CA2329076A1 - Human hydrolase homologs: n-terminal asparagine amidohydrolase, glycosyl hydrolase, glucohydrolase, biotinidase, and n-acetylglucosamine 6-p deacetylase - Google Patents

Abstract

The invention provides human hydrolase homologs (HHH) and polynucleotides which identify and encode HHH. 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 HHH.

Description

HUMAN HYDROLASE HOMOLOGS: N-TERMINAL ASPARAGINE AMIDOHYDROLASE, GLYCOSYL HYDROLASE, GLUCOHYDROLASE, BIOTINIDASE, AND N

TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human hydrolase homologs and to the use of these sequences in the diagnosis, treatment. and prevention of reproductive. carbohydrate metabolism. and cell proliferation disorders.
BACKGROUND OF THE INVENTION
to Hydrolysis is a common enzymatic mechanism. There are numerous enzymes whose catalytic mechanism involves breaking a covalent bond in a substrate by the addition of a molecule of water across the bond. The reaction involves a nucleophilic attack by the water molecule's oxygen atom on a target bond within the substrate. which results in a splitting of the water molecule across the target bond, thereby breaking the 15 bond and generating two product molecules. This general mechanism applies to a wide variety of enzymes. including phosphatases, glycosyl hydrolases, lysophospholipases, peptidases and amidohydrolases.
The protein phosphorylation/dephosphorylation cycle is one of the major regulatory mechanisms employed by eukaryotic cells to control cellular activities. During ~0 protein phosphorylation, phosphate groups are transferred from adenosine triphosphate molecules to a protein by protein kinases. During protein dephosphoryiation.
phosphate groups are removed from a protein by protein phosphatases. using a hydrolytic mechanism. In this manner. phosphatases are involved in the control of many cellular signaling events that regulate cell growth and differentiation. cell-to-cell contact, the cell 25 cycle, and oncogenesis. (Cohen P. (1989) Annu. Rev. Biochem. 58:453-508.) Glycosyl hydrolases are a large group of hydrolase enzymes that cleave the glycosidic bond between two carbohydrates. or between a carbohydrate and a non-carbohydrate moiety. Based on sequence similarity. these enzymes have been classified into at least ~4 families that are named according to their substrate specificities. Examples 30 of glycosyl hy~drolases include glucosidases. glucanases. xylanases.
amylases.
galactosidases. sialidases. and mannosidases. Disorders of carbohydrate metabolism are frequently associated with glycosyl hydrolases. For example. mutations in a-1.-glucosidase allow the accumulation of glycogen resulting in Pompe's disease, while Krabbe disease results from the accumulation of galactocerebroside due to non-functional galactosylceramidase. The buildup of mannose-containing oligosaccharides, or mannosidosis, results from a-mannosidase insufficiency. Thus, the glycosyl hydrolases are involved in disorders of carbohydrate metabolism. (Davies G. and Henrissat B. (1995) Structure 3:853-859; PROSITE documents PDOC 00621 and PDOC 00511.) Lysophospholipases (LPLs) are widely distributed enzymes that metabolize intracellular lipids, and occur in numerous isoforms. These isoforms vary in molecular mass, the substrate metabolized, and the optimum pH required for activity. Small isoforms.
approximately 15-30 kD, function as hydrolases; large isoforms, those exceeding 60 kD. function both as hydrolases and transacylases. A particular substrate for LPLs, lysophosphatidylcholine, causes lysis of cell membranes when it is formed or imported into a cell. LPLs are regulated by lipid factors including acylcarnitine. arachidonic acid, and phosphatidic acid. These lipid factors are signaling molecules important in numerous pathways, including the inflammatory response.
(Anderson, R.
et al. (1994) Toxicol. Appl. Pharmacol. 125:176-183; Selle, H. et al. (1993);
Eur. J. Biochem.
212:411-416.) Hydrolases are involved in many protein degradation pathways. Peptidases, such as pyroglutamyl peptidase and N-terminal asparagine amidohydrolase. hydrolyze peptide bonds and therefore participate in protein degradation. For example, N-terminal asparagine amidohydrolase cleaves the N-terminal asparagine from target proteins, thereby conferring metabolic instability by tagging the target for further degradation. Other hydrolases participate in scavenging non-protein cofactors during protein degradation. Biotin is an essential water-soluble vitamin that acts as a cofactor for various carboxvlases involved in normal metabolism. Biotinidase, or biotin-amide amidohydrolase, cleaves the biotin-lysine bond, allowing the recycling of biotin during protein 35 degradation. Biotinidase deficiency, or multiple carboxylase deficiency, can result from insufficient recycling of biotin. (Rawlings N.D. and Barrett A.J. ( 1993) Biochem. J. 290:205-218;
Barrett A.J. and Rawlings N.D. ( 1995) Arch. Biochem. Biophys. 318:247-250:
Pomponio R.J. et al. (1997) Hum. Molec. Genet. 6:?39-745.) The discovery of new human hydrolase homologs and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of reproductive, carbohydrate metabolism. and cell proliferation disorders.
SUMMARY OF THE INVENTION

The invention features substantially purifed polypeptides, human hydrolase homologs, referred to collectively as "NHH" and individually as ''HHH-1", "HHH-2", "HHH-3". "HHH-4", "HHH-S". "HHH-6" and "HHH-7." In one aspect. the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID
NO:1. SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4. SEQ ID NO:S. SEQ ID N0:6. and SEQ
ID
N0:7 (SEQ ID NO:1 through SEQ ID N0:7) and fragments thereof.
The invention further provides a substantially purified variant having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1 through SEQ ID N0:7.
or to a fragment of any of these sequences. The invention also provides an isolated and purified polynucleotide encoding the poiypeptide comprising an amino acid sequence selected from the croup consisting of SEQ ID NO: l through SEQ ID N0:7 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 through SEQ ID N0:7 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID
N0:7 and fragments thereof, as well as 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 through SEQ ID
N0:7 and fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:8, SEQ
ID N0:9, SEQ
ID NO:10, S~Q ID NO:11, SEQ iD N0:12, SEQ ID N0:13, and SEQ ID N0:14 (SEQ ID
N0:8 through SEQ ID N0:14) and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:8 through SEQ ID N0:14 and fragments there of, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:8 through SEQ ID N0:14 and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucieotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:I through SEQ ID N0:7 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 comprising the amino acid sequence selected from the group consisting of SEQ ID NO: I through SEQ
ID N0:7 and fragments thereof. the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide: and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ
ID NO:1 through SEQ ID N0:7 and fragments thereof in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ
ID NO:1 through SEQ ID N0:7 and fragments thereof. as well as a purified agonist and a purified antagonist to the polypeptide. The invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof. The invention further provides a method for treating or preventing an carbohydrate metabolism disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof. The invention also provides a method for treating or preventing a cell proliferation disorder. the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID
NO:1 through SEQ ID N0:7 and fragments thereof, in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID N0:7 and fragments thereof. to at least one of the nucleic acids of the biological 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 encoding the polypeptide in the biological sample. In one aspect. the nucleic acids of the biological sample are amplified by the polyrnerase chain reaction prior to the hybridizing step.
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 methodology, protocols. cell lines, vectors, and reagents 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,"
l0 "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 methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies 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 pnor mvent~on.
DEFINITIONS
''HHH," as used herein, refers to the amino acid sequences of substantially purified HHH
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," as used herein. refers to a molecule which, when bound to HHH, increases or prolongs the duration of the effect of HHH. Aeonists may include proteins, nucleic acids, carbohydrates. or any other molecules which bind to and modulate the effect of HHH.
An "allelic variant," as this term is used herein. is an alternative form of the gene encoding HHH. 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.
Anv given natural or recombinant eene may have none. one. or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding HHH. as described herein, include those sequences with deletions, insertions. or substitutions of different nucleotides, resulting in a polynucleotide the same as HHH or a polypeptide with at least one functional characteristic of HHH. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HHH, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the poiynucleotide sequence encoding HHH. 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 HHH.
Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, andlor the amphipathic nature of the residues, as long as the biological or immunological activity of HHH is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine: serine and threonine; and phenylalanine and tyrosine.
The terms "amino acid" or ''amino acid sequence," as used herein, 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 HHH which are preferably at least 5 to about 1~ amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunologicai activity of HHH. Where "amino acid sequence" is recited herein 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," as used herein, relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerise chain reaction (PCR) technologies well known in the art. (See, e.g.. Dieffenbach, C.W. and G.S.
Dveksier ( 1995) PCR
Primer. a Laboratory Manual, Cold Spring Harbor Press. Plainview, NY, pp.i-5.) The term "antagonist." as it is used herein. refers to a molecule which, when bound to HHH, decreases the amount or the duration of the effect of the biological or immunological activity of HHH. Antagonists may include proteins. nucleic acids.
carbohydrates. antibodies, or any other molecules which decrease the effect of HHH.
As used herein, 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 HHH 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 conjueated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (1CLH). The coupled peptide is then used l0 to immunize the animal.
The term "antigenic determinant," as used herein, refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (given regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense." as used herein, refers to any composition containing a nucleic acid sequence which is complementary to the ''sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation.
The designation "negative" can refer to the antisense strand, and the designation "positive'' can refer to the sense strand.
As used herein, 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 HHH, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms ''complementary" or ''complementarily," as used herein, refer to the natural binding of polynucleotides by base pairing. For example, the sequence "A-G-T"
binds to the complementary sequence "T-C-A." 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 design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition comprising a given amino acid sequence." as these terms are used herein, 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 HHH or fragments of HHH 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," as used herein. refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction, and resequenced. or which has been assembled from the overlapping t5 sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEWTM Fragment Assembly system (GCG, Madison, WI). Some sequences have been both extended and assembled to produce the consensus sequence.
As used herein, 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 HHH. by Northern analysis is indicative of the presence of nucleic acids encoding HHH in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding HHH.
A "deletion," as the term is used herein. 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," as used herein. 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," as used herein, refers to a degree of complementariy.
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 trom hybridizing to a target nucleic acid is referred to as ''substantially similar." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot. solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarily (e.g., less than about 30%
similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlignT"' program (DNASTAR, Inc., Madison WI). The MegAlignT'" program can create alignments between two or more sequences according to different methods, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
The percentage similarity between two amino acid sequences, e.g., sequence A
and sequence B, is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. ( 1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
"Human artificial chromosomes'" (HACs), as described herein. 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.
(See, e.g., Harrington, J.J. et al. ( 1997) Nat Genet. 15:345-3~~.) The term "humanized antibody." as used herein, 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." as the term is used herein. refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
As used herein. the term ''hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds bet,veen complementary bases. A hybridization complex may be formed in solution (e.g., Cot or R"t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper. membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" or "addition," as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, 10 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.
IS The term "microarray," as used herein, refers to an arrangement of distinct polynucleotides arrayed on a substrate, e.g., paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
The terms ''element" or "array element" as used herein in a microarray context. refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate," as it appears herein, refers to a change in the activity of HHH. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics. or any other biological, functional, or immunological properties of HHH.
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, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated' or "operably linked." as used herein, refer to functionally related nucleic acid sequences. A promoter is operably associated or operable linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame. certain genetic elements, e.g., repressor genes. are not contiQUOUSIy linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
The term "oligonucleotide." as used herein. refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides.
and most preferably about 20 to 25 nucleotides. which can be used in PCR amplification or in a hybridization assay or microarray. As used herein. the term "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), as used herein. refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about ~ nucleotides in length linked to a l0 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. (See, e.g., Nielsen. P.E. et al. {1993) Anticancer Drug Des. 8:53-63.) The term "sample," as used herein. is used in its broadest sense. A biological sample I S suspected of containing nucleic acids encoding HHH, or fragments thereof, or HHH 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 solid support; a tissue: a tissue print; etc.
As used herein, the terms "specific binding" or "specifically binding'' refer to that 20 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 25 amount of labeled A that binds to the antibody.
As used herein, 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 30 by reducing the concentration of salt. increasing the concentration of forcnamide. or raising the hybridization temperature.
The term "substantially purified," as used herein. 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 7~% free, and most preferably about 90% free from other 35 components with which they are naturally associated.

A "substitution," as used herein. refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
"Transformation." as defined herein, 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 eukarvotic 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 HHH polypeptides. as used herein. 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 (eucine 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. LASERGENET"' software.
The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to HHH. 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 polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms'' (SNPs) in which the polynucleotide sequence varies by one 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 hydrolase homologs (HHH), the polynucleotides encoding HHH, and the use of these compositions for the diagnosis, treatment, or prevention of reproductive, carbohydrate metabolism. and cell proliferation disorders. In Table l, columns 1 and 2 show the sequence identification numbers (SEQ ID NO:) of the amino acid and nucleic acid sequence. respectively. Column 3 shows the Clone ID of the Incyte Clone in which nucleic acids encoding each HHH .were first identified. and column 4, the cDNA
library of this clone. Column ~ describes fragments, and shows the Incyte clones (and libraries) and shotgun sequences useful as fragments, e.g., in hybridization technologies, and which are part of the consensus nucleotide sequence of each HHH.
The columns of Table 2 show various properties of the polypeptides of the invention:
column l references the SEQ ID NO; column 2 shows the number of amino acid residues: column 3, potential phosphorylation sites; column ~, potential glycosylation sites:
column ~, the identity of the protein; and column 6; analytical methods used to identify the protein through sequence homologies and protein motifs.
The columns of Table 3 show the tissue expression of each nucleic acid sequence by northern analysis, diseases or disorders associated with this tissue expression, and the vector into which each cDNA was cloned.

Protein NucleotideClone Library Fragments lD

NO: NO:

321510H1 (EOSIHET02) 2593359H1 (LUNGNOT22) 1 8 321510 EOSIHET03 290233776 (DRGCNOTO1 ) 3016872Ei1 (MUSCNOT07) 3513795F6 (ENDINOT02) 56227786 (NEUTLPTO1 ) 634343H1 (NEUTGMTO1) 634343X12 (NEUTGMTOI) 2 9 634343 NEUTGMT01 634343X 14 (NEUTGMT01 ) 634343X 17 (NEUTGMT01 ) 121775671 (7VEUTGMT01 ) 406420H1 (EOS1HET02) 75566986 (BRAITUT02) 75566976 (BRAITUT02) 3 10 2017918 THP1NOT01 915932H1 (BRSTNOT04) 2017918H1 (THPINOT01) 201791876 (THP1NOT01 ) 3t 19919F6 (LUNGTUT13) 121549271 (BRSTTUT01) 1269841F1 (BRAINOT09) 133996971 (COLNTUT03) 1513403F1 (PANCTUT01) 1528049F1 (UCMCLSTO1) 165234976 (PROSTUT08) 4 I l 2175072 ENDCNOT03 1657538F6 (URETTUT01 ) 1812758F6 (PROSTUT12) 1856319F6 (PROSNOT18) 2175072H1 (ENDCNOT03) 2613966H 1 (THYRNOT09) 3030372H1 (HEARFET02) 3773551H1 (BRSTNOT25) 121:148681 ( BRSTTUT01 ) ! 517312 F6 ( PANCTUT01 ) 2197153H1 (SPLNFET02) 12 2403107 ENDANOT01 '_'_6276376 (UTRSNOT02) 2403107H1 (ENDANOT01) 1284408F6 (COLNNOT16) i362518F6 (LUNGNOT12) 1514414F6 (PANCTUT01 ) 6 13 3069540 UTRSNOR01 1578848F6 (DUODNOTO1 ) 3069540H I (UTRSNOR01 ) 3257058HI (OVARTUNO1) 030600H 1 (THP INOBOI
) 2016607H 1 (UTRSNOT08) 7 14 4182350 BRAUNOTO? ?445385F6 (THP1NOT03) 2812186F6 (OVARNOT10) -1182350H 1 (BRAUNOT02) Amino Potential Potential Analytical Protein Acid PhosphorylationGlycosylationIdentificationMethod SEQ 1D ResiduesSites Sites NO:

S15 S58 N-terminal 5111 5145 asparaeine BLAST

1 310 5216 S58 N252 amidohydrolaseGI 1373365 T246 S302 N39 N273 Vanin-1 BLAST

5_0 -X26 T336 N357 N41 GI 4138229 S370 l N468 T326 S74 N133 Glycosyl hydrolaseBLOCKS

3 346 T232 5269 (BL00592) S308 GIYcosyl hydrolase S61 (BL00775) 5166 T241 GlucohydrolaseBLAST

386 S113 S'l2 acetylglucosamineB

_ 6-P deacetvlase 7 206 T128 S 132 Glycosyl hydrolasep~TS

(PR00746) Tissue Expression DiseaselClass Seq ID Vector NO:

(Fraction of Total)(Fraction of Total) Reproductive (0.205)Cancer (0.432) 1 Gastrointestinal Inflammation (0.364)pBluescript (0.182) Cardiovascular (0.136)Fetal (0.136) Hematopoietic/lmmuneInflammation (0.474) (0.421 }

2 Gastrointestinal Cancer (0.421 ) pSPORTI
(0.316) Reproductive (0.158) Hematopoietic/ImmuneCancer (0.500) (0.333) 3 Cardiovascular (0.250)Inflammation (0.500)pINCY

Nervous (0.250) Fetal (0.167) Reproductive (0.340)Cancer (0.538) 4 Hematopoietic/lmmuneInflammation (0.292)pINCY
(0.142) Gastrointestinal Fetal (0.179) (0.132) Reproductive (0.242)Fetal (0.333) Cardiovascular (0.212)Inflammation (0.242)pINCY

