320 gttcttaagt gaatgttaat ttctgaggtt tgggactttg tggtggtttt tttcttcttt 1380 tcttttccat tcttctttct ttctttttat gttgtttgct gtaaatgctg cacatccaga 1440 ttgcatatca ggacattggt tattttatgc tttcttggat ataaccatga tcagagtgcc 1500 atggccacta ccccactgtt tgctctcctg caaatcaact gcttttaatt tacacttaaa 1560 caaattgttt tgagtgttag ctactgcctt tctagatatt agtcatttgg aataaaaatt 1620 caatttcact gaaaaaaaaa a 1641 <210> 24 <211> 849 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2012492 <400> 24 aaaatgaatg aagaatgagg acattccacc actctccaag ttatcaagat gaagacccaa 60 gatggtggca ttcactctga aggtgcagca gctgagcatt ccaaattcgg gaaccaccag 120 aaaggctggc ctctcttcaa catgggatct tctggacttt tgagcctcct ggtgctattc 180 gtcctcttag cgaatgtcca gggacctggt ctgactgatt ggttatttcc caggagatgt 240 cccaaaatca gagaagaatg tgaattccaa gaaagggatg tgtgtacaaa ggacagacaa 300 tgccaggaca acaagaagtg ttgtgtcttc agctgcggaa aaaaatgttt agatctcaaa 360 caagatgtat gcgaaatgcc aaaagaaact ggcccctgcc tggcttattt tcttcattgg 420 tggtatgaca agaaagataa tacttgctcc atgtttgtct atggtggctg ccagggaaac 480 aataacaact tccaatccaa agccaactgc ctgaacacct gcaagaataa acgctttccc 540 tgattggata aggatgcact ggaagaactg ccagaatgtg gctcatgctc tgagtactgt 600 tcctgtacct gacggatgct ccagactggc ttccagtttc actctcagca ttccaagatc 660 ttagcccttc ccagaacaga acgcttgcat ctacctcctc ttcctccatc tttggctctt 720 ttgatgcaca atatccatcc gttttgattt catctttatg tcccctttat ctccaacttc 780 tagaactccc agtttatacc tgtgtcactc tcaatttttt ccagtaaagt acttgatgta 840 aaaaaaaaa 849 <210> 25 <211> 2166 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2309875 <400> 25 ggaccaacaa agatggcggc ggcccctgcg gcgggagcga tctgggcaac ggctgcggct 60 aaagctgcag ccgggcccac gggggggctg cacgggggta gtagggggtg gccctgaact 120 ggggcctggc cctggctggc ctctcccgcc gcctcactgg gggacaggtc cagcctgtgg 180 tgtccacaat gccccaggcc tctgagcacc gcctgggccg tacccgagag ccacctgtta 240 atatccagcc ccgagtggga tccaagctac catttgcccc cagggcccgc agcaaggagc 300 gcagaaaccc agcctctggg ccaaacccca tgttacgacc tctgcctccc cggccaggtc 360 tgcctgatga acggctcaag aaactggagc tgggacgggg acggacctca ggccctcgtc 420 ccagaggccc ccttcgagca gatcatgggg ttcccctgcc tggctcacca cccccaacag 480 tggctttgcc tctcccatct cggaccaact tagcccgttc caagtctgtg agcagtgggg 540 acttgcgtcc aatggggatt gccttgggag ggcaccgtgg caccggagag cttggggctg 600 cactgagccg cttggccctc cggcctgagc cacccacttt gagacgtagc acttctctcc 660 gccgcctagg gggctttcct ggacccccta ccctgttcag catacggaca gagccccctg 720 cttcccatgg ctccttccac atgatatccg cccggtcctc tgagcctttc tactctgatg 780 acaagatggc tcatcacaca ctccttctgg gctctggtca tgttggcctt cgaaacctgg 840 gaaacacgtg cttcctgaat gctgtgctgc agtgtctgag cagcactcga cctcttcggg 900 acttctgtct gagaagggac ttccggcaag aggtgcctgg aggaggccga gcccaagagc 960 tcactgaagc ctttgcagat gtgattggtg ccctctggca ccctgactcc tgcgaagctg 1020 tgaatcctac tcgattccga gctgtcttcc agaaatatgt tccctccttc tctggataca 1080 gccagcagga tgcccaagag ttcctgaagc tcctcatgga gcggctacac cttgaaatca 1140 accgccgagg ccgccgggct ccaccgatac ttgccaatgg tccagttccc tctccacccc 1200 gccgaggagg ggctctgcta gaagaacctg agttaagtga tgatgaccga gccaacctaa 1260 tgtggaaacg ttacctggag cgagaggaca gcaagattgt ggacctgttt gtgggccagt 1320 tgaaaagttg tctcaagtgc caggcctgtg ggtatcgctc cacgaccttc gaggtttttt 1380 gtgacctgtc cctgcccatc cccaagaaag gatttgctgg gggcaaggtg tctctgcggg 1440 attgtttcaa ccttttcact aaggaagaag agctagagtc ggagaatgcc ccagtgtgtg 1500 accgatgtcg gcagaaaact cgaagtacca aaaagttgac agtacaaaga ttccctcgaa 1560 tcctcgtgct ccatctgaat cgattttctg cctcccgagg ctccatcaaa aaaagttcag 1620 taggtgtaga ctttccactg cagcgactga gcctagggga ctttgccagt gacaaagccg 1680 gaagtcctgt ataccagctg tatgcccttt gcaaccactc aggcagcgtc cactatggcc 1740 actacacagc cctgtgccgg tgccagactg gttggcatgt ctacaatgac tctcgtgtct 1800 cccctgtcag tgaaaaccag gtggcatcca gcgagggcta cgtgctgttc taccaactga 1860 tgcaggagcc accccggtgc ctgtgacacc tctaagctct ggcacctgtg aagcccttta 1920 aacaccctta agccccaggc tccccgttta cctcagagac gtctattttt gtgtcttttt 1980 aatcggggag gggggagggg gtggttgtag ctccattatt ttttttatta aaaaataccc 2040 ttccacctgg aggctccctt gtctcccagc cccatgtaca aagctcacca agcccctgcc 2100 catgtacagc ccccagaccc tctgcaatat cactttttgt gaataaattt attaagaaaa 2160 aaaaaa <210> 26 <211> 2069 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2479394 <400> 26 gacttgagtc actctcagac tctttataaa tacagcttga ctcagccact gtatgactga 60 ctccccgggg acatgaggtg gatactgttc attggggccc ttattgggtc cagcatctgt 120 ggccaagaaa aattttttgg ggaccaagtt ttgaggatta atgtcagaaa tggagacgag 180 atcagcaaat tgagtcaact agtgaattca aacaacttga agctcaattt ctggaaatct 240 ccctcctcct tcaatcggcc tgtggatgtc ctggtcccat ctgtcagtct gcaggcattt 300 aaatccttcc tgagatccca gggcttagag tacgcagtga caattgagga cctgcaggcc 360 cttttagaca atgaagatga tgaaatgcaa cacaatgaag ggcaagaacg gagcagtaat 420 aacttcaact acggggctta ccattccctg gaagctattt accacgagat ggacaacatt 480 gccgcagact ttcctgacct ggcgaggagg gtgaagattg gacattcgtt tgaaaaccgg 540 ccgatgtatg tactgaagtt cagcactggg aaaggcgtga ggcggccggc cgtttggctg 600 aatgcaggca tccattcccg agagtggatc tcccaggcca ctgcaatctg gacggcaagg 660 aagattgtat ctgattacca gagggatcca gctatcacct ccatcttgga gaaaatggat 720 attttcttgt