EP1196574A2

EP1196574A2 - Human proteins involved in detoxification

Info

Publication number: EP1196574A2
Application number: EP00948589A
Authority: EP
Inventors: Y. Tom Tang; Henry Yue
Original assignee: Incyte Genomics Inc
Current assignee: Incyte Corp
Priority date: 1999-07-07
Filing date: 2000-07-06
Publication date: 2002-04-17
Also published as: AU6206500A; CA2379133A1; WO2001004305A3; WO2001004305A2; JP2003517292A

Abstract

The invention provides human detoxification proteins (DETX) and polynucleotides which identify and encode DETX. 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 DETX.

Description

HUMAN PROTEINS INVOLVED IN DETOXIFICATION

TECHNICAL FIELD

This invention relates to nucleic acid and amino acid sequences of human detoxification proteins and to the use of these sequences in the diagnosis, treatment, and prevention of autoimmune/inflammatory disorders, and cell proliferative disorders, including cancer

BACKGROUND OF THE INVENTION

Detoxification is the metabolic conversion ot pharmacologically active, often toxic, molecules to pharmacologically less active molecules The enzymes that catalyze detoxification reactions are often found in peroxisomes and the membrane of smooth endoplasmic reticulum (ER)

Peroxisomes can contain one or more enzymes that catalyze the removal ot oxygen trom organic substrates, producing hydrogen peroxide (H₂0₂). The enzyme catalase uses H₂0₂ to oxidize a variety of other substrates, e.g., formic acid, formaldehyde, phenol, and alcohol, by a peroxidative reaction which converts the H₂0₂ to water by the removal of hydrogens from the substrate. The peroxisomes of liver and kidney cells detoxity a variety of toxic molecules that enter the bloodstream by means of such an oxidative reaction

Another mapr function of oxidative reactions in peroxisomes is the degradation of fatty acid molecules. Beta oxidaϋon, the sequential shortening of the alkyl chains of fatty acids, occurs in the peroxisomes The two carbon atom blocks removed from the alkyl chains are converted to acetyl CoA and exported to the cytosol and reused in biosynthetic reactions.

The smooth ER is usually prominent in cells involved in lipid metabolism, e.g., cells that synthesize steroid hormones from cholesterol The membrane of the smooth ER contains enzymes involved in the detoxification of pid-soluble drugs and various harmful metabolites. (Reviewed in Alberts, B et al. (1994) Molecular Biology of the Cell, Garland Publishing, New York, NY, pp. 575- 580 )

Oxidative stress involves increased production of reactive oxygen species (ROS). ROS have been shown to cause neuronal apoptosis and other harmful effects. Oxidative stress is thought to be associated with various pathologies, including emphysema, Down syndrome, cataracts, adult respiratory distress, cancer, neural disorders, atherosclerosis, and aging. In response to oxidative stress, the expression of a number of genes is modulated Some of the modulated genes are catalase, superoxide dismutase, glutathione reductase, NAD(P)H-dependent alkyl hydroperoxide reductase, ox\R, endonuclease IV, and glucose-6-phosphate dehydrogenase These genes encode ROS detoxifying enzymes This suggests that one defense against the harmful and/or toxic effects of oxidative stress is the increase of detoxification enzymes to remove ROS (See Crawford, D R. et al. (1997) Arch Biochem Biophys 342(1) 6-12 )

The most extensively studied detoxification reactions are those catalyzed by enzymes in the cytochrome P450 family Cytochrome P450 enzymes have been classified into approximately forty different families P450 proteins are heme proteins, with a conserved cysteine residue in the C-terminus of the polynucleotide that is involved m binding heme (PROSITE PDOC00081 at http // www expasy ch cgi-bin on May 13, 1999) Cytochrome P450 enzymes catalyze reactions in which water-insoluble drugs are made sufficiently water-soluble to leave the cell and be excreted in the urine Metabolites that would otherwise accumulate to toxic levels in the cell membrane are also rendered water-soluble enough to allow them to leave the cell membrane and be excreted (Alberts, B et al (1994) Molecular Biology of the Cell. Garland Publishing. New York NY, p 579)

The discovery of new human detoxification proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of autoimmune/inflammatory disorders, and cell proliferative disorders, including cancer

SUMMARY OF THE INVENTION The invention features purified polypeptides, human detoxification proteins, referred to collectively as "DETX^"' and individually as "'DETX-1 " and "DETX-2 " In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1 -2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and d) an lmmunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1 -2 In one alternative, the invention provides an isolated polypeptide comprising the ammo acid sequence of SEQ ID NO 1-2 The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, b) a naturally occurring amino acid sequence having at least 909c sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-2. c) a biologically active tragment ot an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and d) an lmmunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2 In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO 1-2 In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO 3-4 Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

The invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.

Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-2.