Developmental (0.182)Cancer (0.212) Reproductive (0.290)Cancer (0.677) 6 Gastrointestinal Inflammation (0.226)pINCY
(0.226) Cardiovascular (0.129)Fetal (0.129) Reproductive (0.308)Cancer (0.500) 7 Hematopoietic/ImmuneFetal (0.192} pINCY
(0.192) Nervous (0.192) Inflammation (0.192) The invention also encompasses HHH variants. A preferred HHH 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 HHH amino acid sequence, and which contains at least one functional or structural characteristic of HHH.
The invention also encompasses polynucleotides which encode HHH. In a particular embodiment, the invention encompasses polynucleotide sequences comprising the sequences of SEQ ID N0:8, SEQ ID N0:9. SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID
N0:13, and SEQ ID N0:14 which encode HHH-1, HHH-2, HHH-3, HHH-4. HHH-5, HHH-b, and HHH-7, respectively.
The invention also encompasses a variant of a polynucleotide sequence encoding HHH.
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 poiynucleotide sequence encoding HHH. A particular aspect of the invention encompasses a variant of SEQ ID N0:8 which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID
N0:8. The invention further encompasses a polynucleotide variant of SEQ ID N0:9 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:9. The invention further encompasses a polynucleotide variant of SEQ ID NO:10 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence identity to SEQ ID
NO:10. The invention further encompasses a polynucleotide variant of SEQ ID NO:11 having at least about 80%, more preferably at least about 90%. and most preferably at least about 95%
polynucleotide sequence identity to SEQ ID NO:11. The invention further encompasses a polynucleotide variant of SEQ
ID N0:12 having at least about 80%. more preferably at least about 90%. and most preferably at least about 95% polynucleotide sequence identity to SEQ ID N0:12. The invention further encompasses a polynucleotide variant of SEQ ID N0:13 having at least about 80%, more preferably at least about 90%, and most preferably at least about 95%
polynucleotide sequence identity to SEQ ID N0:13. The invention further encompasses a polynucleotide variant of SEQ
ID N0:14 having at least about 80%. more preferably at least about 90%. and most preferably at least about 95% polynucieotide sequence identity to SEQ ID N0:14. Any one of the poiynucieotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HHH.
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 HHH. 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 HHH. and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode HHH and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HHH under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HHH 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 HHH 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 HHH
and HHH 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 HHH 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
N0:8 through SEQ ID N0:14 and fragments thereof, under various conditions of stringency.
(See, e.g., Wahl. G.M. and S.L. Berger ( 1987) Methods Enzymol. 152:399-407;
Kimmel. A.R.
( 1987) Methods Enzymol. 152:507-511.) For example, stringent salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about 500 mM
NaCI and ~0 mM trisodium citrate, and most preferably less than about 250 mM
NaCI and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide. while high stringency hybridization can be obtained in the presence of at least about 35% formamide. and most preferably at least about 50% formamide.
Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time. the concentration of detergent. e.g., sodium dodecyl sulfate (SDS). and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.
In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCI. 7~ mM
trisodium citrate, and I%
SDS. In a more preferred embodiment. hybridization will occur at 37°C
in X00 mM NaCI, 50 mM
trisodium citrate, 1% SDS, 3~% formamide, and 100 ~cg/ml denatured salmon sperm DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium citrate, i% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash stringency conditions can be defined by sail concentration and by temperature.
As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM
NaCI and 3 mM trisodium citrate. and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include temperature of at least about 25°C, more preferably of at least about 42°C. and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C in 1 ~ 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 and analysis are well known and generally available 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 poiymerase I, SEQUENASE~
(Amersham Pharmacia Biotech, Piscataway NJ), Taq polymerase (Perkin Elmer). thetmostable T7 polymerase (Amersham Phatmacia Biotech), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE Amplification System (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the ABI Catalyst 800 (Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton. Reno NV) in combination with thenmal cyclers (for PCR). Sequencing is then carried out using either ABI 373 or 377 DNA sequencers (Perkin Elmer) or the MEGABASE capillary electrophoresis (Molecular Dynamics), and the sequences are analyzed using tools (computer programs and algorithms) which are well known in the art. (Ausubel (1997, unit 7.7); Meyers, R.A. (1995; Molecular Bioiogv and Biotechnolosy, Wiley VCH, lnc, New York NY. p 856-853).
The nucleic acid sequences encoding HHH 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 e~campie. 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 5 fragments comprising a known genomic locus and surrounding sequences. (See, e.g.. Triglia, T. et al. ( 1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR
amplification of LNIA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom. M. et al. (1991) PCR Methods Appiic.
1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an l0 engineered double-stranded sequence into a region of unknown sequence before performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. ( 1991 ) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR, nested primers, and PromoterFinderT'" libraries to walk genomic DNA (Clontech, Palo Alto. CA).
This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions.
15 For all PCR-based methods, primers may be designed using commercially available software, such as OLIGOT"' 4.06 Primer Analysis software (National Biosciences Inc., Plymouth. MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC
content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been 20 size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes. are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. tn 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., GenotyperTM and Sequence NavigatorTM, 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 HHN may be cloned in recombinant DNA molecules that direct expression of HHH, 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 HHH.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HHH-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding HHH may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. ( 1980) Nucl.
Acids Res. Symp. Ser. 21 S-223, and Horn, T. et al. ( 1980) Nucl. Acids Res.
Symp. Ser. 225-232.) Alternatively, HHH itself or a fragment thereof may be synthesized using chemical methods. For l5 example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. (1995) Science 269:202-204.) Automated synthesis may be achieved using the ABI 431A Peptide Synthesizer (Perkin Eimer). Additionally, the amino acid sequence of HHH, 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 and Co., New York, NY.) In order to express a biologically active NHH, the nucleotide sequences encoding HHH 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' untransiated regions in the vector and in polynucleotide sequences encoding HHH. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HHH. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. (n cases where sequences encoding HHH and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or transiational control signals may be needed. However, in cases where only coding sequence. or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See. e.g., Scharf. D. et al. ( 1994) Results Probl. Cell Differ.
20:125-I 62.) Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding HHH 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; and Ausubel. F.M. et al. ( 1995, and periodic supplements) Current Protocols in Molecular BioloQV, John Wiley & Sons, New York. NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be utilized to contain and express sequences encoding HHH. 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 HHH. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding HHH can be achieved using a multifunctional E, coli vector such as Bluescript~
(Stratagene) or pSportlT'"
plasmid (GIBCO BRL). Ligation of sequences encoding HHH 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 HHH are needed. e.g. for the production of antibodies, vectors which direct high level expression of HHH may be used.
For example, vectors containing the strong, inducible TS or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of HHH. A number of vectors containing constitutive or inducible promoters. such as alpha factor. alcohol oxidase. and PGH, may be used in the yeast Saccharomyces cerevisiae or Pichiapastoris. 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, supra; and Grant et al. { 1987) Methods Enzymol. 153:516-54: Scorer. C. A. et al. (1994) Biol1"echnology 12:1$1-184.) Plant systems may also be used for expression of HHH. Transcription of sequences encoding HHH 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., Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technolosy ( 1992) McGraw Hill, New York, NY; pp.
191-196.) 1n 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 HHH
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential Ei or E3 region of the viral genome may be used to obtain infective virus which expresses HHH in host cells. (See, e.g., Logan, J. and T. Shenk ( 1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
SV40 or EBV-based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes.
For long term production of recombinant proteins in mammalian systems, stable expression of HHH in cell lines is preferred. For example, sequences encoding HHH 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 i 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 aeent. 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 api cells. respectively.
(See, e.g., Wigler, M. et S al. ( 1977) Cell 11:223-232: and Lowy, I. et al. ( 1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers resistance to methotrexate: neo confers resistance to the aminoglycosides neomycin and G-418;
and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570;
Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
(See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo Alto, CA), B
glucuronidase and its substrate (3-D-glucuronoside, or luciferase and its substrate fuciferin 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. et al. ( 1995) Methods Mol. Biol. SS:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding HHH is inserted within a marker gene sequence, transformed cells containing sequences encoding HHH can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HHH
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.
2S In general, host cells that contain the nucleic acid sequence encoding HHH
and that express HHH 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 HHH using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site. monoclonal-based immunoassay utilizing 3S monoclonal antibodies reactive to two non-interfering epitopes on HHH is preferred. but a competitive binding assay may be employed. These and other assays are well known in the art.
(See, e.g.. Hampton, R. et al. (1990) Serological Methods. a Laboratory Manual, APS Press. St Paul. MN. Section IV; Coligan, J. E. et al. ( 1997 and periodic supplements) Current Protocols in lmmunoloay, Greene Pub. Associates and Wilev-interscience, New York, NY; and Maddox. D.E.
~ et al. ( 1983) J. Exp. Med. 158:1211-1216).
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 polynucieotides encoding HHH
include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled 10 nucleotide. Alternatively, the sequences encoding HHH. 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 IS Pharmacia & Upjohn (Kalamazoo, MI), Promega (Madison, WI), and U.S.
Biochemical Corp.
(Cleveland, OH). Suitable reporter molecules or labels which may be used for ease of detection include radionuciides, enrymes, fluorescent, chemiluminescent, or chromogenic agents. as well as substrates. cofactors, inhibitors, magnetic particles. and the like.
Host cells transformed with nucleotide sequences encoding HHH may be cultured under 20 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 HHH may be designed to contain signal sequences which direct secretion of HHH through a prokaryotic or eukaryotic cell membrane.
25 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-translationai 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 correct modification and processing of the foreign protein.
In another embodiment of the invention. natural. modified. or recombinant nucleic acid sequences encoding HHH 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 HHH protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HHH
activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include. but are not limited to.
glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Ttx. 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-mvc, and hemagglutinin (HA) enable l0 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 proteolvtic cleavage site located between the HHH
encoding sequence and the heterologous protein sequence, so that HHH may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel, F. M. et at. ( 1995 and periodic supplements) Current Protocols in Molecular Biolosv, John Wiley & Sons, New York, NY, 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 radiolabeied HHH may be achieved in vitro using the TNTT'" rabbit reticulocyte iysate or wheat germ extract systems (Promega, Madison, WI). 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 '3S-methionine.
Fragments of HHH 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 Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of HHH may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity exists between HHH-1 and N-terminal asparagine amidohydrolase from mouse (GI 1373365). In addition. HHH-I is expressed in libraries from reproductive and gastrointestinal tissues. Chemical and structural similarity exists between HHH-2 and vanin-1 from mouse (GI 4138229). In addition. HHH-2 is expressed in libraries from hematopoietic/immune and gastrointestinal tissues. Protein sequence analysis identifies HHH-3 as a glycosyl hydrolase (BL00592). In addition. HHH-3 is expressed in libraries from hematopoietic/immune and cardiovascular tissues. Protein sequence analysis identifies HHH-4 as a glycosyl hydrolase (BL007775). In addition. HHH-4 is expressed in libraries from reproductive and hematopoietic/immune tissues. Chemical and structural similarity exists between HHH-S and glucohydrolase from yeast (G1 728850). In addition. HHH-5 is expressed in libraries from reproductive and cardiovascular tissues. Chemical and structural similarity exists between HHH-6 and N-acetylglucosamine 6-P deacetylase from Bacillus subtiiis (GI 2618856), and HHH-6 contains a signal peptidases signature sequence. In addition, HHH-6 is expressed in libraries from reproductive and gastrointestinal tissues. Protein sequence analysis identifies HHH-7 as a glycosyl hydrolase (PR00746). In addition, HHH-7 is expressed in libraries from reproductive and hematopoietic/immune tissues. Finally, HHH is associated with libraries made from cancerous or proliferative tissues. Therefore. HHH appear to play roles in reproductive.
carbohydrate metabolism, and cell proliferation disorders.
Therefore, in one embodiment, an antagonist of HHH may be administered to a subject to treat or prevent a reproductive disorder. Such a disorder may include, but is not limited to, disorders of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis; disruptions of the estrous cycle. disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea; disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia. prostatitis, Peyronie's disease. carcinoma of the male breast, and gynecomastia. In one aspect, an antibody which specifically binds HHH 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 HHH.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HHH may be administered to a subject to treat or prevent a reproductive disorder including, but not limited to, those described above.
In another embodiment, HHH or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder of carbohydrate metabolism. Such disorders can include, but are not limited to, diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent diabetes mellitus, hypoglycemia, glucagonoma, galactosemia, hereditary fructose intolerance, fructose-l.6-diphosphatase deficiency, obesity. congenital type II
dyserythropoietic anemia.
mannosidosis, neuraminidase deficiency. galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases. fructosuria. pentosuria. and inherited abnormalities of pyruvate metabolism.

In yet another embodiment, a vector capable of expressing HHH or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder of carbohydrate metabolism including, but not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified HHH in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder of carbohydrate metabolism including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of HHH
may be administered to a subject to treat or prevent a disorder of carbohydrate metabolism including, but not limited to, those listed above.
In yet another embodiment, an antagonist of HHH may be administered to a subject to treat or prevent a cell proliferation disorder. Such a disorder may include, but is not limited to, actinic keratosis, arteriosclerosis, atherosclerosis. bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, t5 psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and. in particular, cancers of the adrenal gland, bladder, bone. bone marrow, brain. breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver. lung, muscle. ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid. and uterus.
In one aspect, an antibody which specifically binds HHH 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 HHH.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding HHH may be administered to a subject to treat or prevent a cell proliferation disorder including cancer such as 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 treatmem 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 HHH may be produced using methods which are generally known in the art. In particular. purified HHH may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HHH.
Antibodies to HHH 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 HHH or with any fragment or oligopeptide thereof which has immunogenic properties. Dependine on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's. mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions. KLH, and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corvnebacterium parvum are especially preferable.
It is preferred that the oligopeptides. peptides, or fragments used to induce antibodies to 5 HHH 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 HHH 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 HHH 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 26:495-497;
Kozbor. D. et al.
( 1985) J. Immunol. Methods 81:31-42: Cote, R.J. et al. ( 1983) Proc. Natl.
Acad. Sci.
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison. S.L. et al. ( 1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger. M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. ( 1985) Nature 314:452-4~4.) Alternatively, techniques described for the production of single chain antibodies may be adapted. using methods known in the art, to produce HHH-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition. may be generated by chain shuffling from random combinatorial immunoglobulin 3.~ libraries. (See. e.g., Burton D.R. ( 1991 ) Proc. Natl. Acad. Sci.
88:1013.1-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi. R. et al. ( 1989) Proc.
Natl. Acad. Sci. 86:
3833-3837: and Winter, G. et al. (1991) Nature 319:293-299.) Antibody fragments which contain specific binding sites for HHH may also be venerated.
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.
10 (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 15 HHH and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HHH epitopes is preferred, but a competitive binding assay may also be employed. (Maddox, supra.) In another embodiment of the invention. the polynucleotides encoding HHH, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect. the 20 complement of the polynucleotide encoding HHH may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transfotlrted with sequences complementary to polynucleotides encoding HHH. Thus, complementary molecules or fragments may be used to modulate HHH activity. or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger 25 fragments can be designed from various locations along the coding or control regions of sequences encoding HHH.
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 30 can be used to construct vectors to express nucleic acid sequences complementary to the polynucieotides encoding HHH. (See, e.g., Sambrook. supra; and Ausubel, supra.) Genes encoding HHH can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof.
encoding HHH. 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 HHH. Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +l0 from the start site. are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases. transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See. e.g., Gee, J.E. et al.
( 1994) in Huber, B.E. and B.I. Carr. Molecular and Immunolo ig c Approaches, Futura Publishing Co., Mt. Kisco, NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HHH.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and riborymes 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 oligonucieotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HHH. Such DNA
sequences may be incorporated into a wide varien~ of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly. can be introduced into cell lines. cells. or tissues.
RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule. or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine. queosine. and wybutosine. as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al.
( 1997) Nature Biotechnology 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HHH, antibodies to HHH, and mimetics, agonists, antagonists, or inhibitors of HHH. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound. which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to. oral, intravenous, intramuscular, infra-arterial, intramedullary, intrathecal, intraventricular. transdermal. subcutaneous, intraperitoneal, intranasal, 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~eton's Pharmaceutical Sciences (Maack Publishing Co.. 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 S compounds with solid excipient and processing the resultant mixture of granules (optionally, afrer grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol: starch from corn, wheat, rice, potato. or other plants: cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth: and proteins. such as gelatin and collagen.
If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc.
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and. optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids. such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethy) cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters. such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing.
dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids. including but not limited to. hydrochloric, sulfuric. acetic. lactic.
tartaric. malic. and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. 1n other cases, the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to SO mM histidine, 0.1 % to 2%
sucrose. and 2% to 7% mannitoh at a pH range of 4.S to S.S, 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 HHH, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions 1S 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 ofadministration. 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 HHH or fragments thereof, antibodies of HHH, and agonists, antagonists or inhibitors of HHH, 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 ED5° (the dose therapeutically effective in SO% of the population) or LD,o (the dose lethal to SO% of the population) statistics. The dose ratio of therapeutic to toxic effects is the therapeutic index. and it can be expressed as the ED<°/LDa° 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.
3S 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 5 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 10 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.