tgcctgtggc caatcctgat ggatatgtgt atactcaaac tcaaaaccga 780 ttatggagga agacgcggtc ccgaaatcct ggaagctcct gcattggtgc tgacccaaat 840 agaaactgga acgctagttt tgcaggaaag ggagccagcg acaacccttg ctccgaagtg 900 taccatggac cccacgccaa ttcggaagtg gaggtgaaat cagtggtaga tttcatccaa 960 aaacatggga atttcaaggg cttcatcgac ctgcacagct actcgcagct gctgatgtat 1020 ccatatgggt actcagtcaa aaaggcccca gatgccgagg aactcgacaa ggtggcgagg 1080 cttgcggcca aagctctggc ttctgtgtcg ggcactgagt accaagtggg tcccacctgc 1140 accactgtct atccagctag cgggagcagc atcgactggg cgtatgacaa cggcatcaaa 1200 tttgcattca catttgagtt gagagatacc gggacctatg gcttcctcct gccagctaac 1260 cagatcatcc ccactgcaga ggagacgtgg ctggggctga agaccatcat ggagcatgtg 1320 cgggacaacc tctactaggc gatggctctg ctctgtctac atttatttgt acccacacgt 1380 gcacgcactg aggccattgt taaaggagct ctttcctacc tgtgtgagtc agagccctct 1440 gggtttgtgg agcacacagg cctgcccctc tccagccagc tccctggagt cgtgtgtcct 1500 ggcggtgtcc ctgcaagaac tggttctgcc agcctgctca attttggtcc tgctgttttt 1560 gatgagcctt ttgtctgttt ctccttccac cctgctggct gggcggctgc actcagcatc 1620 accccttcct gggtggcatg tctctctcta cctcattttt agaaccaaag aacatctgag 1680 atgattctct accctcatcc acatctagcc aagccagtga ccttgctctg gtggcactgt 1740 gggagacacc acttgtcttt aggtgggtct caaagatgat gtagaatttc ctttaatttc 1800 tcgcagtctt cctggaaaat attttccttt gagcagcaaa tcttgtaggg atatcagtga 1860 aggtctctcc ctccctcctc tcctgttttt ttttttttga gacagagttt tgctcttgtt 1920 gcccaggctg gagtttaagg gcccgatctg ggctcaccaa aacctttgcc tccggggttc 1980 aagaaatttt cctgcctaag ccttttgagt accttggttt aaaggggcat tcaacaatgc 2040 ctgggaaatt ttgggttttt agaagaaaa 2069 <210> 27 <211> 2490 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2613215 <400> 27 ccgcgggtga tcagctggtc tgcgctcccc tgacgtgggc tggggcacgt caccgccgaa 60 tggcagcctc cagaaagcca ccgcgagtaa gggtgaatca ccaggatttt caactgagaa 120 atttaagaat aattgaacct aacgaggtga cacactcagg agacacaggt gtggaaacag 180 acggcagaat gcctccaaag gtgacttcag agctgcttcg gcagctgaga caagccatga 240 ggaactctga gtatgtgacc gaaccgatcc aggcctacat catcccatcg ggagatgctc 300 atcagagtga gtatattgct ccatgtgact gtcggcgggc ttttgtctct ggattcgatg 360 gctctgcggg cacagccatc atcacagaag agcatgcagc catgtggact gacgggcgct 420 actttctcca ggctgccaag caaatggaca gcaactggac acttatgaag atgggtctga 480 aggacacacc aactcaggaa gactggctgg tgagtgtgct tcctgaaggg tccagggttg 540 gtgtggaccc cttgatcatt cctacagatt attggaagaa aatggccaaa gttctgagaa 600 gtgccggcca tcacctcatt cctgtcaagg agaacctcgt tgacaaaatc tggacagacc 660 gtcctgagcg cccttgcaag cctctcctca cactgggcct ggattacaca ggcatctcct 720 ggaaggacaa ggttgcagac cttcggttga aaatggctga gaggaacgtc atgtggtttg 780 tggtcactgc cttggatgag attgcgtggc tatttaatct ccgaggatca gatgtggagc 840 acaatccagt atttttctcc tacgcaatca taggactaga gacgatcatg ctcttcattg 900 atggtgaccg catagacgcc cccagtgtga aggagcacct gcttcttgac ttgggtctgg 960 aagccgaata caggatccag gtgcatccct acaagtccat cctgagcgag ctcaaggccc 1020 tgtgtgctga cctctcccca agggagaagg tgtgggtcag tgacaaggcc agctatgctg 1080 tgagcgagac catccccaag gaccaccgct gctgtatgcc ttacaccccc atctgcatcg 1140 ccaaagctgt gaagaattca gctgagtcag aaggcatgag gcgggctcac attaaagatg 1200 ctgttgctct ctgtgaactc tttaactggc tggagaaaga ggttcccaaa ggtggtgtga 1260 cagagatctc agctgctgac aaagctgagg agtttcgcag gcaacaggca gactttgtgg 1320 acctgagctt cccaacaatt tccagtacgg gacccaacgg cgccatcatt cactacgcgc 1380 cagtccctga gacgaatagg accttgtccc tggatgaggt gtaccttatt gactcgggtg 1440 ctcaatacaa ggatggcacc acagatgtga cgcggacaat gcattttggg acccctacag 1500 cctacgagaa ggaatgcttc acatatgtcc tcaagggcca catagctgtg agtgcagccg 1560 ttttcccgac tggaaccaaa ggtcaccttc ttgactcctt tgcccgttca gctttatggg 1620 attcaggcct agattacttg cacgggactg gacatggtgt tgggtctttt ttgaatgtcc 1680 atgagggtcc ttgcggcatc agttacaaaa cattctctga tgagcccttg gaggcaggca 1740 tgattgtcac tgatgagccc gggtactatg aagatggggc ttttggaatt cgcattgaga 1800 atgttgtcct tgtggttcct gtgaagacca agtataattt taataaccgg ggaagcctga 1860 cctttgaacc tctaacattg gttccaattc agaccaaaat gatagatgtg gattctctta 1920 cagacaaaga gtgcgactgg ctcaacaatt accacctgac ctgcagggat gtgattggga 1980 aggaattgca gaaacagggc cgccaggaag ctctcgagtg gctcatcaga gagacgcaac 2040 ccatctccaa acagcattaa taaatacctc cccggttttg tttttgtaaa atgctctgga 2100 ggaaggaaga aacgtggcag atccctgaca tctttcccct ttcctttcct tcttccctac 2160 ctcccctttt tactttagac tttaagaaga acagaaaatc ttcttatcct ctttgatatt 2220 ttattgcaaa cactcagtct tttatgattt tttaattgtt gagaacaagc caagaataaa 2280 attgctgcac cagaaggagg gtccctccaa agttgaacac ttggtgaaag gaagatgccc 2340 cgacttcttt ggccagtgat ggggaatcag tgagtgctcc atgatggtca tgttccaggt 2400 gctagtacat cattcatgat caccttaatg ctcatgagac tatatttatg atcagtgaat 2460 aaaaatgtca gaactgtgaa aaaaaaaaaa 2490 <210> 2B