In another embodiment, antibodies which specifically bind HHH may be used for the diagnosis of disorders characterized by expression of HHH, or in assays to monitor patients being treated with HHH or agonists, antagonists, or inhibitors of HHH. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic 20 assays for HHH include methods which utilize the antibody and a label to detect HHH 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.
25 A variety of protocols for measuring HHH. including ELISAs, RIAs, and FACS.
are known in the art and provide a basis for diagnosing altered or abnormal levels of HHH expression.
Normal or standard values for HHH expression are established by combining body fluids or cell extracts taken from normal mammalian subjects. preferably human, with antibody to HHH under conditions suitable for complex formation The amount of standard complex formation may be 30 quantitated by various methods, preferably by photometric means. Quantities of HHH 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 HHH may be used 35 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 HHH may be correlated with disease. The diagnostic assay may be used to determine absence. presence, and excess expression of HHH, and to monitor regulation of HHH levels during therapeutic S intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences. including genomic sequences, encoding HHH or closely related molecules may be used to identify nucleic acid sequences which encode HHH. The specificity of the probe, whether it is made from a highly specific region, e.g., the ~' 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 HHH, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences. and should preferably have at least SO% sequence identity to any of the HHH encoding sequences. The hybridization I 5 probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:8 through SEQ ID N0:14 or from genomic sequences including promoters, enhancers, and introns of the HHH gene.
Means for producing specific hybridization probes for DNAs encoding HHH
include the cloning of polynucleotide sequences encoding HHH or HHH 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
poiymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as'=P or''S. or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding HHH may be used for the diagnosis of a disorder associated with expression of HHH. Examples of such a disorder include. but are not limited to, reproductive disorder such as disorders of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis: disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome.
endometrial and ovarian tumors, uterine fibroids. autoimmune disorders, ectopic pregnancies. and teratogenesis; cancer of the breast, fibrocystic breast disease. and galactorrhea: disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis. cancer of the prostate. benign prostatic hyperplasia, prostatitis. Peyronie's disease. carcinoma of the male breast. and gynecomastia. carbohydrate metabolism disorders such as diabetes, insulin-dependent diabetes mellitus, non-insulin-dependent s diabetes mellitus, hypoglycemia. ~:lucagonoma. galactosemia. hereditaw tW
ctose intolerance, fructose-1.6-diphosphatase deficiency, obesity, congenital type II
dyserythropoietic anemia, mannosidosis. neuraminidase deficiency, galactose epimerase deficiency, glycogen storage diseases, lysosomal storage diseases, fructosuria. pentosuria, and inherited abnormalities of pyruvate metabolism, and cell proliferation disorders, such as actinic keratosis. arteriosclerosis, atherosclerosis. bursitis, cirrhosis, hepatitis. mined connective tissue disease (MCTD), myelofibrosis. paroxysmal nocturnal hemoglobinuria. polycythemia vera, psoriasis, primary thrombocvthemia, and cancers including adenocarcinoma, leukemia. lymphoma, melanoma, myeloma. sarcoma, teratocarcinoma, and, in particular, cancers ofthe adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract. heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus. thyroid, and uterus. The polynucleotide sequences encoding HHH
may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies;
in PCR
technologies: in dipstick, pin, and >rLISA assays: and in microarrays utilizing fluids or tissues from patients to detect altered HHH expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HHH may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding HHH 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 HHH 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 HHH. 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 HHH. 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 a relatively high amount of transcript 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 HHH may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding HHH, or a fragment of a polynucleotide complementary to the IS polynucleotide encoding HHH, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of HHH include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al. ( 1993) J. Immunol.
Methods 159:235-244: and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The microarray can be used to monitor the expression level of large numbers of genes simultaneously and to idemify genetic variants. mutations, and polymorphisms. This information may be used to determine gene function. to understand the genetic basis of a disorder. to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared. used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796: Schena. M. et al. ( 1996) Proc. Natl.
Acad. Sci. 93:10614-10619: Baldeschweiler et al. (1995) PCT application W095/2~ 1116; Shalon.
D. et al. ( 1995) PCT application W095/35505: Heller. R.A. et al. ( 1997) Proc. Natl. Acad. Sci.
94:2150-215: and Heller. M.J. et al. ( 1997) U.S. Patent No. ~.60~.662.) WO 99/61626 PC1'/US99/12021 In another embodiment of the invention, nucleic acid sequences encoding HHH
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., Price, C.M.
(1993) Blood Rev. 7:127-134; and Trask, B.J. (1991) Trends Genet. 7:149-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R.A. (ed.) Molecular Biology and Biotechnoloey, VCH Publishers New York, NY, 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 HHH on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the piacement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to l 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:77-580.) 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. HHH, 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 HHH 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, such as plastic pins or some other surface. The test compounds are reacted with HHH, or fragments thereof. and washed. Bound HHH is then detected by methods well known in the art. Purified HHH can also be coated directly onto plates 5 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 HHH specifically compete with a test compound for binding HHH. In this manner, antibodies can be used to detect the presence of any peptide which 10 shares one or more antigenic determinants with HHH.
In additional embodiments, the nucleotide sequences which encode HHH 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.
IS 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 entire disclosure of all applications, patents, and publications, cited above and below, 20 and of US provisional application 60/087,236 (filed May 29, 1998), is hereby incorporated by reference.
EXAMPLES
I. cDNA Library Construction 25 RNA was purchased from Clontech (Palo Alto. CA) or isolated at Incyte from tissues described in Table 4. The tissue was homogenized and lysed in guanidinium isothiocyanate, and the lysate was centrifuged over a CsCI cushion. Alternatively, the tissue was homogenized and lysed in phenol or a suitable mixture of denaturants such as TRIZOL reagent (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate, and the lysate was extracted with 30 chloroform ( 1:~ v/v). RNA was precipitated from lysates with either isopropanol or sodium acetate and ethanol. Alternatively, RNA was purified from lysates by preparative agarose gel electrophoresis and recovered from Whatman P81 paper (Whatman. Lexington. MA).
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA
purity, and RNA
was maintained in RNase-free solutions. In some cases. RNA was treated with DNase. For most 35 libraries. polv.~+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega.

Madison, WI), Oligotex resin. or the Oligotex kit (QIAGEN Inc, Chatsworth, CA). Alternatively, RNA was isolated directly from tissue lysates using the RNA Isolation kit (Stratagene) or the Ambion PolyA Quick kit (Ambion. Austin. TX).
RNA was used for cDNA synthesis and construction of the cDNA libraries according to procedures recommended in the UNIZAP vector (Stratagene. La Jolla. CA) or Superscript plasmid system (Life Technologies, Inc), both of which are based on methods well known in the art (Ausubel, 1997, units S.1-6.6). Alternatively, cDNA libraries were constructed by Stratagene using RNA provided by Incyte. Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and cDNA
was digested with an appropriate restriction enzyme(s). For most libraries, cDNA was size-selected (300-1000 bp) using Sephacryl S 1000 or Sepharose CL2B or 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 (Stratagene), pSPORT 1 (Life Technologies), pINCY
(Incyte Pharmaceuticals Inc, Palo Aito. CA). pINCY was amplified in JM 109 cells and purified using the QiaQuick column (QIAGEN Inc). Recombinant plasmids were transformed into competent E. coli cells, e.g., XL1-Blue, XL1-Blue MRF, or SOLR (Stratagene) or DHSn, DHI OB, or ElectroMAX
DH l OB (Life Technologies).

TABLE -t Nucleotide SEQ ID Library Library Description NO:

The EOSIHET02 library was constructed using 8 EOSIHET02 ~'' isolated from peripheral blood cells apheresed from a 48-year-old Caucasian male.

Patient history included hypereosinophilia.

The NEUTGMTOI library was constructed using RNA isolated from peripheral blood granulocvtes collected by density gradient centrifugation 9 NEUTGMTO1 through Ficoll-Hypaque. The cells were isolated from huffy coat units obtained from 20 unrelated male and female donors. Cells were cultured in 10 nM GM-CSF for 1 hour before washing and harvesting for RNA preparation.

The THP1NOT01 library was constructed using RNA isolated from THP-1 cells.
THP-1 (ATCC

THPINOTOI TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia.

The ENDCNOT03 library was constructed using 1 I ENDCNOT03 ~A isolated from dermal microvascular endothelial cells removed from a neonatal Caucasian male.

The ENDANOTOI library was constructed using 12 ENDANOTO1 RNA isolated from aortic endothelial cell tissue from an e~cplanted heart removed from a male during a heart transplant.

The UTRSNORO 1 library was constructed using RNA isolated from uterine endometrium tissue 13 UTRSNORO1 removed from a 29-year-old Caucasian female during a vaginal hysterectomy and cystocele repair.

The BRAUNOT02 library was constructed using 14 BRAUNOT02 ~'4 isolated from globus pallidus/substantia innominata tissue removed from the brain of a 35-year-old Caucasian male.

II. Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision ( UniZAP vector system, Stratagene) or by cell lysis. Plasmids v~ere purified using the MAGIC
MINIPREPS DNA
purification system (Promeea, Madison. WI); Miniprep kit (Advanced Genetic Technologies Corporation, Gaithersburg. MD): QIAwell-8 Plasmid, QIAwell PLUS DNA, or QIAwell ULTRA
DNA purification systems: or REAL Prep 96 plasmid kit (QIAGEN Inc) using the recommended protocol. Following precipitation, plasmids were resuspended in 0. I ml of distilled water and stored, with or without lyophilization, at 4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR
(Rao. V.B. (1994) Anal. Biochem. 216:1-14) in a high-throughput format. Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates (Genetix Ltd, Christchurch UK) and concentration of amplified plasmid DNA was quantified fluorometrically using Pico Green Dye (Molecular Probes, Eugene OR) and a Fluoroscan II fluorescence scanner (Labsystems Oy, Helsinki. Finland).
III. Sequencing and Analysis The cDNAs were prepared for sequencing using either an AB1 Catalyst 800 (Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with Pettier Thermal Cyclers (PTC200; MJ Research. Watertown MA). The cDNAs were sequenced on the or 377 DNA Sequencing systems (Perkin Elmer) by the method of Sanger F. and A.R. Coulson (1975; J. Mol. Biol. 94:441-448) using standard ABI protocols, base calling software, and kits.
Alternatively, cDNAs were sequenced using solutions and dyes from Amersham Pharmacia IS Biotech. Reading frame was determined using standard methods (Ausubel, supra).
The cDNA sequences presented in Table 1 and the full length nucleotide and amino acid sequences disclosed in the Sequence Listing were Queried against databases such as GenBank primate (pri), rodent (rod), mammalian (mamp), vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS, and other databases which contain previously identified and annotated motifs and sequences. Algorithms such as Smith Waterman which deal with primary sequence patterns and secondary structure gap penalties (Smith, T. et al. ( 1992) Protein Engineering 5:35-51 ) and programs and algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S.F. ( 1993) J. Mol. Evol 36:290-300: and Altschul et al. ( 1990) J. Mol.
Biol. 215:403-410), and HMM (Hidden Markov Models: Eddy. S.R. (1996) Cur. Opin. Str. Biol. 6:361-36~, and Sonnhammer. E.L.L. et al. ( 1997) Proteins 28:405-420) were used to assemble and analyze nucleotide and amino acid sequences. The databases, programs. algorithms, methods and tools are available, well known in the art. and described in Ausubel (supra. unit 7.7), in Meyers. R.A.

(1995; Molecular BioloQV and BiotechnoloiP.~y, Wiley VCH, Inc, New York NY, p 856-853), in documentation provided with software (Genetics Computer Group (GCG). Madison WI), and on the world wide web (www). Two comprehensive websites which list, describe, and/or link many of the databases and tools are: 1 ) the www resource in practical sequence analysis (http://genome.wustl.edu/eddy/bio5495/online-resources.html), and 2) the bibliography of computational gene recognition (http://linkage.rockefeller.edu/wli/gene/
programs.html). For example, the first website links PFAM as a database (http://genome.wustl.
edu/Pfam/) and as an HMM search tool (http://genome.wustl.edu/eddy/cgi-bin/hmm_page.cgi).
TABLE 5 summarizes the databases and toots used herein. The first column of shows the tool, program, or algorithm: the second column, the database; the third column, a brief description: and the fourth column (where applicable), scores for determining the strength of a match between two sequences (the higher the value, the more homologous).

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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;
and Ausubel, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST are used to search for identical or related molecules in nucleotide databases such as GenBank or LIFESEQT~"
database (Incyte Pharmaceuticals). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar.
The basis of the search is the product score, which is defined as:
sequence identity x % maximum BLAST score The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 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 analysis are reported as a list of libraries in which the transcript encoding HHH occurs. Abundance and percent abundance are also reported.
Abundance directly reflects the number of times a particular transcript is represented in a cDNA
library, and percent abundance is abundance divided by the total number of sequences examined in the cDNA library.
V. Extension of HHH Encoding Polynucleotides The nucleic acid sequences of Incyte Clones 321510, 634343. ?017918, 2175072, 2403107, 3069540, and 4182350 were used to design oligonucleotide primers for extending partial nucleotide sequences to full length. 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 pofynucleotide.] Primers were used to facilitate the ewension 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 OLIGOTM 4.06 (National Biosciences. Plymouth. MN). or another appropriate program. to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more. and to anneal to the 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 (GIBCO BR1.) 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-PCRT'"
kit (Perkin Elmer) and thoroughly mixing the enzyme and reaction mix. PCR was performed using the Pettier Thermal Cycler (PTC200; M.J. Research, Watertown, MA), beginning with 40 pmol of each primer and the recommended concentrations of all other components of the kit, with the following parameters:
Step 1 94 C for I min (initial denaturation) l0 Step 2 65 C for 1 min Step 3 68 C for 6 min Step 4 94 C for 15 sec Step ~ 65 C for 1 m i n Step 6 68 C for 7 min Step 7 Repeat steps 4 through 6 for an additional 15 cycles Step 8 94 C for 15 sec Step 9 65 C for 1 min Step 10 68 C for 7:1 S min Step I 1 Repeat steps 8 through 10 for an additional 12 cycles Step 12 72 C for 8 min Step 13 4 C (and holding) A 5 ~1 to 10 ul 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 QIAQUICKT"' (QIAGEN Inc.). and trimmed of overhangs using Klenow enzyme to facilitate religation and cloning.
After ethanol precipitation, the products were redissolved in 13 ul of ligation buffer, lul T4-DNA lipase ( 15 units) and lul T4 poiynucleotide kinase were added, and the mixture was incubated at room temperature for 2 to 3 hours. or overnight at l6° C.
Competent E. colt cells (in ~I of appropriate media) were transformed with 3 ul of ligation mixture and cultured in 80 ~cl of S(~C medium. (See, e.g., Sambrook, supra. Appendix A, p. 2.) After incubation for one hour at 37°C, the E. colt mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook. sUDra, Appendix A. p. 1 ) containing carbenicillin (2t carb). The following day, several colonies were 35 randomly picked from each plate and cultured in 1 ~0 ul of liquid LBI2x carb medium placed in an individual welt of an appropriate commercially-available sterile 96-well microtiter plate. The following day. ~ ul of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1:10 with water, 5 ~cl from each sample was transferred into a PCR array.
For PCR amplification. 18 ul of concentrated PCR reaction mix (3.3x) containing 4 units 40 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 94 C for 60 sec Step 94 C for 20 sec Step >j C for 30 sec Step 7'_' C for 90 sec Step Repeat steps 2 through 4 for an additional ~ 29 cycles Step 72 C for 180 sec Step 4 C (and holding) Aliquots of the PCR reactions were run on agarose gels together with molecular weight markers. The sizes of the PCR products were compared to the original partial cDNAs. and appropriate clones were selected. ligated into plasmid, and sequenced.
In like manner. the nucleotide sequence of SEQ ID N0:8 through SEQ ID N0:14 are I S used to obtain 5' regulatory sequences using the procedure above.
oligonucleotides designed for 5' extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:8 through SEQ ID N0:14 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 OLIGOT"' 4.06 software (National Biosciences) and labeled by combining SO pmol of each oligomer, 250 uCi of {y-'=P] adenosine triphosphate (Amersham, Chicago, IL), and T4 polynucleotide kinase (DuPont NEN~', Boston, MA). The labeled oligonucleotides are substantially purified using a Sephade~cT"' G-25 superfine size exclusion dextran bead column (Pharmacia & Upjohn, Kalamazoo. MI). An aliquot containing 10'counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA
digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I. Xbal, or Pvu II
(DuPont NEN, Boston, MA).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus. Schleicher & Schuell, Durham, NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals. blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTS' film (Kodak. Rochester. NY) is exposed to the blots to film for several hours, hybridization patterns are compared visually.

VII. rlicroarrays 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 LASERGENET~'. Full-length cDNAs.
ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying.
(See. e.g., Schena, M. et al. ( 1995) Science 270:467-470; and Shalom D. et al. ( 1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides Sequences complementary to the HHH-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HHH.
Although use of oligonucleotides comprising from about t 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 OLIGOT"' 4.06 software and the coding sequence of HHH. To inhibit transcription, a complementary oligonucleotide is designed from the most unique ~' 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 HHH-encoding transcript.
IX. Expression of HHH
Expression and purification of HHH is achieved using bacterial or virus-based expression systems. For expression of HHH in bacteria. cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA tr~attscription. 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 HHH upon induction with isopropyl beta-D-5 thiogaiactopyranoside (IPTG). Expression of HHN in eukaryotic cells is achieved by infecting insect or mammalian celi lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HHH by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is 10 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 al. ( 1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.) 15 In most expression systems, HHH is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
GST, a 26-kilodalton enzyme from Schistosoma iaponicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and 20 antigenicity (Pharmacia, Piscataway, NJ). Following purification, the GST
moiety can be proteolytically cleaved from HHH at specifically engineered sites. FLAG, an 8-amino acid peptide. enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester. NY). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN Inc, 25 Chatsworth, CA). Methods for protein expression and purification are discussed in Ausubel, F. M.
et al. (1995 and periodic supplements) Current Protocols in Molecular Bioloev, John Wiley &
Sons, New York, NY, ch 10, 16. Purified HHH obtained by these methods can be used directly in the following activity assay.
30 X. Demonstration of HHH Activity For purposes of example, an assay measuring the ~i-glucosidase activity of an HHH
molecule is described. Varying amounts of HHH are incubated with 1 mM 4-nitrophenyl (i-D-glycopyranoside (a substrate) in ~0 mM sodium acetate buffer, pH 5.0, for various times (typically 1-5 minutes) at 37°C. The reaction is halted by heating to 100°C
for 2 minutes. The absorbance 35 is measured spectrophotometrically at 410 nm. and is proportional to the activity of HHH in the sample. (Htmova, M. et al. (1998) J. Biol. Chem. 273:11134-l 1143.) XI. Functional Assays HHH function is assessed by expressing the sequences encodine HHH 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 SPORTT"~ (Life Technologies, Gaithersburg, MD) and pCRT"s 3.1 (Invitrogen, Carlsbad, CA, both of which contain the cytomegalovirus promoter. 5-10 ug of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 ~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, Palo Alto, CA), CD64, or a I S CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter, down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. ( 1994) Flow Cvtometry, Oxford, New York, NY.
The influence of HHH on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HHH 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 (1gG). 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 HHH and other genes of interest can be analyzed by Northern analysis or microarray techniques.

XII. Production of HHH Specific Antibodies HHH substantially purified using polyacrvlamide 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 urine standard protocols.
Alternatively. the HHH amino acid sequence is analyzed using LASERGENET'"
software (DNASTAR Inc.) to determine regions of high immunogenicity, and a corresponding ofigopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel supra, ch. I 1.) Typically. oligopeptides I S residues in length are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis, MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel suyra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
XIII. Purification of Naturally Occurring HHH Using Specific Antibodies Naturally occurring or recombinant HHH is substantially purified by immunoaffinity chromatography using antibodies specific for HHH. An immunoaffinity column is constructed by covaiently coupling anti-HHH antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing HHH are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HHH (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/HHH 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 HHH is collected.
XIV. Identification of Molecules Which Interact with HHH
HHH. or biologically active fragments thereof: are labeled with'=51 Bolton-Hunter reagent. (See, e.e., Bolton et al. ( 1973) Biochem. J. 133:~?9.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HHH.
washed, and anv wells with labeled HHH complex are assayed. Data obtained using different concentrations of HHH are used to calculate values for the number. affinity. and association of HHH 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.

SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
BANDMAN, Olga HILLMAN, Jennifer L.
YUE, Henry t_nr., Preeti CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra BAUGHN, Mariah R.
<120> HUMAN HYDROLASE HOMOLOGS
<130> PF-0534 PCT
<140> To Be Assigned <141> Herewith <150> 60/087,236 <151> 1998-05-29 <160> 54 <170> PERL Program <210> 1 <211> 310 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 321510 <400> 1 Met Pro Leu Leu Val Glu Gly Arg Arg Val Arg Leu Pro Gln Ser Ala Gly Asp Leu Val Arg Ala His Pro Pro Leu Glu Glu Arg Ala Arg Leu Leu Arg Gly Gln Ser Val Gln Gln Val Gly Pro Gln Gly Leu Leu Tyr Val Gln Gln Arg Glu Leu Ala Val Thr Ser Pro Lys Asp Gly Ser Ile Ser Ile Leu Gly Ser Asp Asp Ala Thr Thr Cys His Ile Val Val Leu Arg His Thr Gly Asn Gly Ala Thr Cys Leu Thr His Cys Asp Gly Thr Asp Thr Lys Ala Glu Val Pro Leu Ile Met Asn Ser Ile Lys Ser Phe Ser Asp His Ala Gln Cys Gly Arg Leu Glu Val His Leu Val Gly Gly Phe Ser Asp Asp Arg Gln Leu Ser Gln Lys Leu Thr His Gln Leu Leu Ser Glu Phe Asp Arg Gln Glu Asp Asp Ile His Leu Val Thr Leu Cys Val Thr Glu Leu Asn Asp Arg Glu Glu Asn Glu Asn His Phe Pro Val Ile Tyr Gly Ile Ala Val Asn Ile Lys Thr Ala Glu Ile Tyr Arg Ala Ser Phe Gln Asp Arg Gly Pro Glu Glu Gln Leu Arg Ala Ala Arg Thr Leu Ala Gly Gly Pro Met Ile Ser Ile Tyr Asp Ala Glu Thr Glu Gln Leu 215 2'20 225 Arg Ile Gly Pro Tyr Ser Trp Thr Pro Phe Pro His Val Asp Phe Trp Leu His Gln Asp Asp Lys Gln Ile Leu Glu Asn Leu Ser Thr Ser Pro Leu Ala Glu Pro Pro His Phe Val Glu His Ile Arg Ser Thr Leu Met Phe Leu Lys Lys His Pro Ser Pro Ala His Thr Leu Phe Ser Gly Asn Lys Ala Leu Leu Tyr Lys Lys Asn Glu Asp Gly Leu Trp Glu Lys Ile Ser Ser Pro Gly Ser <210> 2 <211> 520 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 634343 <400> 2 Met Val Thr Ser Ser Phe Pro Ile Ser Val Ala Val Phe Ala Leu Ile Thr Leu Gln Val Gly Thr Gln Asp Ser Phe Ile Ala Ala Val Tyr Glu His Ala Val Ile Leu Pro Asn Lys Thr Glu Thr Pro Val Ser Gln Glu Asp Ala Leu Asn Leu Met Asn Glu Asn Ile Asp Ile Leu Glu Thr Ala Ile Lys Gln Ala Ala Glu Gln Gly Ala Arg Ile Ile Val Thr Pro Glu Asp Ala Leu Tyr Gly Trp Lys Phe Thr Arg Glu Thr Val Phe Pro Tyr Leu Glu Asp Ile Pro Asp Pro Gln Val Asn Trp Ile Pro Cys Gln Asp Pro His Arg Phe Gly His Thr Pro Val Gln Ala Arg Leu Ser Cys Leu Ala Lys Asp Asn Ser Ile Tyr Val Leu Ala Asn Leu Gly Asp Lys Lys Pro Cys Asn Ser Arg Asp Ser Thr Cys Pro Pro Asn Gly Tyr Phe Gln Tyr Asn Thr Asn Val Val Tyr Asn Thr Glu Gly Lys Leu Val Ala Arg Tyr His Lys Tyr His Leu Tyr Ser Glu Pro Gln Phe Asn VaI Pro Glu Lys Pro Glu Leu val Thr Phe Asn Thr Ala Phe Gly Arg Phe Gly Ile Phe Thr Cys Phe Asp Ile Phe Phe Tyr Asp Pro Gly Val Thr Leu Val Lys 2i5 220 225 Asp Phe His Val Asp Thr Ile Leu Phe Pro Thr Ala Trp Met Asn Val Leu Pro Leu Leu Thr Ala Ile Glu Phe His Ser Ala Trp Ala Met Gly Met Gly Val Asn Leu Leu Val Ala Asn Thr His His Val Ser Leu Asn Met Thr Gly Ser Gly Ile Tyr Ala Pro Asn Gly Pro Lys Val Tyr His Tyr Asp Met Lys Thr Glu Leu Gly Lys Leu Leu Leu Ser Glu Val Asp Ser His Pro Leu Ser Ser Leu Ala Tyr Pro Thr Ala Val Asn Trp Asn Ala Tyr Ala Thr Thr Ile Lys Pro Phe Pro Val Gln Lys Asn Thr Phe Arg Gly Phe Ile Ser Arg Asp Gly Phe Asn Phe Thr Glu Leu Phe Glu Asn Ala Gly Asn Leu Thr Val Cys Gln Lys Glu Leu Cys Cys His Leu Ser Tyr Arg Met Leu Gln Lys Glu Glu Asn Glu Val Tyr Val Leu Gly Ala Phe Thr Gly Leu His Gly Arg Arg Arg Arg Glu Tyr Trp Gln Val Cys Thr Met Leu Lys Cys Lys Thr Thr Asn Leu Thr Thr Cys Gly Arg Pro Val Glu Thr Ala Ser Thr Arg Phe Glu Met Phe Ser Leu Ser Gly Thr Phe Gly Thr Glu Tyr Val Phe Pro Glu Val Leu Leu Thr Glu Ile His Leu Ser Pro Gly Lys Phe Glu Val Leu Lys Asp Gly Arg Leu Val Asn Lys Asn Gly Ser Ser Gly Pro Ile Leu Thr Val Ser Leu Phe Gly Arg Trp Tyr Thr Lys Asp Ser Leu Tyr ser Ser Cys Gly Thr Ser Asn Ser Ala Ile Thr Tyr Leu Leu Ile Phe Ile Leu Leu Met Ile Ile Ala Leu Gln Asn Ile Val Met Leu <210> 3 <211> 346 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2017918 <400> 3 Met Gln Ala His Val Ser Ile Ile Leu Pro Val His Asn Ala Glu Pro Trp Leu Asp Glu Cys Leu Arg Ser Val Leu Gln Gln Asp Phe Glu Gly Thr Met Glu Leu Ser Val Phe Asn Asp Ala Ser Lys Asp Lys Ser Gly Ala Ile Ile Glu Lys Trp Arg Val Lys Leu Glu Asp Ser Gly Val His Val Ile Ile Gly Gly His Asp Ser Pro Ser Pro Arg Gly Val Gly Tyr Ala Lys Asn Gln Ala Val Ala Gln Ser Ser Gly Ser Tyr Leu Cys Phe Leu Asp Ser Asp Asp Val Met Met Pro Gln Arg Val Arg Leu Gln His Glu Ala Ala Val Gln His Pro Ser Ser Ile Ile Gly Cys Arg Val Arg Arg Asp Pro Pro Asn Ser Thr Glu Arg Tyr Thr Arg Trp Ile Asn Gln Leu Thr Pro Glu Gln Leu Leu Thr Gln Val Phe Thr Ser Asn Gly Pro Thr Val Ile Met Pro Thr Trp Phe Cys Ser Arg Ala Trp Phe Ser His Val Gly Pro Phe Asn Glu Gly Gly Gln Gly Val Pro Glu Asp Leu Leu Phe Phe Tyr Glu His Leu Arg Lys Gly Gly Gly Val Ile Arg Val Asp Gln Ser Leu Leu Leu Tyr Arg His His Pro Gln Ala Ala Thr His Cys Val Leu Glu Thr Thr Ile Trp Thr His Arg Val Arg Phe Leu Glu Glu Gln Ala Leu Pro Arg Trp Ala Ala Phe Thr Ile Trp Asn Ala Gly Lys Gln Gly Arg Arg Leu Tyr Arg Ser Leu Thr Ala Gly Ser Gln Arg Lys Val Val Ala Phe Cys Asp Val Asp Glu Asn Lys Ile Arg Lys Gly Phe Tyr Cys His Glu Asp Ser Gln Glu Arg Pro Lys Pro Arg Ile Pro Ile Leu His Phe Arg Ala Ala Arg Pro Pro Phe Val Ile Cys Val Lys Leu Asp Leu Thr Gly Gly Thr Phe Glu Asp Asn Leu Arg Ser Leu His Leu Gln Glu Gly Gln Asp Phe Leu His Phe Ser <210> 4 <211> 401 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2175072 <400> 4 Met Pro Val Gln Leu Ser Glu His Pro Glu Trp Asn Glu Ser Met His Ser Leu Arg Ile Ser Val Gly Gly Leu Pro Val Leu Ala Ser Met Thr Lys Ala Ala Asp Pro Arg Phe Arg Pro Arg Trp Lys Val Ile Leu Thr Phe Phe Val Gly Ala Ala Ile Leu Trp Leu Leu Cys Ser His Arg Pro Ala Pro Gly Arg Pro Pro Thr His Asn Ala His Asn Trp Arg Leu Gly Gln Ala Pro Ala Asn Trp Tyr Asn Asp Thr 80 85 9p Tyr Pro Leu Ser Pro Pro Gln Arg Thr Pro Ala Gly Ile Arg Tyr Arg Ile Ala Val Ile Ala Asp Leu Asp Thr Glu Ser Arg Ala Gln Glu Glu Asn Thr Trp Phe Ser Tyr Leu Lys Lys Gly Tyr Leu Thr Leu Ser Asp Ser Gly Asp Lys Val Ala Val Glu Trp Asp Lys Asp His Gly Val Leu Glu Ser His Leu Ala Glu Lys Gly Arg Gly Met Glu Leu Ser Asp Leu ile Val Phe Asn Gly Lys Leu Tyr Ser Val Asp Asp Arg Thr Gly Val Val Tyr Gln Ile Glu Gly Ser Lys Ala Val Pro Trp Val Ile Leu Ser Asp Gly Asp Gly Thr Val Glu Lys Gly Phe Lys Ala Glu Trp Leu Ala Val Lys Asp Glu Arg Leu Tyr Val Gly Gly Leu Gly Lys Glu Trp Thr Thr Thr Thr Gly Asp Val Val Asn Glu Asn Pro Glu Trp Val Lys Val Val Gly Tyr Lys Gly Ser Val Asp His Glu Asn Trp Val Ser Asn Tyr Asn Ala Leu Arg Ala Ala Ala Gly Ile Gln Pro Pro Gly Tyr Leu Ile His Glu Ser Ala Cys Trp Ser Asp Thr Leu Gln Arg Trp Phe Phe Leu Pro Arg Arg Ala Ser Gln Glu Arg Tyr Ser Glu Lys Asp Asp Glu Arg Lys Gly Ala Asn Leu Leu Leu Ser Ala Ser Pro Asp Phe Gly Asp Ile Ala Val Ser His Val Gly Ala Val Val Pro Thr His Gly Phe Ser ~/~.i Ser Phe Lys Phe Ile Pro Asn Thr Asp Asp Gln Ile Ile Val Ala Leu Lys Ser Glu Glu Asp Ser Gly Arg Val Ala Ser Tyr Ile Met Ala Phe Thr Leu Asp Gly Arg Phe Leu Leu Pro Glu Thr Lys Ile Gly Ser Val Lys Tyr Glu Gly Ile Glu Phe Ile <210> 5 <211> 506 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2403107 <400> 5 Met Lys Arg Arg Ser Val Thr Met Thr Asp Gly Leu Thr Ala Asp Lys Val Thr Arg Ser Asp Gly Cys Pro Thr Ser Thr Ser Leu Pro Arg Pro Arg Asp Ser Ile Arg Ser Cys Ala Leu Ser Met Asp Gln Ile Pro Asp Leu His Ser Pro Met Ser Pro Ile Ser Glu Ser Pro Ser Ser Pro Ala Tyr Ser Thr Val Thr Arg Val His Ala Ala Pro Ala Ala Pro Ser Ala Thr Ala Leu Pro Ala Sex Pro Val Ala Arg Arg Ser Ser Glu Pro Gln Leu Cys Pro Gly Ser Ala Pro Lys Thr His Gly Glu Ser Asp Lys Gly Pro His Thr Ser Pro Ser His Thr Leu Gly Lys Ala Ser Pro Ser Pro Ser Leu Ser Ser Tyr Ser Asp Pro Asp Ser Gly His Tyr Cys Gln Leu Gln Pro Pro Val Arg Gly Ser Arg Glu Trp Ala Ala Thr Glu Thr Ser Ser Gln Gln Ala Arg Ser Tyr Gly Glu Arg Leu Lys Glu Leu Ser Glu Asn Gly Ala Pro Glu Gly Asp Trp Gly Lys Thr Phe Thr Val Pro Ile Val Glu Val Thr Ser Ser Phe Asn Pro Ala Thr Phe Gln Ser Leu Leu Ile Pro Arg Asp Asn Arg Pro Leu Glu Val Gly Leu Leu Arg Lys Val Lys Glu Leu Leu Ala Glu Val Asp Ala Arg Thr Leu Ala Arg His Val Thr Lys Val Asp Cys Leu Val Ala Arg Ile Leu Gly Val Thr Lys Glu Met Gln Thr Leu Met Gly Val Arg Trp Gly Met Glu Leu Leu Thr Leu Pro His Gly Arg Gln Leu Arg Leu Asp Leu Leu Glu Arg Phe His Thr Met Ser Ile Met Leu Ala Val Asp Ile Leu Gly Cys Thr Gly Ser Ala Glu Glu Arg Ala Ala Leu Leu His Lys Thr Ile Gln Leu Ala Ala Glu Leu Arg Gly Thr Met Gly Asn Met Phe Ser Phe Ala Ala Val Met Gly Ala Leu Asp Met Ala Gln Ile Ser Arg Leu Glu Gln Thr Trp Val Thr Leu Arg Gln Arg His Thr Glu Gly Ala Ile Leu Tyr Glu Lys Lys Leu Lys Pro Phe Leu Lys Ser Leu Asn Glu Gly Lys Glu Gly Pro Pro Leu Ser Asn Thr Thr Phe Pro His Val Leu Pro Leu Ile Thr Leu Leu Glu Cys Asp Ser Ala Pro Pro Glu Gly Pro Glu Pro Trp Gly Ser Thr Glu His Gly Val Glu Val Val Leu Ala His Leu Glu Ala Ala Arg Thr Val Ala His His Gly Gly Leu Tyr His Thr Asn Ala Glu Val Lys Leu Gln Gly Phe Gln Ala Arg Pro Glu Leu Leu Glu Val Phe Ser Thr Glu Phe Gln Met Arg Leu Leu Trp Gly Ser Gln Gly Ala Ser Ser Ser Gln Ala Arg Arg Tyr Glu Lys Phe Asp Lys Val Leu Thr Ala Leu Ser His Lys Leu Glu Pro Ala Val Arg Ser Ser Glu Leu <210> 6 <211> 386 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3069540 <400> 6 Met Arg Gly Glu Gln Gly Ala Ala Gly Ala Arg Val Leu Gln Phe Thr Asn Cys Arg Ile Leu Arg Gly Gly Lys Leu Leu Arg Glu Asp Leu Trp Val Arg Gly Gly Arg Ile Leu Asp Pro Glu Lys Leu Phe Phe Glu Glu Arg Arg Val Ala Asp Glu Arg Arg Asp Cys Gly Gly Arg Ile Leu Ala Pro Gly Phe Ile Asp Val Gln Ile Asn Gly Gly Phe Gly Val Asp Phe Ser Gln Ala Thr Glu Asp Val Gly Ser Gly Val Ala Leu Val Ala Arg Arg Ile Leu Ser His Gly Val Thr Ser Phe Cys Pro Thr Leu Val Thr Ser Pro Pro Glu Val Tyr His Lys Val Val Pro Gln Ile Pro Val Lys Ser Gly Gly Pro His Gly Ala Gly Val Leu Gly Leu His Leu Glu Gly Pro Phe Ile Ser Arg Glu Lys Arg Gly Ala His Pro Glu Ala His Leu Arg Ser Phe Glu Ala Asp Ala Phe Gln Asp Leu Leu Ala Thr Tyr Gly Pro Leu Asp Asn Val Arg Ile Val Thr Leu Ala Pro Glu Leu Gly Arg Ser His Glu Val Ile Arg Ala Leu Thr Ala Arg Gly Ile Cys Val Ser Leu Gly His Ser Val Ala Asp Leu Arg Ala Ala Glu Asp Ala Val Trp Ser Gly Ala Thr Phe Ile Thr His Leu Phe Asn Ala Met Leu Pro Phe His His Arg Asp Pro Gly Ile Val Gly Leu Leu Thr Ser Asp Arg Leu Pro Ala Gly Arg Cys Ile Phe Tyr Gly Met Ile Ala Asp Gly Thr His Thr Asn Pro Ala Ala Leu Arg Ile Ala His Arg Ala His Pro Gln Gly Leu Val Leu Val Thr Asp Ala Ile Pro Ala Leu Gly Leu Gly Asn Gly Arg His Thr Leu Gly Gln Gln Glu Val Glu Val Asp Gly Leu Thr Ala Tyr Val Ala Gly Cys Ser Met Glu Ser Ala Leu Glu Ala Ala Ser Leu His Pro Ala Gln Leu Leu Gly Leu Glu Lys Ser Lys Gly Thr Leu Asp Phe Gly Ala Asp Ala Asp Phe Val Val Leu Asp Asp Ser Leu His Val Gln Ala Thr Tyr Ile Ser Gly Glu Leu Val Trp Gln Ala Asp Ala Ala Arg Gln <210> 7 <211> 206 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 4182350 <400> 7 Met Ala Arg Gly Gly Trp Arg Arg Leu Arg Arg Leu Leu Ser Ala Gly Gln Leu Leu Phe Gln Gly Arg Ala Leu Leu Val Thr Asn Thr Leu Gly Cys Gly Ala Leu Met Ala Ala Gly Asp Gly Val Arg Gln Ser Trp Glu IIe Arg Ala Arg Pro Gly Gln Val Phe Asp Pro Arg Arg Ser Ala Ser Met Phe Ala Val Gly Cys Ser Met Gly Pro Phe Leu His Tyr Trp Tyr Leu Ser Leu Asp Arg Leu Phe Pro Ala Ser Gly Leu Arg Gly Phe Pro Asn Val Leu Lys Lys Val Leu Val Asp Gln Leu Val Ala Ser Pro Leu Leu Gly Val Trp Tyr Phe Leu Gly Leu Gly Cys Leu Glu Gly Gln Thr Val Gly Glu Ser Cys Gln Glu Leu Arg Glu Lys Phe Trp Glu Phe Tyr Lys Ala Asp Trp Cys Val Trp Pro Ala Ala Gln Phe Val Asn Phe Leu Phe Val Pro Pro Gln Phe Arg Val Thr Tyr Ile Asn Gly Leu Thr Leu Gly Trp Asp Thr Tyr Leu Ser Tyr Leu Lys Tyr Arg Ser Pro Val Pro Leu Thr Pro Pro Gly Cys Val Ala Leu Asp Thr Arg Ala Asp <210> 8 <211> 1126 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 321510 <400> 8 cgcctccgcg atgccgctgc tcgtcgaggg gcggcgagtg cggctgccgc agtcagccgg 60 ggacctcgtc cgagcccacc cgcctttgga ggaaagagcc agacttctca gaggtcagtc 120 tgttcaacaa gtgggacccc agggccttct gtatgttcag caaagagagc ttgcagtgac 180 ctccccaaag gatggctcca tctccattct gggttctgat gatgccacta cttgtcacat 240 tgtggtcctg aggcacacag gtaatggggc cacctgcttg acacattgtg acggaaccga 300 caccaaagct gaggtcccct tgatcatgaa ctccataaaa tccttttctg accacgctca 360 atgtggaagg ctggaagtac accttgttgg aggcttcagt gacgacaggc agttgtcaca 420 aaaactcact catcaacttc ttagtgaatt tgacaggcaa gaagatgaca ttcacttagt 480 gacattatgt gtgacagaat taaatgaccg ggaagaaaac gaaaaccact ttccagtaat 540 atatggcatt gctgtcaaca ttaagactgc agagatttac agagcatcct ttcaagatcg 600 gggtccggag gagcagcttc gtgctgcgcg aactttagca ggaggaccaa tgattagcat 66o ttatgatgca gagacagaac aacttcgtat aggaccgtac tcctggacac catttccaca 720 tgtggatttc tggttgcacc aagatgacaa gcaaatacta gagaatcttt ccacttcgcc 780 tctggctgag ccaccccact ttgttgaaca tattagatct accttgatgt ttttaaaaaa 840 acacccatct ccagctcaca cactgttttc tggaaataaa gccctactct acaaaaaaaa 900 tgaagatggc ttgtgggaaa agatctcttc tccaggaagt taaaaaacat gaattaccaa 960 agaaagcacc ttcttggcct gacagaccat tggtggggct ggcacgaatc cagatctgga 1020 tcctacatct gttgggtctt aggcctcctt ccctcctcag tgtctttcaa atgactttca 1080 tcaaatgact ttcaaaataa aaccttattt tggcaaaaaa aaaaaa 1126 <210> 9 <211> 2260 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 634343 <400> 9 cccccttgac taaagctcca aggacagaga aaaacatcca gatttgggaa cacaataaca 60 gatatgattg tccccacttc tactgccaaa attataaaac tgttaactcc tcctcatcag 220 cttacctgac tacttaaaag caaaagagtt aattaagtat tactaattgg tgatactaga 180 tcaatgaaga gaaagcagct gagatccaga ggagtggaag gtcccccttg actaaagcta 240 aatcactaaa ccttggccat ggtcacttcc tcttttccaa tctctgtggc agtttttgcc 300 ctaataaccc tgcaggttgg tactcaggac agttttatag ctgcagtgta tgaacatgct 360 gtcattttgc caaataaaac agaaacacca gtttctcagg aggatgcctt gaatctcatg 420 aacgagaata tagacattct ggagacagcg atcaagcagg cagctgagca gggtgctcga 480 atcattgtga ctccagaaga tgcactttat ggatggaaat ttaccaggga aactgttttc 540 ccttatctgg aggatatccc agaccctcag gtgaactgga ttccgtgtca agacccccac 600 agatttggtc acacaccagt acaagcaaga ctcagctgcc tggccaagga caactctatc 660 tatgtcttgg caaatttggg ggacaaaaag ccatgtaatt cccgtgactc cacatgtcct 720 cctaatggct actttcaata caataccaat gtggtgtata atacagaagg aaaactcgtg 780 gcacgttacc ataagtacca cctgtactct gagcctcagt ttaatgtccc tgaaaagccg 840 gagttggtga ctttcaacac cgcatttgga aggtttggca ttttcacgtg ctttgatata 900 ttcttctatg atcctggtgt taccctggtg aaagatttcc atgtggacac catactgttt 960 cccacagctt ggatgaacgt tttgcccctt ttgacagcta ttgaattcca ttcagcttgg 1020 gcaatgggaa tgggagttaa tcttcttgtg gccaacacac atcatgtcag cctaaatatg 1080 acaggaagtg gcatttatgc accaaatggt cccaaagtgt atcattatga catgaagaca 1140 gagttgggaa aacttctcct ttcagaggtg gattcacatc ccctatcctc gcttgcctac 1200 ccaacagctg ttaattggaa tgcctacgcc accaccatca aaccatttcc agtacagaaa 1260 aacactttca ggggatttat ttccagggat gggttcaact tcacagaact ttttgaaaat 1320 gccggaaacc ttacagtctg tcaaaaggag ctttgctgtc atttaagcta cagaatgtta 1380 caaaaagaag agaatgaagt atacgttcta ggagctttta caggattaca tggccgaagg 1440 agaagagagt actggcaggt ctgcacaatg ctgaagtgca aaactactaa tttgacaact 1500 tgtggacggc cagtagaaac tgcttctaca agatttgaaa tgttctccct cagtggcaca 1560 tttggaacag agtatgtttt tcctgaagtg ctacttaccg aaattcatct gtcacctgga 1620 aaatttgagg tgctgaaaga tgggcgtttg gtaaacaaga atggatcatc tgggcctata 1680 ctaacagtgt cactctttgg gaggtggtac acaaaggact cactttacag ctcatgtggg 1740 accagcaatt cagcaataac ttacctgcta atattcatat tattaatgat catagctttg 1800 caaaatattg taatgttata gggcgtctct ttatcactca gcttctgcat catatgcttg 1860 gctgaatgtg. tttatcggct tcccaagttt actaagaaac tttgaagggc tatttcagta 1920 gtatagacca gtgagtccta aatatttttt ctcatcaata attatttttt aagtattatg 1980 ataatgttgt ccattttttt ggctactctg aaatgttgca gtgtggaaca atggaaagag 2040 cctgggtgtt tgggtcagat aaatgaagat caaactccag ctccagcctc atttgcttga 2100 gactttgtgt gtatggggga cttgtatgta tgggagtgag gagtttcagg gccattgcaa 2160 acatagctgt gcccttgaag agaatagtaa tgatgggaat ttagaggttt atgactgaat 2220 tccctttgac attaaagact atttgaattc aaaaaaaaaa 2260 <210> 10 <211> 1675 <212> DNA
<213> Homo Sapiens 10/3 ~