<211> 3148 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 001528 <400> 28 gaagatggcg gaaggcggag cggcggatct ggacacccag cggtctgaca tcgcgacgct 60 gctcaaaacc tcgctccgga aaggggacac ctggtaccta gtcgatagtc gctggttcaa 120 acagtggaaa aaatatgttg gctttgacag ttgggacaaa taccagatgg gagatcaaaa 180 tgtgtatcct ggacccattg ataactctgg acttctcaaa gatggtgatg cccagtcact 240 taaggaacac cttattgatg aattggatta catactgttg ccaactgaag gttggaataa 300 acttgtcagc tggtacacat tgatggaagg tcaagagcca atagcacgaa aggtggttga 360 acagggtatg tttgtaaagc actgcaaagt agaagtatat ctcacagaat tgaagctatg 420 tgaaaatgga aacatgaata atgttgtaac tcgaagattt agcaaagctg acacaataga 480 tacaattgaa aaggaaataa gaaaaatctt cagtattcca gatgaaaagg agaccagatt 540 gtggaacaaa tacatgagta acacatttga accactgaat aaaccagaca gcaccattca 600 ggatgctggt ttataccaag gacaggtatt agtgatagaa cagaaaaatg aagatggaac 660 acggccaagg ggtccttcta ctcctaatgt gaaaaactca aattactgtc ttccatcata 720 taccgcttat aagaactatg attattcgga acctggaaga aacaatgaac agccaggcct 780 ctgtggccta agtaacttgg gaaatacgtg tttcatgaac tcagctattc agtgtttgag 840 caacacacct ccacttactg agtatttcct caatgataag tatcaagaag aactgaattt 900 tgacaatccc ttaggaatga gaggtgaaat agctaaatct tatgccgaac tgatcaagca 960 aatgtggtct ggaaagttta gctacgtcac cccaagagcc tttaagacac aggtaggacg 1020 ttttgcacct cagttctctg gatatcagca gcaagactgt caagaactgt tagctttcct 1080 attagatgga ttacatgagg atttgaatag aattaggaaa aaaccatata tacaattaaa 1140 agatgcagat ggaaggccag ataaggtggt tgccgaagaa gcctgggaaa accatttaaa 1200 acgaaatgat tctatcatag tagatatatt tcatggcctt ttcaaatcaa ctttagtttg 1260 tcctgagtgt gctaagattt cagtaacatt tgatcctttt tgttacttga cacttccatt 1320 gcccatgaaa aaagaacgca ccttggaagt ttacttagtt agaatggatc cacttaccaa 1380 acctatgcag tacaaagtgg ttgtccccaa aattggaaac atattagatc tttgtacagc 1440 attgtctgct ttgtcaggaa tacctgcaga taagatgata gttactgata tatacaatca 1500 tagatttcac agaatattcg ctatggatga aaaccttagt agtattatgg aacgggatga 1560 tatttatgtg tttgaaatta acatcaatag gacagaagat acagagcacg tgattattcc 1620 tgtttgccta agagaaaaat tcagacactc gagttatacc caccatactg gttcttcact 1680 ttttggtcag ccctttctta tggctgtacc acgaaacaat actgaagaca aactttataa 1740 tctcctgctc ttgagaatgt gccgatatgt caaaatatct actgaaactg aagaaactga 1800 aggatcccta cactgctgta aggaccaaaa tattaatggg aatggcccaa atggcataca 1860 tgaagaaggc tcaccaagtg aaatggaaac agatgagcca gatgatgaat ccagccagga 1920 tcaagaactt ccctcagaga atgaaaacag tcagtctgaa gattcagttg gaggagataa 1980 tgattctgaa aatggattat gtactgagga tacttgcaaa ggtcaactca cgggacacaa 2040 aaaacgattg tttacattcc agttcaacaa cttaggcaat actgatatca actacatcaa 2100 agatgatacc aggcatataa gatttgatga taggcagctt aggctagatg aaagatcttt 2160 tcttgctttg gattgggatc ctgatttgaa aaaaagatat tttgatgaaa atgctgctga 2220 ggactttgaa aaacatgaaa gtgtggagta taaacctcct aaaaaaccct ttgtgaaatt 2280 aaaagattgc attgaacttt ttacaacaaa agaaaagcta ggtgctgaag atccctggta 2340 ttgtccgaat tgtaaagaac atcagcaagc cacaaagaaa ttggatttat ggtccctgcc 2400 tccagtactt gtagtacatc tcaagcgatt ttcttacagt cgatacatga gagacaagtt 2460 ggatacctta gttgattttc ctatcaatga cttggatatg tcggaattct taattaatcc 2520 aaatgcaggt ccttgccgct ataatctgat tgctgtttcc aaccactatg gagggatggg 2580 aggaggacac tatactgctt ttgcaaaaaa taaagatgat ggaaaatggt actattttga 2640 tgacagtagt gtctccactg catctgaaga ccaaattgtg tccaaagcag catatgtact 2700 cttctaccag agacaagaca ctttcagtgg aactggcttt tttcctcttg accgagaaac 2760 taaaggtgct tcagctgcca ctggcatccc attagaaagt gatgaagata gcaatgataa 2820 tgacaatgat atagaaaatg aaaactgtat gcacactaac taatgaaagt cctagaagcc 2880 ataaaagaga cactttcctg ctggtggtat ctatggaaat gatgaagtta cccaccacat 2940 taaaacaaaa gtctgagatg gggagtttca gataaccgaa tgtaaatcct ttatcagatt 3000 ttaacttgtg cagtacttga agtgaaacac aatgaaaact ttaacagaaa ttgtctctta 3060 atacatttac agtcttgtat ttacaagcta aatatatata ggaaatcaca aataaatccc 3120 ttttaagttt gctgctgttt tgattaaa 3148 <210> 29 <211> 855 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 747 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 998626 <400> 29 caggcaggca tcccccggag ttgtctcttt tcatgccagc gccaacagga ggctgtctgg 60 acacactgat tactcactca ccagcctcct tcttttgtcc accagccccc ctcttttgtc 120 caccagccca gcctgactcc tggagattgt gaatagctcc atccagcctg agaaacaagc 180 cgggtggctg agccaggctg tgcacggagc gcctgacggg cccaacagac ccatgctgca 240 tccagagacc tcccctggcc gggggcatct cctggctgtg ctcctggccc tccttggcac 300 cgcctgggca gaggtgtggc caccccagct gcaggagcag gctccgatgg ccggagccct 360 gaacaggaag gagagtttct tgctcctctc cctgcacaac cgcctgcgca gctgggtcca 420 gccccctgcg gctgacatgc ggaggctgga ctggagtgac agcctggccc aactggctca 480 agccagggca gccctctgtg gaatcccaac cccgagcctg gcgtccggcc tgtggcgcac 540 cctgcaagtg ggctggaaca tgcagctgct gcccgcgggc ttggcgtcct ttgttgaagt 600 ggtcagccta tggtttgcag aggggcagcg gtacagccac gcggcaggag agtgtgctcg 660 caacgccacc tgcacccact acacgcagct cgtgtgggcc acctcaagcc agctgggctg 720 tgggcggcac tagtgtctgc aggccangag catagaagct ttgtctgtgc tactccccgg 780 aggcactggg agtcacggga gacatcatcc tataagaggg tgctgtgtcg tctgacagca 840 tgttcagctg ctcaa 855 <210> 30 <211> 1912 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1393301 <400> 30 agaattcggc acgagggtta gaggcggctt gtgtccacgg gacgcgggcg gatcttctcc 60 ggccatgagg aagccagccg ctggcttcct tccctcactc ctgaaggtgc tgctcctgcc 120 tctggcacct gccgcagccc aggattcgac tcaggcctcc actccaggca gccctctctc 180 tcctaccgaa tacgaacgct tcttcgcact gctgactcca acctggaagg cagagactac 240 ctgccgtctc cgtgcaaccc acggctgccg gaatcccaca ctcgtccagc tggaccaata 300 tgaaaaccac ggcttagtgc ccgatggtgc tgtctgctcc aacctccctt atgcctcctg 360 gtttgagtct ttctgccagt