WO 99/b1b26 PCT/US99/12021 <220>
<221> misc_feature <223> Incyte Clone No: 2017918 <400> 10 ccaggccatg caggcccacg tgtctattat cctcccagtc cacaacgctg aaccgtggct 60 ggacgaatgt ttgaggtctg ttttgcaaca ggactttgaa ggtaccatgg agctgtctgt 120 tttcaatgat gccagtaagg acaagtctgg ggctatcatt gaaaaatgga gagtgaagct 180 ggaagattct ggtgtccacg tgatcattgg ggggcacgat tctccctctc ctagaggcgt 240 cggatacgct aaaaatcaag cagttgccca gagctcaggg tcttaccttt gctttttgga 300 ttcggatgac gtcatgatgc cccagcgggt gaggctgcaa cacgaggctg ccgttcagca 360 cccgtcgagc atcattggtt gcagagtgag gagagatccc cctaactcca ccgaacgata 420 cacacgttgg atcaaccagc tgacgccgga gcagctccta acccaggttt tcacctcaaa 480 tggccccacg gtgatcatgc ccacctggtt ctgctcgcga gcgtggttct cccacgtggg 540 cccctttaac gagggaggtc agggcgtccc ggaggacctg ctgttcttct acgagcacct 600 caggaagggc ggcggcgtca tccgcgtgga ccagagtctc ctgctgtatc gccaccaccc 660 acaggcggcc acgcactgcg tcctcgagac gaccatctgg acccaccgcg tccgcttcct 720 ggaagagcag gccctgcccc gctgggcggc cttcaccatc tggaacgctg gcaagcaggg 780 gcgccggctg taccgcagct tgactgccgg cagccagcgc aaggtggtgg cattctgtga 840 cgtggacgag aacaagatca ggaaaggctt ctattgccac gaggactctc aggaaagacc 900 caagccccga atccccatcc tgcacttccg agccgcccgg ccacccttcg tcatctgcgt 960 gaagctggac ctcacagggg gcacctttga ggncaacctg aggtcactgc acttgcagga 1020 gggccaggac ttccttcact tcagctgacg gacccatggc agctgctccc agtgcatccc 1080 aggccagcag gagctttgtg agctgcaggc atggcgatgg tgcgcctgtt ccacacccag 1140 caggcgcaac cagagtctcg tgtgtgccga ccacaggagc caagcctttt ccactgtgtg 1200 gactcatgtg gccaaggcta ggcctggtca cccaggaccc tcaccacgtg accccagcca 1260 atcgggacag ttcaaggagg aggagacccc tattacacag gttggaataa aatatttaaa 1320 tctcgtagaa taaaggacta ggggtgatag ggggagtatg ggataggagg aatgggtggg 1380 cgggaaaaaa gaaagggcgc ccgcttctgg ggggtccgag tttatccgtc cgcttggatg 1440 cggaactgca gagcccttct aatggggggc acctaaattt tcaatctcct gggcccggcg 1500 gttttacaga gctgttggct ggggaaaaca cctggggggt tacgccaagt tgaaatcgcc 1560 ttgggaggag aagtccccct ttgtgggcaa cttggggggt aaatggcgag aagggccccg 1620 gacgcgattg ggcctttccc aaagtgtttg cgccggcctt gattgggggg gaggg 1675 <210> 11 <211> 3203 <212> DNA
<2I3> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2175072 <400> 11 gagatggagt aattttgctg tggaaagact tcacgtcttg ccgaatgaaa tcagcctcca 60 gcgcctgcag cttccggaac taagatgtga ctgggctgta attttgctgt ggaaagactt 120 cacgtcttgc cgaatgaaag tcccgcctgt ctgtcacgct gatgcccgtg cagctgtctg 180 agcacccgga atggaatgag tctatgcact ccctccggat cagtgtgggg ggccttcctg 240 tgctggcgtc catgaccaag gccgcggacc cccgcttccg cccccgctgg aaggtgatcc 300 tgacgttctt tgtgggtgct gccatcctct ggctgctctg ctcccaccgc ccggcccccg 360 gcaggccccc cacccacaat gcacacaact ggaggctcgg ccaggcgccc gccaactggt 420 acaatgacac ctaccccctg tctcccccac aaaggacacc ggctgggatt cggtatcgaa 480 tcgcagttat cgcagacctg gacacagagt caagggccca agaggaaaac acctggttca 540 gttacctgaa aaagggctac ctgaccctgt cagacagtgg ggacaaggtg gccgtggaat 600 gggacaaaga ccatggggtc ctggagtccc acctggcgga gaaggggaga ggcatggagc 660 tatccgacct gattgttttc aatgggaaac tctactccgt ggatgaccgg acgggggtcg 720 tctaccagat cgaaggcagc aaagccgtgc cctgggtgat tctgtccgac ggcgacggca 780 ccgtggagaa aggcttcaag gccgaatggc tggcagtgaa ggacgagcgt ctgtacgtgg 840 gcggcctggg caaggagtgg acgaccacta cgggtgatgt ggtgaacgag aacccggagt 900 gggtgaaggt ggtgggctac aagggcagcg tggaccacga gaactgggtg tccaactaca 960 acgccctgcg ggctgctgcc ggcatccagc cgccaggcta cctcatccat gagtctgcct 1020 gctggagtga cacgctgcag cgctggttct tcctgccgcg ccgcgccagc caggagcgct 1080 acagcgagaa ggacgacgag cgcaagggcg ccaacctgct gctgagcgcc tcccctgact 1140 tcggcgacat cgctgtgagc cacgtcgggg cggtggtccc cactcacggc ttctcgtcct 1200 tcaagttcat ccccaacacc gacgaccaga tcattgtggc cctcaaatcc gaggaggaca 1260 gcggcagagt cgcctcctac atcatggcct tcacgctgga cgggcgcttc ctgttgccgg 1320 agaccaagat cggaagcgtg aaatacgaag gcatcgagtt catttaactc aaaacggaaa 1380 cactgagcaa ggccatcagg actcagcttt tataaaaaca agaggagtgc acttttgttt 1440 tgttttgttc tttttggaac tgtgcctggg ttggaggtct ggacagggag cccagtcccg 1500 ggccccatag tggtgcgggg cactgggacc cccgggcccc acggaggccg cggtctgaac 1560 tgctttccat gctgccatct ggtggtgatt tcggtcactt caggcattga ctcaaggcct 1620 gcctaactgg ctgggtcgtt tcttccatcc gacctcgttt cttttctttc ctatgttctt 1680 ttgttcagtg aatatcccta gagctcctac catatgtcag gccctatgcc tcaccctgag 1740 aacgcagtat gcatgaggtg gacctgtttg ctgggaaccc caggtcaccc ccttttcttc 1800 ctactctgtg cctggagcat catgtccacc cctgcagatc cttggaaaag aaaatgttta 1860 tgttgcaggg tattgcatgg tcacgagtga gggcaggccc ctgggggaca catctgccca 1920 cagctgcaca ggccagggcg caggcacatc tgttggttct caggcctcag ataaaaccat 1980 ctccgcatca tatggccagt gaccgctttc tcccttcaag aaaattctgt ggctgtgcag 2040 tactttgaag ttttaattat taacctgctt taattaaagc agtttccttt cttataaagt 2100 ggaatcacca aatcttatca cacagagcac agtcctgtag ttacccagcc cgctccagca 2160 gtgcgggaga ttgtaaggaa gcggtggcgg ctggtgaagc aagtctcaca tgtcggtgtt 2220 cttggccaat ggatacaaag ataaagaaaa tgttgccttt ttctaggaac tgtcagaaat 2280 cctcatgcct ttcaagactt ctgtgaatga cttgaatttt ttattccctg cctagggtct 2340 gtgaacgagg cctgtctctt ccctggggtt tctttccatg gcctttattt ctcctcttcc 2400 agtgggagtt ttgcaggctc ttctctgtgg aaacttcacg agcgttggct gggcctcggc 2460 ttcgctggag tgtactccag ggtgaaggca gagtgggatt tgagacccag gttaggcacg 2520 acccaggtct gagaagggac gtttccatca ttcacagtgc cctccccaca gcactacctc 2580 accccgaccc ccaccctcac tcctacccca ccccgcgatc gtcaggggtg ccacggtggg 2640 ccggagggtg ccggctctgg ctgtccctgt gccggtccct cacaaacctc tccccctttg 2700 aaactcaagc acagctgcga ggagggcagc gaggagggac ccctctctca tggttgtctc 2760 tttcccccgc tatgtcatag gtagtggagg aagcgaagga agtgaacgct gaatgtgacg 2820 catttctgaa gagctcagct gtcaccgggc atagcctgga agccccaagt ctgttctgac 2880 tttgcctggc tgtctccttg acccgcctcc tagatcattg tccttgatgt ccaggctggg 2940 tcatttaaaa tagagatgca atcaggaagg ttgggggact tgggactgtg gctgaattga 3000 gaccttgctg atgtattcat gtcagcacct gagtcacagc ccaggtgccc ggaagcagcc 3060 tcttcgcata ggcagtgatt tgcgattact ttaaagctca ccttttttct tcccctctct 3120 gttcgctgct gtcagcataa tgattgtgtt ccttccctat gggatccatc tgttttgtaa 3180 acaataaagc gtctgaggga tgt 3203 <210> 12 <211> 2648 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2403107 <400> 12 cggtggccct caggaggaag gaaggaaaaa cagccgtctc tggcctctgg cctctccagg 60 ctctccaccc tgggggcaga atcaattctg tagccctcta gcaccggcag tggctccgca 120 gtcagctggg cagtggccgt gactgggtca ctctacccta ggaagtgagt gcaggggctg 180 ctggagagcc agaggctggc agcgactatg tgaagttctc caaggagaag tacatcctgg 240 actcatcgcc agagaaactc cacaaggaat tggaggagga gctcaaactc agcagcacgg 300 atctccgcag ccatgcctgg taccatggcc gcatcccccg agaggtctcg gagaccttgg 360 tacaacgcaa cggcgacttc ctcatccggg actcactcac cagcctgggc gactatgtgc 420 tcacgtgctg ctggcgcaac caggccttgc acttcaagat caacaaggtg gtggtgaagg 480 caggcgagag ctacacacac atccagtacc tgtttgagca ggagagcttt gaccacgtgc 540 ccgccctcgt gcgctatcat gtgggcagcc gcaaggctgt gtcagagcag agtggtgcca 600 tcatctactg cccggtgaac cgcaccttcc cactgcgcta cctcgaggcc agctatggcc 660 tgggacaggg gagtagcaag cctgctagcc ccgtcagccc ctcaggcccc aagggcagcc 720 acatgaagcg gcgcagcgtc accatgaccg atgggctcac tgctgacaag gtcacccgca 780 gcgatggctg ccccaccagt acgtcgctgc cccgccctcg ggactccatc cgcagctgtg 840 ccctcagcat ggaccagatc ccagacctgc actcacccat gtcgcccatc tccgagagcc 900 ctagctcccc tgcctacagc actgtaaccc gtgtccatgc cgcccctgca gccccttctg 960 ccacagcatt gcctgcctcc cctgtcgccc gccgttccag tgagccccag ctgtgtcccg 1020 gaagtgcccc aaagacccat ggggagtcag acaagggccc ccacaccagc ccctcccaca 1080 cccttggcaa ggcctccccg tcaccatcac tcagcagcta cagtgacccg gactctggcc 1140 actactgcca gctccagcct cccgtgcgtg gcagccgaga gtgggcagcg actgagacct 1200 ccagccagca ggccaggagc tatggggaga ggctaaagga actgtcagaa aatggggccc 1260 ctgaagggga ctggggcaag accttcacag tccccatcgt ggaagtcact tcttccttca 1320 acccggccac cttccagtca ctactgatcc ccagggataa ccggccactg gaggtgggcc 1380 ttctgcgcaa ggtcaaggag ctgctggcag aagtggatgc ccggacgctg gcccggcatg 1440 tcaccaaggt ggactgcctg gttgctagga tactgggcgt taccaaggag atgcagaccc 1500 taatgggagt ccgctggggc atggaactgc tcaccctccc ccatggccgg cagctacgcc 1560 tagacctgct ggaaaggttc cacaccatgt ccatcatgct ggccgtggac atcctgggct 1620 gcaccggctc tgcggaggag cgggcagcgc tgctgcacaa gaccattcag ctggcggccg 1680 agctgcgggg gactatgggc aacatgttca gcttcgcggc ggtcatgggt gccctggaca 1740 tggctcagat ttctcggctg gagcagacat gggtgaccct gcggcagcga cacacagagg 1800 gtgccatcct gtacgagaag aagctcaagc cttttctcaa gagcctcaac gagggcaaag 1860 aaggcccgcc gctgagcaac accacgtttc ctcatgtgct gcccctcatc accctgctgg 1920 agtgtgactc ggccccacca gagggccctg agccctgggg cagcacggag cacggcgtgg 1980 aggtggtgct ggctcacctg gaggccgccc gcacagtggc acaccacgga ggcctgtacc 2040 acaccaatgc tgaagtcaag ctgcaggggt tccaggcccg gccggagctc ctggaggtgt 2100 tcagcacgga gttccagatg cgccttctct ggggcagtca gggtgccagc agcagccagg 2160 cccggcgcta tgagaagttc gacaaggtcc tcactgccct gtcccacaag ctggaacctg 2220 ctgtccgctc cagcgagctg tgaccccagg gacatttccc ctctgcagct gcggacagcg 2280 tcaggggcag aggggcacac aactttcccc agagcacccc aaggacactg tgatcaaccc 2340 gagaatgttc tgggttcaac tcaagcatct cccttgcacc tccagggtcc tgcgtggacc 2400 ctgggttcca tcccaactgc tacaagctca acaggtctcc attgatggag cacaggaacg 2460 gcggttcccc caccagcctt tgctgcttcc cttcctgctg tgggttcctg gtttcggacc 2520 ctcggaagcc aggtgcttca ggctccctca atctctgtgc tggacctcta caaagatcaa 2580 agtctccatt ttaaatgtga aataagtctc tttgtctgga aaattgatgt tcagaaaggg 2640 ggaccccg 2648 <210> 13 <211> 1361 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 1321, 1333, 1354, 1355 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 3069540 <400> 13 ggggctccgg agccgctcgc tcccgacacg gctcacgatg cgcggcgagc agggcgcggc 60 gggggcccgc gtgctccagt tcactaactg ccggatcctg cgcggaggga aactgctcag 120 ggaggatctg tgggtgcgcg gaggccgcat cttggaccca gagaagctgt tctttgagga 180 gcggcgcgtg gccgacgagc ggcgggactg cgggggccgc atcttggctc ccggattcat 240 cgacgtgcag atcaacggtg gatttggtgt tgacttctct caagccacgg aggacgtggg 300 ttcgggggtt gccctcgtgg cccggaggat cctgtcgcac ggcgtcacct ccttctgccc 360 caccctggtc acttccccac cggaggttta tcacaaggtt gttcctcaga tccctgtgaa 420 gagtggtggt ccccatgggg caggggtcct cgggctgcac ctggagggcc ccttcatcag 480 ccgggagaag cggggcgcgc accccgaggc ccacctccgc tccttcgagg ccgatgcctt 540 ccaggacttg ctggccacct acgggcccct ggacaatgtc cgcatcgtga cgctggcccc 600 agagttgggc cgtagccacg aagtgatccg ggcgctgacg gcccgtggca tctgcgtgtc 660 cctagggcac tcagtggctg acctgcgggc ggcagaggat gctgtgtgga gcggagccac 720 cttcatcacc cacctcttca acgccatgct gcctttccac caccgcgacc caggcatcgt 780 ggggctcctg accagcgacc ggctgcccgc aggccgctgc atcttctatg ggatgattgc 840 agatggcacg cacaccaacc ccgccgccct gcggatcgcc caccgtgccc atccccaggg 900 gctggtgctg gtcaccgatg ccatccctgc cttgggcctg ggcaacggcc ggcacacgct 960 gggacagcag gaagtggaag tggacggtct gacggcctac gtggcaggct gcagcatgga 1020 gtcggccctg gaggctgcat ccctgcaccc cgcccagttg ctggggctgg agaagagtaa 1080 ggggaccctg gactttggtg ctgacgcaga cttcgtggtg ctcgacgact cccttcacgt 1140 ccaggccacc tacatctcgg gtgagctggt gtggcaggcg gacgcagcta ggcagtgaca 1200 aggacctcgg ctgagaggac acctggccgc agcgggatgc catcagggcc gggtggttgg 1260 ggagctggtc tccagggagt gagtcgggag ccctgctgga ttgatgccca tggcctgtgc 1320 ngtgccctgg agncggtggc tgggataaac gtgnncccag c 1361 <210> 14 <211> 1401 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 4182350 <400> 14 gcaatttggg atcgacagtg actcgcgact ggtcggcgcg gcgaaagcag agcggcgcgc 60 cggttccttg gttcctgagg gcgatggcgc ggggtggctg gcgccggcta cgccgcctgt 120 tatccgcggg gcagcttcta ttccagggcc gcgcgctgct cgtcactaac acgctgggct 180 gcggcgcgct catggcggcc ggtgatggcg tgcgccagtc ctgggagatc cgcgcccggc 240 ccggccaggt tttcgaccca cggcgctccg cgagcatgtt tgcggtgggc tgcagcatgg 300 gtcccttcct gcactactgg tacttgtcgc tggaccgcct attccctgcg tctggcctcc 360 gaggcttccc aaatgtcctc aagaaggtcc tcgtggatca gctggtagcc tctccattgc 420 tgggcgtctg gtacttcttg ggccttggct gcctggaggg tcagacagtg ggtgagagct 480 gccaggagct gcgggagaag ttctgggaat tctacaaggc agactggtgc gtgtggcctg 540 ctgcgcagtt cgtgaacttc ctcttcgtgc ccccccaatt tcgagtcacc tacatcaacg 600 gcctgacgct gggctgggac acgtacctgt cctacttgaa gtaccggagc ccagttcctc 660 tgacaccccc aggctgtgtg gccctggaca cccgagcaga ctgaactgtc tgcttcctgg 720 accagatgca agactgtctc ctggcggacc accccctctg acagaagggg aatgggctcc 780 tgcagcaagc tcgggtcttg agccacgtcc cagcaccact tcagctccgg agcattgggc 840 tgagccgccc tttccaagct cacttctggg actgagtttc ctcaaccgga acacacccat 900 gaagatggat gatcatcccc tagcccttct cagcaggaac ctcatgcgac ctgtgaccaa 960 gatgtcccat cctcagcaca gggcccactc tgccaaccag tctcaagcac cagcccctca 1020 acactgccat ccacctggct ctgggccaag ccaccaatcc agagctccct caggtcctgg 1080 gactaaggcg gggacatgac tgatcccctc agagcaggct caggcctgga gtcggccccc 1140 aaaagtttca catagggcca ggcagcctct gtgtttcttt ccctggtcct gaactgtgga 1200 aatgccatta aactctctct ataatgtaac tgaaactgct ggctgggcgc agtggctccc 1260 acctgtaatc tcagcccttc ccgaggctga ggtggaagga ttgctcgagg ccaggtgttc 1320 gagaccagcc tgggcaacat agcaagaccc tgtctctatt tatattgaaa aataaaaata 1380 acaccagagg aataaaaaaa a 1401 <210> 15 <211> 223 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 95, 182 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 030600H1 <400> 15 ccggagccca gttcctctga cacccccagg ctgtgtggcc ctggacaccc gagcagactg 60 aactgtctgc ttcctggacc agatgcaaga ctgtntcctg gcggaccacc ccctctgaca 120 gaaggggaat gggctcctgc agcaagctcg ggtcttgagc cacgtcccag caccacttca 180 gntccggagc attgggctga gccgcccttt ccaagctcac ttt 223 <210> 16 <211> 530 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 94, 414, 437, 527 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 121448681 <400> 16 gcggcagcga cacacagagg gtgccatcct gtacgagaag aagctcaagc cttttctcaa 60 gagcctcaac gagggcaaag aaggcccgcc gcanagcaac accacgtttc ctcatgtgct 120 gcccctcatc accctgctgg agtgtgactc ggccccacca gagggccctg agccctgggg 180 cagcacggag cacggcgtgg aggtggtgct ggctcacctg gaggccgccc gcacagtggc 240 acaccacgga ggcctgtacc acaccaatgc tgaagtcaag ctgcaggggt tccaggcccg 300 gccggagctc ctggaggtgt tcagcacgga gttccagatg cgccttctct ggggcagtca 360 gggtgccagc agcagccagg cccggcgcta tgagaagttc gacaaggtcc tcantgccct 420 gtcccacaag ctggaanctg ctgtccgctc cagcgagctg tgaacccagg gacatttccc 480 ctgcgcagtt cgtgaacttc ctcttcgtgc ccccccaatt tcg ctctgcagct gggacaggtc aggggcaaag ggcaacaatt tcccagngcc 530 <210> 17 <211> 691 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 531, 540, 588, 612, 665 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 121549271 <400> 17 gctttattgt ttacaaaaca gatggatccc atagggaagg aacacaatca ttatgctgac 60 agcagcgaac agagagggga agaaaaaagg tgagctttaa agtaatcgca aatcactgcc 120 tatgcgaaga ggctgcttcc gggcacctgg gctgtgactc aggtgctgac atgaatacat 180 cagcaaggtc tcaattcagc cacagtccca agtcccccaa ccttcctgat tgcatctcta 240 ttttaaatga cccagcctgg acatcaagga caatgatcta ggaggcgggt caaggagaca 300 gccaggcaaa gtcagaacag acttggggct tccaggctat gcccggtgac agctgagctc 360 ttcagaaatg cgtcacattc agcgttcact tccttcgctt cctccactac ctatgacata 420 gcgggggaaa gagacaacca tgagagaggg gtccctcctc gctgccctcc tcgcagtgtg 480 cttgagtttc aaagggggag aggtttgtga gggaccggca cagggacagc nagagccggn 540 accctccggc ccaccgtggc acccctgacg attcccgggg tggggtanga attaagggtg 600 ggggtcgggg tnaaggaagt cctttgggga gggcacttgt taattattgg aaaacgtccc 660 ttttnagctt gggtcgtgcc taaccttggg t 691 <210> 18 <211> 615 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 568, 571 <223> a or g or c or t, unknor~m, or other <220>
<221> misc_feature <223> Incyte Clone No: 121775671 <400> 18 atagtcttta atgtcaaagg gaattcagtc ataaacctct aaattcccat cattactatt 60 ctcttcaagg gcacagctat gtttgcaatg gccctgaaac tcctcactcc catacataca 120 agtcccccat acacacaaag tctcaagcaa atgaggctgg agctggagtt tgatcttcat 180 ttatctgacc caaacaccca ggctctttcc attgttccac actgcaacat ttcagagtag 240 ccaaaaaaat ggacaacatt atcataatac ttaaaaaata attattgatg agaaaaaata 300 tttaggactc actggtctat actactgaaa tagcccttca aagtttctta gtaaacttgg 360 gaagccgata aacacattca gccaagcata tgatgcagaa gctgagtgat aaagagacgc 420 cctataacat tacaatattt tgcaaagcta tgatcattaa taatatgaat attagcaggt 480 aagttattgc tgaattgctg gtcccacatg agctgtaaag tgagtccttt gtgtaccacc 540 tcccaaagag tgacactggt tagtatangg ncccagatgg ttccattcct ggtttaccaa 600 acggcccatt ttttc 615 <210> 19 <211> 547 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 22, 40, 460, 468, 488, 541 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1269841F1 <400> 19 ggcccctggg gacacatctg cncacagctg cacaggccan ggcgcaggca catctgttgg 60 ttctcaggcc tcagataaaa ccatctccgc atcatatggc cagtgaccgc tttctccctt 120 caagaaaatt ctgtggctgt gcagtacttt gaagttttaa ttattaacct gctttaatta 180 aagcagtttc ctttcttata aagtggaatc accaaatctt atcacacaga gcacagtcct 240 gtagttaccc agcccgctcc agcagtgcgg gagattgtaa ggaagcggtg gcggctggtg 300 aagcaagtct cacatgtcgg cgttcttggc caatggatac aaagataaag aaaatgttgc 360 ctttttctag gaactgtcag aaatcctcga tgcctttcaa gacttctgtg aatgactgaa 420 ttttttattc cctgcctagg gtctgtgaac gaagcctgtn tcttcccngg ggggtttctt 480 tccatggnct ttattctcct cttccagtgg gaatttttgc aggcgccttc gcgtgggaac 540 ntcacga 547 <210> 20 <211> 502 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 89, 460 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1284408F6 <400> 20 gcggcgcgtg gccgacgagc ggcgggactg cgggggccgc atcttggctc ccggattcat 60 cgacgtgcag atcaacggtg gatttggtnt tgacttctct caagccacgg aggacgtggg 120 ttcgggggtt gccctcgtgg cccggaggat cctgtcgcac ggcgtcacct ccttctgccc 180 caccctggtc acttccccac cggaggttta tcacaaggtt gttcctcaga tccctgtgaa 240 gagtggtggt ccccatgggg caggggtcct cgggctgcac ctggagggcc ccttcatcag 300 ccgggagaag cggggcgcgc accccgaggc ccacctccgc tccttcgagg ccgatgcctt 360 ccaggattgc tggccaccta cgggcccctg gacaatgtcc gcatcgtgac gctggcccag 420 atttgggcgt aaccacgaag taattccggg cgctgacggn ccgtggcatc tgcgtgttcc 480 taaggaatca attggttgac tt <210> 21 <211> 707 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 3, 7, 21, 25, 34, 36, 53, 454, 495, 499, 549, 552, 596, 617, 623, 654, 656, 661, 678, 681, 683 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 133996971 <400> 21 atnaatntat agatggatcc natanggaag gaananaatc attatgctga cancagcgaa 60 cagagagggg aagaaaaaag gtgagcttta aagtaatcgc aaatcactgc ctatgcgaag 120 aggctgcttc cgggcacctg ggctgtgact caggtgctga catgaataca tcagcaaggt 180 ctcaattcag ccacagtccc aagtccccca accttcctga ttgcatctct attttaaatg 240 acccagcctg gacatcaagg acaatgatct aggaggcggg tcaaggagac agccaggcaa 300 agtcagaaca gacttggggc ttccaggcta tgcccggtga cagctgagct cttcagaaat 360 gcgtcacatt cagcgttcac ttccttcgct tcctccacta cctgggtctc aaatcccact 420 ctgccttcac cctggagtac actccagcga agcngaggcc cagccaacgc tcgtgaagtt 480 tccacagaga agagnctgna aaactcccac tggaagagga gaaataaagg ccatggaaag 540 aaaccccang gnagagacag gcctcgttca cagaccctaa ggcagggaat aaaaantcca 600 gtcattcaca gaagtcntga aangcatgag ggatttctga cattcctaga aaangncaca 660 nttccttaac cttggaanca ntngccaaga agccgacatg tgagact 707 <210> 22 <211> 577 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 324, 412, 459, 468, 486, 514, 526, 530, 543, 544, 568, 576 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1362518F6 <400> 22 ccgcgaccca ggcatcgtgg ggctcctgac cagcgaccgg ctgcccgcaa gccgctgcat 60 cttctatggg atgattgcag atggcacgca caccaacccc gccgccctgc ggatcgccca 120 ccgtgcccat ccccaggggc tggtgctggt caccgatgcc atccctgcct tgggcctggg 180 caacggccgg cacacgctgg gacagcagga agtggaagtg gacggtctga cggcctacgt 240 ggcaggctgc agcatggagt cggccctgga ggctgcatcc ctgcaccccg cccagttgct 300 ggggctggag aagagtaagg ggancctggg atttggtgct gacgcagatt cgtggtgctc 360 gacgactccc ttcaacgtcc aaggccaact acatctcggg tgaactggtg tnggcaaggc 420 ggacgaagct aggcagtgac aaggacttcg ggttgagang acaacttngg ccgcaacggg 480 ggattncatt aagggcccgg ttggttttgg gaanttggtc ctccangggn ttgagttccg 540 gannccctgc ttggatttga atgcccangg ggcctnt 577 <210> 23 <211> 424 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 92 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1513403F1 <400> 23 agcttataaa gtggaatcac caaatcttat cacacagagc acagtcctgt agttacccag 60 cccgctccag cagtgcggga gattgtaagg angcggtggc ggctggtgaa gcaagtctca 120 catgtcggcg ttcttggcca atggatacaa agataaagaa aatgttgcct ttttctagga 180 actgtcagaa atcctcatgc ctttcaagac ttctgtgaat gacttgaatt ttttattccc 240 tgcctagggt ctgtgaacga ggcctgtctc ttccctgggg tttctttcca tggcctttat 300 ttctcctctt ccagtgggag ttttgcaggc tcttctctgt ggaaacttca cgagcgttgg 360 ctgggcctcg gcttcgctgg agtgtactcc agggtgaagc agaatgggat ttgagacccc 420 aggt 424 <210> 24 <211> 524 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 71, 319, 351, 400, 409, 415, 430, 453, 466, 490, 507, 519 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1514414F6 <400> 24 tggcatctgc gtgtccctag ggcactcagt ggctgacctg cgggcggcag aggatgctgt 60 gtggagcgga ncaccttcat cacccacctc ttcaacgcca tgctgccttt ccaccaccgc 120 gacccaggca tcgtggggct cctgaccagc gaccggctgc ccgcaggccg ctgcatcttc 180 tatgggatga ttgcagatgg cacgcacacc aaccccgccg ccctgcggat cgcccaccgt 240 gcccatcccc aggggctggt gctggtcacc gatgccatcc ctggccttgg gcctgggcaa 300 cggccggcac acgctgggna aacaggaagt ggaagtggac ggtctgacgg ntacgtggca 360 ggcaccaaga cgctgagtgg cagccatagc cccaatggan gtctgtgtnc gggantttct 420 gcaaggccan aaggttgcag gaatggagtc ggncctggag ggctgnattc ctgcaacccg 480 gcccaaattn ctgggggttg gagaagngta aaggggganc cctt 524 <210> 25 <211> 684 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 44, 173, 314, 318, 350, 381, 383, 385, 389, 398, 403, 418, 423, 424, 427, 434, 445, 455, 459, 462, 481, 498, 500, 506, 516, 518, 535, 541, 544, 556, 564, 566, 579, 586, 590, 598, 606, 611, 612, 625, 629, 639, 642, 644, 648, 671, 677 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1517312F6 <400> 25 gccgctgggt ttaggaggtc cccgggttgc cggcggcgac agcnggggaa gcatgactgc 60 tgtgggccga agtgccccgc gctggggtcc cgaggggctg ctggagagcc agaggtggca 120 gcgactatgt gaagttctcc aaggagaagt acatcctgga ctcatcgcca ganaaactcc 180 acaaggaatt ggaggaggag ctcaaactca gcagcacgga tctccgcagc catgcctggt 240 accatggccg catcccccga gaggtctcgg agaccttggt acaacgcaac gggcgattct 300 catccgggac tcantcanca gcctggggga ctatgtgctc aagtgccggn tgggggcaac 360 caaggccttt gcactttcaa ngntnaacna aggtggtngt ttnaagggca aggcgaanaa 420 gtnncancac cacnttccaa gttancttgt ttttnaaanc anggaaaaaa gctttttgaa 480 nccaaaggtt ggcccggncn cctttnggtg gggggntnaa ttcaattttt ggggnaaaaa 540 nccnggcaaa agggtnttgt gttntnaagg aaggccaana aatttnggtn tgccccantg 600 aaattnttaa nntttgcccc cgggnttgna aaacggggna ancntttncc cccaaattgg 660 ggggtttaaa ntttcgngag gggg 684 <210> 26 <211> 490 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 125, 484 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1528049F1 <400> 26 gctgcgagga gggcagcgag gagggacccc tctctcatgg ttgtctcttt cccccgctat 60 gtcataggta gtggaggaag cgaaggaagt gaacgctaaa tgtgacgcat ttctgaagag 120 ctcanctgtc accgggcata gcctggaagc cccaagtctg ttctgacttt gcctggctgt 180 ctccttgacc cgcctcctag atcattgtcc ttgatgtcca ggctgggtca tttaaaatag 240 agatgcaatc aggaaggttg ggggacttgg gactgtggct gaattgagac cttgctgatg 300 tattcatgtc agcacctgag tcacagccca ggtgcccgga agcagcctct tcgcataggc 360 agtgatttgc gattacttta aagctcacct tttttcttcc cctctctgtt cgctgctgtc 420 agcataatga ttgtgttcct tccctatggg atccatctgt tttgtaaaca ataaagcgtc 480 tganggatgt 490 <210> 27 <211> 556 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 444, 471, 479, 496, 518, 544 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1578848F6 <400> 27 ggggctccgg agccgctcgc tcccgacacg gctcacgatg cgcggcgagc agggcgcggc 60 gggggcccgc gtgctccagt tcactaactg ccggatcctg cgcggaggga aactgctcag 120 ggaggatctg tgggtgcgcg gaggccgcat cttggaccca gagaagctgt tctttgagga 180 gcggcgcgtg gccgacgagc ggcgggactg cgggggccgc atcttggctc ccggattcat 240 cgacgtgcag atcaacggtg gatttggtgt tgattctctc aagccacgga ggacgtgggt 300 tcgggggttg ccctcgtggc ccggaagatc ctgtcgcaag gcgtcaactc cttctgcccc 360 aacctggtca tttcccaccg gagtttatca caaagttgtt cctcagattc ctgtgaagaa 420 tggtggtccc atggggcaag ggtnctcggg ctgcactgga ggcccttata nccggaaanc 480 gggcggcacc cgaggncact tcgtcctcga gcgatgcntc aaacttctgc actaagggcc 540 tganaattcg atgtaa 5S6 <210> 28 <211> 594 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 232, 376, 419, 521, 523, 533, 541, 555, 561, 571, 577 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 165234976 <400> 28 tctttgtttt tataaaagct gagtcctgat ggccttgctc agtgtttccg ttttgagtta 60 aatgaactcg atgccttcgt atttcacgct tccgatcttg gtctccggca acaggaagcg 120 cccgtccagc gtgaaggcca tgatgtagga ggcgactctg ccgctgtcct cctcggattt 180 gagggccaca atgatctggt cgtcggtgtt ggggatgaac ttgaaggacg anaagccgtg 240 agtggggacc accgccccga cgtggctcac agcgatgtcg ccgaagtcag gggaggcgct 300 cagcagcagg ttggcgccct tgcgctcgtc gtccttctcg ctgtagcgct cctggctggc 360 gcggcgcggc aggaanaacc agcgctgcag cgtgtcactc cagcaggcag actcatggnt 420 gaggtagcct ggcggctgga tgccggcagc aacccgcagg tgtgtagttg gacacccagt 480 tctcgtggtc cacgctgcct tgtagcccac cacttcacca ntncggttcc gtnacacata 540 nccgtagtgt gtcantcttg ncaggcgcca ntaaacntgt cttaattcag catc 594 <210> 29 <211> 514 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 19, 361, 409, 439, 450, 482, 484, 493, 496, 497, 513 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1657538F6 <400> 29 ctgaaaaagg gctacctgna ccctgtcaga cagtggggac aaggtggccg tggaatggga 60 caaagaccat ggggtcctac gagtcccacc tggcggagaa ggggagaggc atggagctat 120 ccgacctgat tgttttcaat gggaaactct actccgtgga tgaccggacg ggggtcgtct 180 accagatcga aggcagcaaa gccgtgccct gggtgattct gtccgacggc gacggcaccg 240 tggagaaagg cttcaaggcc gaatggtggc agtgaaggac gagcgtctgt acgtgggcgg 300 cctgggcaag gagtggacga ccactacggg tgatgtggtg aacgagaacc cggagtgggt 360 naaggtggtg ggctacaagg gcagcgtgga ccacgagaac tgggtgtcna actacaacgc 420 cctgcgggtt gctgccggna tccagccgcn agttaccttc atccatgagt tgcctgctgg 480 antnaaacct tgnagnntgg ttcttcttgc cgnc 514 <210> 30 <211> 524 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 361, 369, 488 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1812758F6 <400> 30 gagcaaggcc atcaggactc agcttttata aaaacaagag gagtgcactt ttgttttgtt 60 ttgttctttt tggaactgtg cctgggttgg aggtctggac agggagccca gtcccgggcc 120 ccatagtggt gcgggcactg gacccccggg ccccacggag gccgcggtct gaactgcttt 180 ccatgctgcc atctggtggt gatttcggtc acttcaggca ttgactcaag gcctgcctaa 240 ctggctgggt cgtttcttcc atccgacctc gtttcttttc tttcctatgt tcttttgttc 300 agtgaatatc cctagagctc ctaccatatg tcaggcccta tgctcaccct gagaacgcat 360 nagcatgang tggactgttt gctgggaacc caggtcaccc cttttcttcc tatctgtgct 420 ggagatcatg tcaaccctgc agatcctgga aaagaaatgt tatgttgcaa ggtattgcat 480 gtcacgantg aaggcaggcc ctggggaaca tctgcccaca gctg 524 <210> 31 <211> 568 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 41, 169, 170, 182, 253, 256. 257, 301, 340, 359, 374, 386, 394, 415, 445, 473, 474, 481, 509, 519, 533, 542, 557, 559 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 1856319F6 <400> 31 gtaattttgc tgtggaaaga cttcacgtct tgccgaatga nagtcccgcc tgtctgtcac 60 gctgatgccc gtgcagtgtc tgagcacccg gaatggaatg agtctatgca ctccctccgg 120 atcagtgtgg ggggccttcc tgtgctggcg tccatgacca aggccgcgnn cccccgcttc 180 cngccccgct ggaaggtgat cctgacgttc tttgtgggtg ctgccatcct ctggctgctc 240 tgctcccacc gcncgnnccc cggcaggccc cccacccaca atgcacacaa ctggaggctc 300 ngccaggcgc ccgccaactg gtacaatgac acctaccccn tgtctccccc acaaaggana 360 ccggctggga ttcngtatcg aatcgngtta tcgnagacct ggacacagag tcaanggccc 420 aagaggaaaa cacctggtta gttanctgaa aagggtactg accctgtaag aanntgggga 480 naaggtggcc gtggaatgga caaaacatng ggtctgganc caactggcga aanggggaag 540 gntggagctt cgactgntnt ttaatgga 568 <210> 32 <211> 282 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 64, 85, 126, 134, 165, 178, 182, 191, 226, 246, 249, 269, 270, 279 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 2016607H1 <400> 32 gtgactcgcg actggtcggc gcggcgaaag cagagcggcg cgccggttcc ttggttcctg 60 aggncgatgg cgccgggtgg ctggngcggc tacgccgcct gttatccgcg gggcagcttc 120 tattcnaggg ccgngcgctg ctcgtcacta acacgctgag ctgcngcgcg ctcatggngg 180 cnggtgatag ngtgcgccag tcctggcgag atccgcggcc cggcancggc caggttttcg 240 acccanggng ggtccacgaa catgtttgnn gtgggctgna gc 282 <210> 33 <211> 650 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 6, 11, 21, 22, 26, 31, 44, 46, 55, 56, 64, 72, 74, 77, 79, 83, 85, 86, 102, 160, 172, 178, 210. 280, 307, 319, 322, 344. 388, 394, 444, 462, 503, 521, 526, 549, 561, 567. 570, 599, 603, 609. 618, 626, 635, 648 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 201791876 <400> 33 atactnctac nccaacctgt nnaaanggag nctcctcctc cttnancttt cccgnnaggg 60 tggngtcacg tngngangnt ccngnntgac caggcctagc cntggccaca tgagtccaca 120 cagtggaaaa ggcttggctc ctgtggtcgg cacacacgan actctggttg cncctgcngg 180 gtgtggaaca ggcgcaccat cgccatgccn gcagctcaca aagctcctgc tggcctggga 240 tgcactggga gcagctgcca tgggtccgtc agctgaagtn aaggaagtcc tggccctcct 300 gcaagtncag tgacctcang tngtcctcaa aggcgccccc tgtnaggtcc agcttcacgc 360 agatgacgaa gggtggccgg gcggctcnga agtncaggat ggggattcgg ggcttgggtc 420 tttcctgaga gtcctcgtgg caanagaagc ctttcctgat cntggttctc gtccacggtc 480 acagaatgcc accaccttgc gcngggctgc cggcagtcaa nctgcngtac agccggcgcc 540 cctgctttnc cagcgttcca natggtnaan ggccgcccca gcggggcaag ggccttgcnc 600 ttncaaggna atcggacncc gttggntccc aaatngtcgt ctcccaanga 650 <210> 34 <211> 247 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2197153H1 <400> 34 cggtggccct caggaggaag gaaggaaaaa cagccgtctc tggcctctgg cctctccagg 60 ctctccaccc tgggggcaga atcaattctg tagccctcta gcaccggcag tggctccgca 120 gtcagctggg cagtggccgt gactgggtca ctctacccta ggaagtgagt gcaggggctg 180 ctggagagcc agaggctggc agcgactatg tgaagttctc caaggagaag tacatcctgg 240 actcatc 247 <210> 35 <211> 581 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 564 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 226276376 <400> 35 gagatattta ttaacagatg ggggtgctgg tgtgggggcc cttgtctgac tccccatggg 60 tctttggggc acttccggga cacagctggg gctcactgga acggcgggcg acaggggagg 120 caggcaatgc tgtggcagaa ggggctgcag gggcggcatg gacacgggtt acagtgctgt 180 aggcagggga gctagggctc tcggagatgg gcgacatggg tgagtgcagg tctgggatct 240 ggtccatgct gagggcacag ctgcggatgg agtcccgagg gcggggcagc gacgtactgg 300 tggggcagcc atcgctgcgg gtgaccttgt cagcagtgag cccatcggtc atggtgacgc 360 tgcgccgctt catgtggctg cccttggggc ctgaggggct gacggggcta gcaggcttgc 420 tactcccctg tcccaggcca tagctggcct cgaggtagcg cagtgggaag gtgcggttca 480 ccgggcagta gatgatggca ccactctgct ctgacacagc ttgcggctgc ccacatatag 540 cgcacgaggc ggcacgtggt caanctctct gctcaaacag g 581 <210> 36 <211> 553 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 24, 63, 334, 377, 409, 415, 435, 439, 444, 456, 459, 466, 473, 498, 504, 511, 521, 527, 535, 549, 552 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 2445385F6 <400> 36 cggcgctccg cgagcatgtt tgcngtgggc tgcagcatgg gtcccttcct gcactactgg 60 tanttgtcgc tggaccgcct attccctgcg tctggcctcc gaggcttccc aaatgtcctc 120 aagaaggtcc tcgtggatca gctggtagcc tctccattgc