tcactcacta ccgttgctcc aaccacgtct actatgccaa 420 gagagtcctg tgttcccagc cagtctctat tctctcacct aacactctca aggagataga 480 agcttcagct gaagtctcac ccaccacgat gacctccccc atctcacccc acttcacagt 540 gacagaacgc cagaccttcc agccctggcc tgagaggctc agcaacaacg tggaagagct 600 cctacaatcc tccttgtccc tgggaggcca ggagcaagcg ccagagcaca agcaggagca 660 aggagtggag cacaggcagg agccgacaca agaacacaag caggaagagg ggcagaaaca 720 ggaagagcaa gaagaggaac aggaagagga gggaaagcag gaagaaggac aggggactaa 780 ggagggacgg gaggctgtgt ctcagctgca gacagactca gagcccaagt ttcactctga 840 atctctatct tctaaccctt cctcttttgc tccccgggta cgagaagtag agtctactcc 900 tatgataatg gagaacatcc aggagctcat tcgatcagcc caggaaatag atgaaatgaa 960 tgaaatatat gatgagaact cctactggag aaaccaaaac cctggcagcc tcctgcagct 1020 gccccacaca gaggccttgc tggtgctgtg ctattcgatc gtggagaata cctgcatcat 1080 aacccccaca gccaaggcct ggaagtacat ggaggaggag atccttggtt tcgggaagtc 1140 ggtctgtgac agccttgggc ggcgacacat gtctacctgt gccctctgtg acttctgctc 1200 cttgaagctg gagcagtgcc actcagaggc cagcctgcag cggcaacaat gcgacacctc 1260 ccacaagact ccctttgtca gccccttgct tgcctcccag agcctgtcca tcggcaacca 1320 ggtagggtcc ccagaatcag gccgctttta cgggctggat ttgtacggtg ggctccacat 1380 ggacttctgg tgtgcccggc ttgccacgaa aggctgtgaa gatgtccgag tctctgggtg 1440 gctccagact gagttcctta gcttccagga tggggatttc cctaccaaga tttgtgacac 1500 agactatatc cagtacccaa actactgttc cttcaaaagc cagcagtgtc tgatgagaaa 1560 ccgcaatcgg aaggtgtccc gcatgagatg tctgcagaat gagacttaca gtgcgctgag 1620 ccctggcaaa agtgaggacg ttgtgcttcg atggagccag gagttcagca ccttgactct 1680 aggccagttc ggatgagctg gcgtctattc tgcccacacc ccagcccaac ctgcccacgt 1740 tctctattgt tttgagaccc cattgctttc aggctgcccc ttctgggtct gttactcggc 1800 ccctactcac atttccttgg gttggagcaa cagtcccaga gagggccatg gtgggagctg 1860 cgccctcctt aaaagatgac tttacataaa atgttgatct tcaaaaaaaa as 1912 <210> 31 <211> 768 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1444055 <400> 31 taagtgttac aatttaaagc caaaggcata ccaaatatta aagcaacaca acataaggaa 60 tcccattcac cacacaccat ttaaacacaa atgatggatt ctgttattta tcaaacccat 120 ttaatgtttt gtgtagtgtt ttcatggatt ccaggaatgg cttccctttc tcaaatgatc 180 aatttcaagt cttattctcc atatttccca tctgattcca ctatgagcag tgccattgca 240 tgccctacta gaactgtgga tacgctttaa aacaagtgga agatgatagg gtgggactca 300 ttacgtctaa tcttggggaa cactgataac gtgtccagga gagacagcac aaggggctcc 360 atcttcatca cacaactcat cgcatgcttc cagagatatt cctggcgctg ccacctagag 420 gaagtatttt ggaaggttca gcaagcattt gaaagtccgg aggcaacagt ccaaatgccc 480 accatagaac gagtgtccat gacaagatat ttctacctct ttcctggcaa ctgaaaatgg 540 ttaagcattg agagttgttg gtggtgtatg aaataaatga aagtgtgata ttggagcagg 600 aaaccacaag cagcccagcc ctcctttatc aacttcaaga aacaccttta ctagtacaga 660 ttgaatgctt aacattttga atttcaataa aggtgaagac aaatgaaaaa aataaaaaaa 720 aaacaaaaac aaaaaacaaa caaaaaaaca aaaaaaaaat aaaaacgg 768 <210> 32 <211> 2069 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 1650177 <400> 32 gcacaggggc cggcaccacg gggttatcga agcagctgtc aagatgctgg ggtccctggt 60 gttgaggaga aaagcactgg cgccacggct actcctccgg ctgctcaggt ccccaacgct 120 ccggggccat ggaggtgctt ccggccggaa tgtgactact gggagtctcg gggagccgca 180 gtggctgagg gtagccaccg gggggcgccc tggaacatcg ccggccttgt tctccggacg 240 tggggcagcc accggggggc gccagggagg acgcttcgat accaaatgcc tcgcggctgc 300 cacttgggga cgccttcctg gtcccgaaga aacactccca ggacaggaca gctggaacgg 360 ggtccccagc agggccggac tgggcatgtg cgccctggcc gcagcgctgg tggttcattg 420 ctacagcaag agtccgtcca acaaggatgc agccctgttg gaagctgccc gtgccaacaa 480 tatgcaagaa gtcagcagtg tggtacaggt cctgcttgct gctggggctg atccaaacct 540 tggagatgat ttcagcagtg ttttcaagac tgccaaggaa cagggaatcc attctttgga 600 agtcctgatc acccgagagg atgacttcaa caacaggctg aacaaccgcg ccagtttcaa 660 gggctgcacg gccttgcact atgctgttct tgctgatgac taccgcactg tcaaggagct 720 gcttgatgga ggagccaacc ccctgcagag gaatgaaatg ggacacacac ccttggatta 780 tgcccgagaa ggggaagtga tgaagcttct gaggacttct gaagccaagt accaagagaa 840 gcagcggaag cgtgaggctg aggagcggcg ccgcttcccc ctggagcagc gactaaagga 900 gcacatcatt ggccaggaga gcgccatcgc cacagtgggt gctgcgatcc ggaggaagga 960 gaatggctgg tacgatgaag aacaccctct ggtcttcctc ttcttgggat catctggaat 1020 aggaaaaaca gagctggcca agcagacagc caaatatatg cacaaagatg ctaaaaaggg 1080 cttcatcagg ctggacatgt ccgagttcca ggagcgacac gaggtggcca agtttattgg 1140 gtctccacca ggctacgttg gccatgagga gggtggccag ctgaccaaga agttgaagca 1200 gtgccccaat gctgtggtgc tctttgatga agtagacaag gcccatccag atgtgctcac 1260 catcatgctg cagctgtttg atgagggccg gctgacagat ggaaaaggga agaccattga 1320 ttgcaaggac gccatcttca tcatgacctc caatgtggcc agcgacgaga tcgcacagca 1380 cgcgctgcag ctgaggcagg aagctttgga gatgagccgt aaccgtattg ccgaaaacct 1440 gggggatgtc cagataagtg acaagatcac catctcaaag aacttcaagg agaatgtgat 1500 tcgccctatc ctgaaagctc acttccggag ggatgagttt ctgggacgga tcaatgagat 1560 cgtctacttc ctccccttct gccactcgga gctcatccaa ctcgtcaaca aggaactaaa 1620 cttctgggcc aagagagcca agcaaaggca caacatcacg ctgctctggg accgcgaggt 1680 ggcagatgtg ctggtcgacg gctacaatgt gcactatggc gcccgctcca tcaaacatga 1740 ggtagaacgc cgtgtggtga accagctggc agcagcctat gagcaggacc tgctgccagg 1800 gggctgtact ttgcgcatca cggtggagga ctcagacaag cagctactca aaagcccaga 1860 actgccctca ccccaggctg agaagcgcct ccccaagctg cgtctggaga tcatcgacaa 1920 ggacagcaag actcgcagac tggacatccg ggcaccactg caccctgaga aggtgtgcaa 1980 caccatctag cagccacctg cctgctccta tgtgccctca ccatccaata aaggcccctt 2040 ggctgtggca tggcaaaaaa aaaaaaaaa 2069 <210> 33 <211> 2594 <212> DNA