tgggcgtctg gtacttcttg 180 ggccttggct gcctggaggg tcagacagtg ggtgagagct gccaggagct gcgggagaag 240 ttctggggaa ttctacaagg cagactggtg cgtgtggcct gctgcgcagt tcgtgaactt 300 cctcttcgtg cccccccaat ttcgagtcac ctanatcaac ggcctgacgc tgggctgggg 360 acacgtacct gtcctanttg aagtaccgga gcccagttcc tctgacacnc cccangctgt 420 gtggccctgg acacncganc agantgaact gtctgnttnc tggacnagat ggnagactgt 480 ctcctggggg acaccccntc tggncagaaa ngggatgggg ntcctgnagc aaagntcggg 540 gtcttgagnc ang <210> 37 <211> 269 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 2593359H1 <400> 37 ccagctcaca cactgttttc tggaaataaa gccctactct acaaaaaaaa tgaagatggc 60 ttgtgggaaa agatctcttc tccaggaagt taaaaaacat gaattaccaa agaaagcacc 120 ttcttggcct gacagaccat tggtggggct ggcacgaatc cagatctgga tcctacatct 180 gttgggtctt aggcctcctt ccctcctcag tgtctttcaa atgactttca tcaaatgact 240 ttcaaaataa aaccttattt tggcaaaag 269 <210> 38 <211> 234 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 101, 233 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 2613966H1 <400> 38 gagatggagt aattttgctg tggaaagact tcacgtcttg ccgaatgaaa tcagcctcca 60 gcgcctgcag cttccggaac taagatgtga ctgggcttgc ngaggcgctg actcctctcc 120 tccctccctg gctgtgcagg tcccgcctgt ctgtcacgct gatgcccgtg cagctgtctg 180 agcacccgga atggaatgag tctatgcact ccctccggat cagtgtgggg ggnt 234 <210> 39 <211> 604 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 306, 335, 377, 383, 388, 400, 402, 409, 434, 463, 467, 471, 473, 476, 477, 479, 480, 482, 490, 495, 506, 516, 520, 523, 525, 532, 539, 541, 543, 545, 552, 553, 556, 557, 558, 559, 564, 566, 573, 575, 579, 584, 585, 587, 589, 590, 593, 598 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 2812186F6 <400> 39 gtttcctcaa ccggaacaca cccatgaaga tggatgatca tcccctagcc cttctcagca 60 ggaacctcat gtgacctgtg accaagatgt cccatcctca gcacagggcc cactctgcca 120 accagtctca agcaccagcc cctcaacact gccatccacc tggctctggg ccaagccacc 180 aatccagagc tccctcaggt cctgggacta aggcggggac atgactgatc ccctcagagc 240 aggctcaggc ctggagtcgg cccccaaaag tttcacatag ggccaggcag cctctgtgtt 300 tctttncctg gtctgaactg tggaaatgcc attanactct ctctaatgta actgaaactt 360 gctggctggg ggcgcantgg gcntcccnac cttgtaattn tngggcccnt ttccccggag 420 ggctttaaag gttngaaaag gggaattggc ctccaaaggg gcncaanggg ntngtnntnn 480 gnaaaaaacn aagcnccttg gggggngcca atacanttgn ggnanaagaa gnccccccnt 540 ngntnccccc annatnnnnt aaanantttt gtngngaana atannantnn aanattgnta 600 gctt 604 <210> 40 <211> 599 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 21, 58, 60, 117, 489, 504, 510, 512 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 290233776 <400> 40 ccaaaataag gttttatttt naaagtcatt tgatgaaagt catttgaaag acactgangn 60 gggaaggagg cctaagaccc aacagatgta ggatccagat ctggattcgt gccagcncca 120 ccaatggtct gtcaggccaa gaaggtgctt tctttggtaa ttcatgtttt ttaacttcct 180 ggagaagaga tcttttccca caagccatct tcattttttt tgtagagtag ggctttattt 240 ccagaaaaca gtgtgtgagc tggagatggg tgttttttta aaaacatcaa ggtagatcta 300 atatgttcaa caaagtgggg tggctcagcc agaggcgaag tggaaagatt ctctagtatt 360 tgcttgtcat cttggtgcaa ccagaaatcc acatgtggaa atggtgtcca ggagtacggt 420 cctatacgaa gtgttctgtc tctgcatcat aaatgctaat cattggtcct cctgctaaag 480 ttcgcgcana cgaagctgct cctncggacn cngatcttga aaggatgctc tgtaaatctc 540 tgcagtctta atgttgacag caatgccata tattactgga aagtgggttt cgttttctt 599 <210> 41 <211> 227 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3016872H1 <400> 41 gccaaaataa ggttttattt tgaaagtcat ttgatgaaag tcatttgaaa gacactgagg 60 agggaaggag gcctaagacc caacagatgt aggatccaga tctggattcg tgccagcccc 120 accaatggtc tgtcaggcca agaaggtgct ttctttggta attcatgttt tttaacttcc 180 tggagaagag atcttttccc acaagccatc ttcatttttt ttgtaga 227 <210> 42 <211> 309 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 115 <223> a or g or c or t, unknown, or other <220>
<221> misc feature <223> Incyte Clone No: 3030372H1 <400> 42 gtcgtttctt ccatccgacc tcgtttcttt tctttcctat gttcttttgt tcagtgaata 60 tccctagagc tcctaccata tgtcaggccc tatgcctcac cctgagaacg cagtncgcat 120 gaggtggacc tgtttgctgg gaaccccagg tcaccccctt ttcttcctac tctgtgcctg 180 gagcatcatg tccacccctg cagatccttg gaaaagaaaa tgtttatgtt gcagggtatt 240 gcatggtcac gagtgagggc aggcccctgg ggacacatct gcccacagct gcacaggcca 300 gggcgcagg 309 <210> 43 <211> 574 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 209, 414, 471, 476, 506, 563 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 3119919F6 <400> 43 aggactttga aggtaccatg gagctgtctg ttttcaatga tgccagtaag gacaagtctg 60 gggctatcat tgaaaaatgg agagtgaagc tggaagattc tggtgtccac gtgatcattg 120 gggggcacga ttctccctct cctagaggcg tcggatacgc taaaaatcaa gcagttgccc 180 agagctcagg gtcttacctt tgctttttng attcggatga cgtcatgatg ccccagcggg 240 tgaggctgca acacgaggct gccgttcagc acccgtcgag catcattggt tgcagagtga 300 ggagagatcc ccctaactcc accgaacgat acacacgttg gatcaaccag ctgacgccgg 360 agcagctcct aacccagcac gttccaagag ccaggctgct gcatgcgatg ttgnacaaca 420 aggtgtcctc ggcaatggac tgtgttctgt gagactggac acgggggccg nccgcngttg 480 ccgccttcgg ggtatgactt ggctgncgtc ttgaagggaa ggggactttg cccgttgcca 540 aattgggttg gcttgtccct tgncatccct cttg 574 <210> 44 <211> 240 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 237, 239 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 3257058H1 <400> 44 ctcagctctc ggctggggtt cgtcactggg cgcgggattt ggccgccgcg gggctccgga 60 gccgctcgct cccgacacgg ctcacgatgc gcggcgagca gggcgcggcg ggggcccgcg 120 tgctccagtt cactaactgc cggatcctgc gcggagggaa actgctcagg gaggatctgt 180 gggtgcgcgg aagccgcatc ttggacccag agaagctgtt ctttgaggag cggcgcntng 240 <210> 45 <211> 591 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 40, 46, 150, 159, 217, 295, 362, 372, 401, 405, 409, 415, 422, 450, 457, 458, 473, 496, 498, 502, 521, 534, 563, 565, 583, 584 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 3513795F6 <400> 45 cgcctccgcg atgccgctgc tcgtcgaggg gcggcgagtn cggctnccgc agtcagccgg 60 ggacctcgtc cgagcccacc cgcctttgga ggaaagagcc agacttctca gaggtcagtc 120 tgttcaacaa gtgggacccc agggccttcn gtatgttcng caaagagagc ttgcagtgac 180 ctccccaaag gatggctcca tctccattct gggttcngat gatgccacta cttgtcacat 240 tgtggtcctg aggcacacag gtaatggggc cacctgcttg acacattgtg acggnaccga 300 caccaaagct gaagtcccct tgatcatgaa ctccataaaa tcttttctga ccacgctcaa 360 tntggaaagc tngaagtaca ccttgttgga agctcagtga nggcnagcnt tgttncaaaa 420 antccctcat caaattctta gtggatttgn caaggcnnga aggtggccat tcnccttagt 480 gggcattatg tgtggncnga anttaaattg accggggaag naaacgaaaa accncttttc 540 cagggaaaat attggcattt gcntngtcca aaattttaaa acnnggccga g 591 <210> 46 <211> 304 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte Clone No: 3773551H1 <300>
<400> 46 cgaatcgcag ttatcgcaga cctggacaca gagtcaaggg cccaagagga aaacacctgg 60 ttcagttacc tgaaaaaggg ctacctgacc ctgtcagaca gtggggacaa ggtggccgtg 120 gaatgggaca aagaccatgg ggtcctggag tcccacctgg cggagaaggg gagaggcatg 180 gagctatccg acctgattgt tttcaatggg aaactctact ccgtggatga ccggacgggg 240 gtcgtctacc agatcgaagg cagcaaagcc gtgccctggg tgattctgtc cgacggcgac 300 ggca 304 <210> 47 <211> 227 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte Clone No: 406420H1 <400> 47 gatcaaccag ctgacgccgg agcagctcct aacccaggtt ttcacctcaa atggccccac 60 ggtgatcatg cccacctggt tctgctcgcg agcgtggttc tcccacgtgg gcccctttaa 120 cgaaggaggt cagggcgtcc cggaggacct gctgttcttc tacgagcacc tcaggaaggg 180 cggcggcgtc atccgcgtgg accagagtct cctgctgtat cgccacc 227 <210> 48 <211> 289 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 18 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 56227786 <400> 48 ttatttttta agtattanga taatgttgtc catttttttg gctactctga aatgttgcag 60 tgtggaacaa tggaaagagc ctgggtgttt gggtcagata aatgaagatc aaactccagc 120 tccagcctca tttgcttgag actttgtgtg tatgggggac ttgtatgtat gggagtgagg 180 agtttcaggg ccattgcaaa catagctgtg cccttgaaga gaatagtaat gatgggaatt 240 tagaggttta tgactgaatt ccctttgaca ttaaagacta tttgaattc 289 <210> 49 <211> 756 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 192, 459, 650, 657, 683, 689, 696, 699, 744, 749, 755 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 634343X12 <400> 49 gggaatggga gttaatcttc ttgtggccaa cacacatcat gtcagcctaa atatgacagg 60 aagtggtatt tatgcaccaa atggtcccaa agtgtatcat tatgacatga agacagagtt 120 gggaaaactt ctcctttcag aggtgggttc acatccccta tcctcgcttg cctacccaac 180 agctgttaat tngaatgcct acgccaccac catcaaacca tttccagtac agaaaaacac 240 tttcagggga tttatttcca gggatgggtt caacttcaca gaactttttg aaaatgcagg 300 aaaccttaca gtctgtcaaa aggagctttg ctgtcattta agctacagaa tgttacaaaa 360 agaagagaat gaagtatacg ttctaggagc ttttacagga ttacatggaa ggagaagaga 420 gtactggcag gtctgcacaa tgctgaagtg caaactacna atttgacaac ttgtggacgg 480 ccagtagaaa ctgcttctac aagatttgaa atgttctccc tcagtggcac atttggacac 540 agagtatgtt tttcctgaag tgctacttac cgaagattca tctgtcacct ggaaaatttg 600 aggtgctgaa agatggggcg tttggtaaac aagaatggac tcatctgggn ctatacnaac 660 cagtgtcact ccttggggag gtngtacana aagggntcnc tttacagctc atgtggggac 720 cagcaattcc gcccttaacc ttancccgng ggtant 756 <210> 50 <211> 740 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 24, 533, 6I2, 615, 617, 639, 655, 660, 668, 679, 687, 692, 710, 719, 722, 734 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 634343X14 <400> 50 ctcgtggcac gttaccataa gtancacctg tactctgagc ctcagtttaa tgtccctgaa 60 aagccggagt tggtgacttt caacaccgca tttggaaggt ttggcatttt cacgtgcttt 120 gatatattct tctatgatcc tggtgttacc ctggtgaaag atttccatgt ggacaccata 180 ctgtttccca cagcttggat gaacgttttg ccccttttga cagctattga attccattca 240 gcttgggcaa tgggaatggg agttaatctt cttgtggcca acacacatca tgtcagccta 300 aatatgacag gaagtggtat ttatgcacca aatggtccca aagtgtatca ttatgacatg 360 aagacagagt tgggaaaact tctcctttca gaggtggatt cacatcccct atcctcgctt 420 gcctacccaa cagctgttaa ttggaatgcc tacgccacca ccatcaaacc atttccagta 480 cagaaaaaca ctttcagggg atttatttcc agggatgggt tcaacttcac agnacttttt 540 gaaaatgcag gaaaccttac agtctgtcaa aaggggcttt gctgtcattt aagctacaga 600 atgttaccaa angangngaa tgaagtatac gttctaggng ctttacaggg ttacngggan 660 ggggaagngg tacctggcng tctgcanatg cnccagtgca aaaccaccan ttggccacnt 720 gnggagggcc ctanaaactg 740 <210> 51 <211> 889 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 622, 701, 771, 773, 794, 810, 831. 841, 853, 858, 882, 886, 887, 888 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 634343X17 <400> 51 cccccttgac taaagctcca aggacagaga aaaacatcca gatttgggaa cacaataaca 60 gatatgattg tccccacttc tactgccaaa attataaaac tgttaactcc tcctcatcag 120 cttacctgac tacttaaaag caaaagagtt aattaagtat tactaattgg tgatactaga 180 tcaatgaaga aatcactaaa ccttggccat ggtcacttcc tcttttccaa tctctgtgtc 240 agtttttgcc ctaataaccc tgcaggttgg tactcaggac agttttatag ctgcagtgta 300 tgaacatgct gtcattttgc caaataaaac agaaacacca gtttctcagg aggatgcctt 360 gaatctcatg aacgagtgga gacagcgatc aagcaggcag ctgagcaggg tgctcgaatc 420 attgtgactc cagaagatgc actttatgga tggaaattta ccagggaaac tgttttccct 480 tatctggagg atatcccaga ccctcaggtg aactggattc cgtgtcaaga cccccacaga 540 tttggtcaca caccagtaca agcaagactc agctgcctgg ccaaggacaa ctctatctat 600 gtcttggcaa atttggggga cnaaaagcca tgtaattccc gtgactccac gtgtcctcct 660 aatgggctac tttcaataca taccatgtgg tgtataatac ngaaggaaaa ctcgtggcac 720 gttacccata agtacccacc tgtaactctg agccctcagt ttaatgtccc ngnaaaagcc 780 ggagtttggt gacnttcaac accgcatttn ggaaggttgg gcatttcacg ngcttggaaa 840 nattctccag atnccggngt accccggtga aagatttcct gngggnnnt 889 <210> 52 <211> 496 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 63, 65, 88, 274, 278, 279. 280, 281, 283, 284, 288, 292, 294, 298, 300, 301, 302, 306, 309, 311, 314, 317, 322, 323, 324, 329, 335, 342, 344, 345, 347, 351. 353, 363, 371, 374, 376, 382, 389, 390, 398, 400, 402, 406, 416, 417, 418, 430, 435. 446, 449, 464, 465, 466, 470, 482, 485, 486, 487 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 75566986 <400> 52 ggcagctgct cccagtgcat cccaggccag caggagcttt gtgagctgca ggcatggcga 60 tgntncgcct gttccacacc cagcaggngc aaccagagtc tcgtgtgtgc cgaccacagg 120 agccaagcct tttccactgt gtggactcat gtggccaagg ctaggcctgg tcacccagga 180 ccctcaccac gtgaccccag ccaatcggga cagttcaagg aggaggagac ccctattaca 240 caggttggaa taaaatattt aaatctcgta aaanaaannn nanntggnga angngggnan 390 nngtgnatng nggnaanaga tnnnaagcna aaaanaaggg gngnncngct ntnagggttc 360 cgngttttgt ntancngttg cntgcggann tccagagncn cnttcnaaag ggggannncc 420 taaagttttn aattnccacg gggccngtng gttttgaaaa aggnnnttgn cctggggaaa 480 anccnnnggg ggttta 496 <210> 53 <211> 252 <2I2> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 58, 71, 125, 166, 172, 175, 183, 185, 187, 196, 198, 208, 226, 234, 244, 249 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 755669T6 <400> 53 ttattccaac ctgtgtaata ggtgtctcct cctccttgaa ctgtcccgat tggctggngt 60 cacgtggtga nggtcctggg tgaccaggcc tagccttggc cacatgagtc cacacagtgg 120 aaaangcttg gctcctgtgg tcggcacaca cgagactctg gttgcncctg cnggntgtgg 180 aanangngca ccatcnanat tccctgtngc tcacaaagct cctgcnggcc tggnatgcac 240 tggnagcanc tg <210> 54 <211> 297 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> 6, 24, 31, 34, 117, 141, 189, 195, 205, 206, 2I6, 228, 231, 239 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte Clone No: 915932H1 <400> 54 ccaggncatg caggcccacg tgtntattat nctnccagtc cacaacgctg aaccgtggct 60 ggacgaatgt ttgaggtctg ttttgcaaca ggactttgaa ggtaccatgg agctgtntgt 120 tttcaatgat gccagtaagg ncaagtctgg ggctatcatt gaaaaatgga gagtgaagct 180 gggggattnt ggtgnccacg gtggnnattt gggggnacgg ttttgccngt nctagaggng 240 tcggatacgg taaaaatcaa gcagttgccc agagctcagg gtcttacctt tgctttt 297

Claims (21)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:7 and fragments thereof.

2. A substantially purified variant having at least 90% amino acid 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 sequence of claim 3.

7. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:8 through SEQ ID NO:14 and fragments thereof.

8. An isolated and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide of claim 7.

9. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 7.

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

11. A host cell comprising the expression vector of claim 10.

12. A method for producing a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:7 and fragments thereof.
the method comprising the steps of:
a) culturing the host cell of claim 11 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.

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

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

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

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

17. A method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 16.

18. A method for treating or preventing a carbohydrate metabolism disorder, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 13.

19. A method for treating or preventing a cell proliferation disorder, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 16.

20. A method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:7 and fragments thereof, in a biological sample. the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one of the nucleic acids in the biological 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 encoding the polypeptide in the biological sample.

21. The method of claim 20 wherein the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.