<213> Homo sapiens <220>

<221> misc_feature <223> Incyte Clone No: 1902576 <400> 33 ccagcacctg cggggccctc gggcttggaa ggctgggccg gacggtgaac ggtcggcgcg 60 ggccggatcg gcggcggctg actcgccttc tctccggggc tgcgaccccg aggcaaccgg 120 ctgcagatgg gagcccgcgg agccgcggat gcgggcgggc cggggcgcga cgccggcgag 180 ggagctgttc cgggacgccg ccttccccgc cgcggactcc tcgctcttct gcgacttgtc 240 tacgccgctg gcccagttcc gcgaggacat cacgtggagg cggccccagg agatttgtgc 300 cacaccccgg ctgtttccag atgacccacg ggaagggcag gtgaagcagg ggctgctggg 36D
ggattgctgg ttcctgtgtg cctgcgccgc gctgcagaag agcaggcacc tcctggacca 420 ggtcattcct ccgggacagc cgagctgggc cgaccaggag taccggggct ccttcacctg 480 tcgcatttgg cagtttggac gctgggtgga ggtgaccaca gatgaccgcc tgccgtgcct 540 tgcagggaga ctctgtttct cccgctgcca gagggaggat gtgttctggc tccccttact 600 ggaaaaggtc tacgccaagg tccatgggtc ctacgagcac ctgtgggccg ggcaggtggc 660 ggatgccctg gtggacctga ccggcggcct ggcagaaaga tggaacctga agggcgtagc 720 aggaagcgga ggccagcagg acaggccggg ccgctgggag cacaggactt gtcggcagct 780 gctccacctg aaggaccagt gtctgatcag ctgctgcgtg ctcagcccca gagcaggtgc 840 ccgggagctg ggggagttcc atgccttcat tgtctcggac ctgcgggagc tccagggtca 900 ggcgggccag tgcatcctgc tgctgcggat ccagaacccc tggggccggc ggtgctggca 960 ggggctctgg agagaggggg gtgaagggtg gagccaggta gatgcagcgg tagcatctga 1020 gctcctgtcc cagctccagg aaggggagtt ctgggtggag gaggaggagt tcctcaggga 1080 gtttgacgag ctcaccgttg gctacccggt cacggaggcc ggccacctgc agagcctcta 1140 cacagagagg ctgctctgcc atacgcgggc gctgcctggg gcctgggtca agggccagtc 1200 agcaggaggc tgccggaaca acagcggctt tcccagcaac cccaaattct ggctgcgggt 1260 ctcagaaccg agtgaggtgt acattgccgt cctgcagaga tccaggctgc acgcggcgga 1320 ctgggcaggc cgggcccggg cactggtggg tgacagtcat acttcgtgga gcccagcgag 1380 catcccgggc aagcactacc aggctgtggg tctgcacctc tggaaggtag agaagcggcg 1440 ggtcaatctg cctagggtcc tgtccatgcc ccccgtggct ggcaccgcgt gccatgcata 1500 cgaccgggag gtccacctgc gttgtgagct ctcaccgggc tactacctgg ctgtccccag 1560 caccttcctg aaggacgcgc caggggagtt cctgctccga gtcttctcta ccgggcgagt 1620 ctcccttagc gccatcaggg cagtggccaa gaacaccgcc cccggggcag ccctgcctgc 1680 gggggagtgg gggaccgtgc agctacgggg ttcttggaga gtcggccaga cggcgggggg 1740 cagcaggaac tttgcctcat accccaccaa cccctgcttc cccttctcgg tccccgaggg 1800 ccctggcccc cgctgcgtcc gcatcactct gcatcagcac tgccggccca gtgacaccga 1860 gttccacccc atcggcttcc atatcttcca ggtcccagag ggtggaagga gccaggacgc 1920 acccccactg ctgctgcagg agccgctgct gagctgcgtg ccacatcgct acgcccagga 1980 ggtgagccgg ctctgcctcc tgcctgcggg cacctacaag gttgtgccct ccacctacct 2040 gccggacaca gagggggcct tcacagtgac catcgcaacc aggattgaca ggccatccat 2100 tcacagccag gagatgctgg gccagttcct ccaagaggtc tccgtcatgg cagtgatgaa 2160 aacctaacag ggtggccccc tgtgccagct caggtgactg gagcccgagg gcctgacagg 2220 ttcccagcag ctgggccggc cagccttgca ctgtgggggc tggtcctgag tcttggcctg 2280 cctcccagcc ctgccagggg gctgcggcct aggggtccac gggaagcctc cgtcaggaga 2340 gacgcagccc tgggggccag ctggtgctgc aaggaagggt gggaagcttg ctggcttctg 2400 ttgcgccact gagacggcag agaccccagg atcccagagc ttcccaggat ccctcccaga 2460 tcctctgctg actccatatg gaggcctcac acccagaggg tagggcagca gatcttcttt 2520 ataactattt attgttcgaa tcacttttag gatgtaactt tataaataaa catgagcgct 2580 gaaaaaaaaa aggg 2594 <210> 34 <211> 481 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2024210 <400> 34 tgaggtgcga cacacataat tgtcccaatt tttaagattg atggggagca tgaagcattt 60 ttttaatgtg ttggcaggcc ccattaaatg cataaactgc ataggactca tgtggtctga 120 atgtatttta gggctttctg ggaattgtct tgacagagaa cctcagctgg acaaagcagc 180 cttgatctga gtgagctaac tgacacaatg aaactgtcag gcatgtttct gctcctctct 240 ctggctcttt tctgcttttt aacaggtgtc ttcagtcagg gaggacaggt tgactgtggt 300 gagttccagg accccaaggt ctactgcact cgggaatcta acccacactg tggctctgat 360 ggccagacat atggcaataa atgtgccttc tgtaaggcca tagtgaaaag tggtggaaag 420 attagcctaa agcatcctgg aaaatgctga gttaaagcca atgtttcttg gtgacttgcc 480 <210> 35 <211> 3080 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2523109 <400> 35 cgcagcgcgg ctttcaggcc aacatggccg tgctgctgct gctgctccgt gccctccgcc 60 ggggtccagg cccgggtcct cggccgctgt ggggcccagg cccggcctgg agtccagggt 120 tccccgccag gcccgggagg gggcggccgt acatggccag caggcctccg ggggacctcg 180 ccgaggctgg aggccgagct ctgcagagct tacaattgag actgctaacc cctacctttg 240 aagggatcaa cggattgttg ttgaaacaac atttagttca gaatccagtc agactctggc 300 aacttttagg tggtactttc tattttaaca cctcaaggtt gaagcagaag aataaggaga 360 aggataagtc gaaggggaag gcgcctgaag aggacgaaga ggagaggaga cgccgtgagc 420 gggacgacca gatgtaccga gagcggctgc gcaccttgct ggtcatcgcg gttgtcatga 480 gcctcctgaa tgctctcagc accagcggag gcagcatttc ctggaacgac tttgtccacg 540 agatgctggc caagggcgag gtgcagcgcg tccaggtggt gcctgagagc gacgtggtgg 600 aagtctacct gcaccctgga gccgtggtgt ttgggcggcc tcggctagcc ttgatgtacc 660 gaatgcaggt tgcaaatatt gacaagtttg aagagaagct tcgagcagct gaagatgagc 720 tgaatatcga ggccaaggac aggatcccag tttcctacaa gcgaacagga ttctttggaa 780 atgccctgta ctctgtgggg atgacggcag tgggcctggc catcctgtgg tatgttttcc 840 gtctggccgg gatgactgga agggaaggtg gattcagtgc ttttaatcag cttaaaatgg 900 ctcgtttcac cattgtggat gggaagatgg ggaaaggagt cagcttcaaa gacgtggcag 960 gaatgcacga agccaaactg gaagtccgcg agtttgtgga ttatctgaag agcccaaaac 1020 gcttcctcca gcttggcgcc aaggtcccaa agggcgcact gctgctcggc ccccccggct 1080 gtgggaagac gctgctggcc aaggcggtgg ccacggaggc tcaggtgccc ttcctggcga 1140 tggccggccc agagttcgtg gaggtcattg gaggcctcgg cgctgcccgt gtgcggagcc 1200 tctttaagga agcccgagcc cgggccccct gcatcgtcta catcgatgag atcgacgcgg 1260 tgggcaagaa gcgctccacc accatgtccg gcttctccaa cacggaggag gagcagacgc 1320 tcaaccagct tctggtagaa atggatggaa tgggtaccac agaccatgtc atcgtcctgg 1380 cgtccacgaa ccgagctgac attttggacg gtgctctgat gaggccaggc cgactggacc 1440 ggcacgtctt cattgatctc cccacgctgc aggagaggcg ggagattttt gagcagcacc 1500 tgaagagcct gaagctgacc cagtccagca ccttttactc ccagcgtctg gcagagctga 1560 caccaggatt cagtggggct gacatcgcca acatctgcaa tgaggctgcg ctgcacgcgg 1620 cgcgggaggg acacacttcc gtgcacactc tcaacttcga gtacgccgtg gagcgcgtcc 1680 tcgcagggac tgccaaaaag agcaagatcc tgtccaagga agaacagaaa gtggttgcgt 1740 ttcatgagtc gggccacgcc ttggtgggct ggatgctgga gcacacggag gccgtgatga 1800 aggtctccat aacccctcgg acaaacgccg ccctgggctt tgctcagatg ctccccagag 1860 accagcacct cttcaccaag gagcagctgt ttgagcggat gtgcatggcc ttgggaggac 1920 gggcctcgga agcactgtcc ttcaacgagg tcacttctgg ggcacaggac gacctgagga 1980 aggtcacccg catcgcctac tccatggtga agcagtttgg gatggcacct ggcatcgggc 2040 ccatctcctt ccctgaggcg caggagggcc tcatgggcat cgggcggcgc cccttcagcc 2100 aaggcctgca gcagatgatg gaccatgaag caagactgct ggtggccaag gcctacagac 2160 acaccgagaa ggtgctgcag gacaacctgg acaagttgca ggcgctggca aacgcccttc 2220 tggaaaagga agtgataaac tatgaggaca ttgaggctct cattggcccg ccgccccatg 2280 ggccgaagaa aatgatcgca ccgcagaggt ggatcgacgc ccagagggag aaacaggact 2340 tgggcgagga ggagaccgaa gagacccagc agcctccact tggaggcgaa gagccgactt 2400 ggcccaagta gttgggaggt gttggctgca cgtgcgggtg gtccgggaag tgagggctca 2460 ctcagccacc ctgagttgct tttcagctga ggtttgcact tcctctcgcg gccctcagta 2520 gtccctgcac agtgacttct gagatctgtt gattgatgac ccttttcatg attttaagtt 2580 tctctgcaga aactactgac ggagtcctgt gtttgtgagt cgtttcccct atggggaagg 2640 ttatcagtgc ttcccgagtg agcatggaac acttcgagtt cccagggtta tagacagtcg 2700 ttcccagtgt ggctgaggcc acccagaggc agcagagcat tcagactcca aacagacccc 2760 tgttcatgcc gacgcttgca cgaccgcccc agttcctgtg gctccctcgg aatgctaagg 2820 ggatcggaca tgaaaggacc ctgtgagccg attgtcctat ctccagcggc cctgtcatcc 2880 agctcactca tcaatggggc cacacagtca ggcccaggca ctgggctccg gaggactcac 2940 cactgccccc tgctgccatg tggactggtg caagttgagg acttcttgct ggtctagtca 3000 cgcatgcagt gttggggatg ccttggtttt tactgctctg agaattgttg agatacttta 3060 ctaataaact gtgtagttgg 3080 <210> 36 <211> 1154 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2588566 <400> 36 gaactatgtt gtggttgcac agacacggag aaaatcagcg gagttcttgc tccgaatctt 60 cctgtaaatg ccagacagtg acaggcacct gagcagccat ttcaacctca gaatgaaggg 120 aagcccttca gaacatggct cccaacaaag cattttcaac agatatgctc agcagaggct 180 ggacattgat gccacccagc ttcagggcct tctcaaccag gagcttctaa caggacctcc 240 aggggacatg ttctccttag atgagtgccg cagcttggtg gctctgatgg aactgaaagt 300 gaatgggcgg ctagaccaag aggagtttgc gcgactgtgg aagcgccttg ttcactacca 360 gcatgttttc cagaaggttc agacaagccc tggagtcctc ctgagctcgg acttgtggaa 420 ggccatagag aatacagact tcctcagagg gatcttcatc agccgtgagc tgctgcatct 480 ggtgaccctc aggtacagcg acagcgtcgg cagggtcagc ttccccagcc tggtctgctt 540 cctgatgcgg cttgaagcca tggcaaagac cttccgcaac ctctctaagg atggaaaagg 600 actctacctg acagaaatgg agtggatgag cctggtcatg tacaactgaa gcaaagagga 660 aagcagaccc atggctcagg acaagctccc agtgatcact caagaatctg gctctcattc 720 taagaggctg tgctgcccag tatggtggtt gtgataaatc taaaccagcc ctgcatgaaa 780 cagagtccaa gctgtctccc aacagcctgg gttcggtcct tggctggccc aggcccagtt 840 aagcctgtgg ccaccaagca gctcatctga gcactttggg atgtattcag cctacgttgc 900 cctggaaaag gaagcaggag atgtctccct gtgggaaagg agaagagaag ttgtctctga 960 gtcccctgtc accagttgga ttcatttctt ggaagagcca gaatgagcca ctttgaccac 1020 cctcgggtgc tatgggtgac acaagagctg tccactgggt gtttgcagaa taattacact 1080 atcttatgtc tggatcctga tgatttcaca gctaaatggc aaaaataaaa catgtttccc 1140 ataaaaaaaa aaaa 1154 <210> 37 <211> 2827 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 2811 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 2740570 <400> 37 catttcgggg gtattctcag caggatgctc aagaattcct tcgatgttta atggatttgc 60 ttcatgaaga attgaaagag caagtcatgg aagtagaaga agatccgcaa accataacca 120 ctgaggagac aatggaagaa gacaagagcc agtcggatgt agattttcag tcttgtgaat 180 cttgtagcaa cagtgataga gcagaaaatg aaaatggctc tagatgcttt tctgaagata 240 ataatgaaac aacaatgtta attcaggatg atgaaaacaa ttcagaaatg tcaaaggatt 300 ggcaaaaaga gaagatgtgc aataagatta ataaagtaaa ttctgaaggc gaatttgata 360 aagatagaga ctctatatct gaaacagtcg acttaaacaa ccaggaaact gtcaaagtgc 420 aaatacacag cagagcttca gaatatatca ctgatgtcca ttcgaatgac ctgtctacac 480 cacagatcct tccatcaaat gaaggtgtta atccacgttt atcggcaagc cctcctaaat 540 caggcaattt gtggccagga ttggcaccac cacacaaaaa agctcagtct gcatctccaa 600 agagaaaaaa acagcacaag aaatacagaa gtgttatttc agacatattt gatggaacaa 660 tcattagttc agtgcagtgt ctgacttgtg acagggtgtc tgtaaccctc gagacctttc 720 aagatctgtc cttgccaatt cctggcaagg aagaccttgc taagctgcat tcatcaagtc 780 atccaacttc tatagtcaaa gcaggatcat gtggcgaagc atatgctcca caagggtgga 840 tagctttttt catggaatat gtgaagaggt ttgttgtctc atgtgtccct agctggtttt 900 ggggtccagt agtaaccttg caagattgtc ttgctgcctt ctttgccaga gatgaactaa 960 aaggtgacaa tatgtacagt tgtgaaaaat gcaaaaagct gagaaatgga gtgaagtttt 1020 gtaaagtaca aaactttcct gagattttgt gcatccacct taaaagattc agacatgaac 1080 taatgttttc caccaaaatc agtacccatg tttcatttcc gctagaaggc ttggatcttc 1140 agccatttct tgctaaggat agtccagctc aaattgtgac atatgatctt ctgtcagtca 1200 tttgccatca tggaactgca agtagtggac actatatagc ctactgccga aacaatctaa 1260 ataatctctg gtatgaattt gatgatcaga gtgtcactga agtttcagaa tctactgtac 1320 aaaatgcaga agcttacgtt cttttctata ggaagagcag cgaagaggca caaaaagaga 1380 ggagaaggat atcaaattta ttgaacataa tggaaccaag cctccttcag ttttatattt 1440 ctcgacagtg gcttaataaa tttaagacct ttgccgaacc tggccctatt tcaaataatg 1500 actttctttg tattcatgga ggtgttcctc caagaaaagc tggttatatt gaagacctgg 1560 ttttgatgct gcctcagaac atttgggata acctatatag caggtatggt ggaggaccag 1620 ctgtcaacca tctgtacatt tgtcatactt gccaaattga ggcggagaaa attgaaaaaa 1680 gaagaaaaac tgaattggaa atttttattc ggcttaacag agcgttccaa aaagaggact 1740 ctccagctac tttttattgc atcagtatgc agtggtttag agaatgggaa agttttgtga 1800 agggtaaaga tggagatcct ccaggtccta ttgacaatac taagattgca gtcactaaat 1860 gtggtaatgt gatgcttagg caaggagcag attctggcca gatttctgaa gaaacatgga 1920 attttctgca gtctatttat ggtggagggc ctgaagttat cctgcgacct ccggttgttc 1980 atgttgatcc agatatactt caagcagaag aaaaaattga agtagaaact cggtctttgt 2040 aatttttagg atgtagagag ttctaatgag gaatcatttt catgtgccct gacatgtaca 2100 catgcgaaaa cattcctaaa agcgtgttta tttgctttat tttttttcat catttatccc 2160 atttatttct tcttagtggg cattatggaa gaatatatta aaatgtgtaa tataccacag 2220 gttggtatat ttagttttaa atacttacca taaagtcttt cagtgtaatt tttttttgag 2280 acagagtctt gctttgtcac ccaggctgga gtgctgtggt gttacctcag ctcactgcag 2340 cctccacctc ctgggttcaa gcgattctcc tgcctcagcc tctcgagtag ctgggattac 2400 aggcacctgc caccatgccc ggctaatttt tgtattttag tagagatggg gtttcaccat 2460 gttggccagg ctagtctcaa actcctgacc tcaggtgatc cacccacctg ggcctcccaa 2520 agtgctggga ttacaggtgt gagccacagc gcctggctcg tgttctaagg aaatagctac 2580 ctaaggtatc ttattaaaac aaatgaacaa aaagtttccc aaactgtgtt ctaaggaaat 2640 agctacctat cttattaaaa aaaaaacaaa aacaaagttt tatgctctaa taagtttggg 2700 aaatgctagg ttatacaaag ctaaacatga ttctttccag tgggacatct gagagtcttt 2760 aattagctaa cagtgcattg tgattctgca aaggagttca ataattcacc ngtacgcgtg 2820 gtcgacc 2827 <210> 38 <211> 2987 <212> DNA

<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2820384 <400> 38 tgctcaccta cgcagctcct ctcgcccctg cagccccgtc caccaccacg agggccatgc 60 caagctgtct agcagccccc ctcgtgcaag ccccgtgagg gtggcaccgt cgtacgtgct 120 caagaaagcc caggtattga gtgctggcag ccggaggggt aaggagcagc gctaccgcag 180 cgtcatctca gacatctttg acggctccat tcttagcctc gtgcagtgtc tcacctgtga 240 ccgggtatcc accacagtgg aaacgttcca ggacttatca ctgcccattc ctggaaagga 300 ggacctggcc aagctccatt cagccatcta ccagaatgtg ccggccaagc caggcgcctg 360 tggggacagc tatgccgccc agggctggct ggccttcatt gtggagtaca tccgacggtt 420 tgtggtatcc tgtacaccca gctggttttg ggggcctgtc gtcaccctgg aagactgcct 480 tgctgccttc tttgccgctg atgagttaaa gggtgacaac atgtacagct gtgagcggtg 540 taagaagctg cggaacggag tgaagtactg caaagtcctg cggttgcccg agatcctgtg 600 cattcaccta aagcgctttc ggcacgaggt gatgtactca ttcaagatca acagccacgt 660 ctccttcccc ctcgaggggc tcgacctgcg ccccttcctt gccaaggagt gcacatccca 720 gatcaccacc tacgacctcc tctcggtcat ctgccaccac ggcacggcag gcagtgggca 780 ctacatcgcc tactgccaga acgtgatcaa tgggcagtgg tacgagtttg atgaccagta 840 cgtcacagaa gtccacgaga cggtggtgca gaacgccgag ggctacgtac tcttctacag 900 gaagagcagc gaggaggcca tgcgggagcg acagcaggtg gtgtccctgg ccgccatgcg 960 ggagcccagc ctgctgcggt tctacgtgtc ccgcgagtgg ctcaacaagt tcaacacctt 1020 cgcagagcca ggccccatca ccaaccagac cttcctctgc tcccacggag gcatcccgcc 1080 ccacaaatac cactacatcg acgacctggt ggtcatcctg ccccagaacg tctgggagca 1140 cctgtacaac agattcgggg gtggccccgc cgtgaaccac ctgtacgtgt gctccatctg 1200 ccaggtggag atcgaggcac tggccaagcg caggaggatc gagatcgaca ccttcatcaa 1260 gttgaacaag gccttccagg ccgaggagtc gccgggcgtc atctactgca tcagcatgca 1320 gtggttccgg gagtgggagg cgttcgtcaa ggggaaggac aacgagcccc ccgggcccat 1380 tgacaacagc aggattgcac aggtcaaagg aagcggccat gtccagctga agcagggagc 1440 tgactacggg cagatttcgg aggagacctg gacctacctg aacagcctgt atggaggtgg 1500 ccccgagatt gccatccgcc agagtgtggc gcagcgctgg gcccagagaa cctgcacggg 1560 gagcagaaga tcgaagccga gacgcgggcc gtgtgatctg ctgggctagt ctccccatgt 1620 gccccacccc gcggaaggcg tgtttgtgcc cagaagagag gccgggctgc tgcagaaccc 1680 cgccgtgtaa agaggcagaa aagttggttt ggtttgcagt aacgctgcaa ctagaaaata 1740 tatgcacttc aggcttgttg aaacgaccaa gactctgtga cgttaatttg ggtctttgtc 1800 ctggcagtgc ctctgccagt cactgtcatc gttgtgtccc ccacaactgt cctcttgcta 1860 gctcggccca gctttgtccc tggagcccga tgctacccct gtcagacaga ggctgcggcc 1920 tgggccagag tcagggagta gctgctgctt cacggcgtct ccactgtgcg attggcccgg 1980 agccccgaag actcggaggg agctgctcag ggccggtgag cgcaccagaa gccctggcca 2040 gtgaggagct cacaggtcct ccctggtggt cccgccgcac ctctgcatct cctgggcgtc 2100 accaggaagg ctctgaagtc ccgggctgct ctcagcactt ctcctgcaga ctgaagactc 2160 tggactcatt gctgattgga acaccaggag gaggttggat ttctgccagt gggggatgtt 2220 tctggaggca gctggtcccc cacaccgcgt cctgctgagc ctgccccctg gattggctgt 2280 aatttgcctc gaagttcagc agttcatctt catgggaaat ttgctgagcc cccaccaggg 2340 aaccggatga tgaaacaggg atacctcaca gcttggccat ttgaggcaaa ggcagcttcc 2400 cgagctgatg ctaaagaaga cagactttcc cttcctccca gcagcagcag tgcagagccc 2460 gcctggaggg atgtgggggc tgtgcagggt gcagcgctca ggtggatcct gggaagcagc 2520 ctctggatgc tgagtggagg gagccactga gcacagcaag gcaccaaagc ccctggagaa 2580 accgccaggg cgaggtgcga ccatcatcag gatcaaagca gacggggcgt gggtggggaa 2640 ggggctctgg gaccagaccc cccacactac tgcgtctttg tttctatcag tctttgtaga 2700 agcaggtggt ggtggaaatt ccagcaggtg ggtcccgcag aggccctgag gcctcacttt 2760 tcggatcttc tgtcccagat cctgctccct ccctgctgag cctggggttc ccctggcatt 2820 ggccccagcc ttctgaaagc cggcgctgca gccagaggcc gcacgctgca ctgtcgcgac 2880 gcagagaggc ttctgtgcag gctgggatcg ggccccatgt ctgtgctgtc tagtttgtgt 2940 tcaaaatgtc agaataaaca cagaataaat gttaaaaaaa aaaaaaa 2987 <210> 39 <211> 1215 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2990692 <400> 39 agagcacctt agtaggccgg attcggctca gatgagtatg cataaggcaa tgctaatggc 60 tcaagcaatg agggggctca ctctaggagg acaagttaga acatttggga aaaaatgtta 120 taattgtggt caaatcggtc atctgaaaag gagttgccca gtcttaaata aacagaatat 180 aataaatcaa gctattacag caaaaaataa aaagccatct ggcctgtgtc caaaatgtgg 240 aaaaggaaaa cattgggcca atcaatgtca ttctaaattt gataaagatg ggcaaccatt 300 gtcgggaaac aggaagaggg gccagcctca ggccccccaa caaactgggg cattcccagt 360 tcaactgttt gttcctcagg gttttcaagg acaacaaccc ctacagaaaa taccaccact 420 tcagggagtc agccaattac aacaatccaa cagctgtccc gcgccacagc aggcagcgcc 480 acagtagatt tatgttccac ccaaatggtc tctttactcc ctggagagcc cccacaaaag 540 attcctagag gggtatatgg cccgctgcca gaagggaggg taggccttat tttagggaga 600 tcaagtctaa atttgaaggg agtccaaatt catactgggg taatttattc agattataaa 660 gggggaattc agttagtgat cagctccact gttccctgga gtgccaatcc aggtgataga 720 attgctcaat tactgctttt gccttatgtt aaaattgggg aaaacaaaac ggaaagaaca 780 ggagggtttg gaagtaccaa ccctgcagga aaagccactt attgggctaa tcaggtctca 840 gaggatagac ccgtgtgtac agtcactatt ccagggaaag agtttgaagg attagtggat 900 acccaggctg atgtttctat catcggcata ggcaccgcct cagaagtgta tcaaagtgcc 960 atgattttac attgtctagg atctgataat caagaaagta cggttcagcc tatgatcact 1020 tctattccaa tcaatttatg gggccgagac ttgttacaac aatggcatgc agagattact 1080 atcccagcct ccctatacag ccccaggaat caaaaaatca tgactaaaat gggatagctc 1140 cctaaaaagg gactaggaaa gaatgaagat ggcattaaag tcccaactga ggctgaaaaa 1200 aatcaaaaaa aaaaa 1215 <210> 40 <211> 1037 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 4590384 <400> 40 gccatggggc tcgggttgag gggctgggga cgtcctctgc tgactgtggc caccgccctg 60 atgctgcccg tgaagccccc cgcaggctcc tggggggccc agatcatcgg gggccacgag 120 gtgacccccc actccaggcc ctacatggca tccgtgcgct tcgggggcca acatcactgc 180 ggaggcttcc tgctgcgagc ccgctgggtg gtctcggccg cccactgctt cagccacaga 240 gacctccgca ctggcctggt ggtgctgggc gcccacgtcc tgagtactgc ggagcccacc 300 cagcaggtgt ttggcatcga tgctctcacc acgcaccctg actaccaccc catgacccac 360 gccaacgaca tctgcctgct gcggctgaac ggctctgctg tcctgggccc tgcagtgggg 420 ctgctgaggc tgccagggag aagggccagg ccccccacag cggggacacg gtgccgggtg 480 gctggctggg gcttcgtgtc tgactttgag gagctgccgc ctggactgat ggaggccaag 540 gtccgagtgc tggacccgga cgtctgcaac agctcctgga agggccacct gacacttacc 600 atgctctgca cccgcagtgg ggacagccac agacggggct tctgctcggc cgactccgga 660 gggcccctgg tgtgcaggaa ccgggctcac ggcctcgttt ccttctcggg cctctggtgc 720 ggcgacccca agacccccga cgtgtacacg caggtgtccg cctttgtggc ctggatctgg 780 gacgtggttc ggcggagcag tccccagccc ggccccctgc ctgggaccac caggccccca 840 ggagaagccg cctgagccac aaccttgcgg catgcaaatg agatggccgc tccaggcctg 900 gaatgttccg tggctgggcc ccacgggaag cctgatgttc agggttgggg tgggacgggc 960 agcggtgggg cacacccatt ccacatgcaa agggcagaag caaacccagt aaaatgttaa 1020 ctgacgaaaa aaaaaaa 1037

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:1-20, 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 90%
polynucleotide sequence identity to the polynucleotide of claim 3.

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

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

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

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

9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:21-40, and fragments thereof.

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

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

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

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

14. A method for producing a polypeptide, the method comprising the steps of:
a) culturing the host cell of claim l3 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 PPRG, 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 PPRG, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 18.