The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO:3-4, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO:3-4, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides. Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 3-4, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO.3-4, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d) The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides

The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO.3-4, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 3-4, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d) The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof

The invention further provides a pharmaceutical composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO.1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and a pharmaceutically acceptable excipient In one embodiment, the pharmaceutical composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO 1-2 The invention additionally provides a method ol treating a disease or condition associated with decreased expression ot functional DETX, comprising administering to a patient in need of such treatment the pharmaceutical composition The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2 The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agomst activity in the sample In one alternative, the invention provides a pharmaceutical composition comprising an agomst compound identified by the method and a pharmaceutically acceptable excipient In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional DETX, compπsing admimsteπng to a patient in need of such treatment the pharmaceutical composition

Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide compπsing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected trom the group consisting of SEQ ID NO 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisUng of SEQ ID NO 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-2 The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagomst activity in the sample In one alternative, the invention provides a pharmaceutical composition compnsing an antagomst compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional DETX, comprising administering to a patient in need of such treatment the pharmaceutical composition

The invention further provides a method of screemng for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1 -2, and d) an immunogenic fragment of an amino acid sequence selected trom the group consisting of SEQ ID NO 1-2 The method compπses a) combimng the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide

The invention further provides a method of screemng for a compound that modulates the activity of a polypeptide compπsing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1-2, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO.1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-2 The method comprises a) combimng the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide. The invention further provides a method for screemng a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO 3-4, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide. The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of I) a polynucleotide sequence selected from the group consisting of SEQ ID NO 3- 4, n) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 3-4. in) a polynucleotide sequence complementary to l), iv) a polynucleotide sequence complementary to n), and v) an RNA equivalent of ι)-ιv) Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting ot SEQ ID NO 3-4. π) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting ot SEQ ID NO 3-4. in) a polynucleotide sequence complementary to I), iv) a polynucleotide sequence complementary to π). and v) an RNA equivalent of ι)-ιv) Alternatively, the target polynucleotide compπses a fragment of the above polynucleotide sequence, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound

BRIEF DESCRIPTION OF THE TABLES

Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding DETX

Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of DETX

Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis, diseases, disorders, or conditions associated with these tissues, and the vector into which each cDNA was cloned

Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding DETX were isolated

Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters

DESCRIPTION OF THE INVENTION Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims It must be noted that as used herein and in the appended claims, the singular forms "a,^" "an,^" and "the^" include plural reference unless the context clearly dictates otherwise Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody'^" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill m the art to which this invention belongs Although any machines, materials, and methods similar or equivalent to those descπbed herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described All publications mentioned herein are cited tor the purpose of describing and disclosing the cell lines. protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention

DEFINITIONS

"DETX" refers to the amino acid sequences ot substantially purified DETX obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, muπne, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant

The term "agonist" refers to a molecule which intensifies or mimics the biological activity of DETX Agomsts may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of DETX either by directly interacting with DETX or by acting on components of the biological pathway in which DETX participates

An "allehc variant^"" is an alternative form of the gene encoding DETX Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered A gene may have none, one, or many allelic variants of its naturally occumng form Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides

Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence

''Altered'^* nucleic acid sequences encoding DETX include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as DETX or a polypeptide with at least one functional characteristic of DETX. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding DETX, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding DETX 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 DETX Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or lmmunological activity of DETX is retained For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and argimne Amino acids with uncharged polar side chains having similar hydrophilicity values may include asparagine and glutamine. and seπne and threonine Amino acids with uncharged side chains having similar hydrophilicity values may include leucine. isoleucine, and valine, glycine and alan e. and phenylalanine and tyrosine

The terms "amino acid^" and "amino acid sequence^" refer to an oligopeptide, peptide. polypeptide. or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules Where "'amino acid sequence^"" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence^" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule

"Amplification'^" relates to the production of additional copies of a nucleic acid sequence Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known

The term '"antagomst^" refers to a molecule which inhibits or attenuates the biological activity of DETX. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of DETX either by directly interacting with DETX or by acting on components of the biological pathway in which DETX participates

The term "antibody^" refers to intact lmmunoglobulm molecules as well as to fragments thereof . such as Fab, F(ab')₂, and Fv fragments, which are capable of binding an epitopic determinant Antibodies that bind DETX polypeptides can be prepared using intact polypeptides or using fragments contaimng small peptides of interest as the immumzing antigen The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired Commonly used earners that are chemically coupled to peptides include bovine serum albumin, thyroglobulm, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the ammal The term "antigenic determinant^" refers to that region of a molecule (I e , an epitope) that makes contact with a particular antibody When a protein or a fragment of a protein is used to immunize a host ammal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigemc determinants (particular regions or three-dimensional structures on the protein) An antigemc determinant may compete with the intact antigen (l e , the immunogen used to elicit the immune response) for binding to an antibody

The term "antisense^" refers to any composition capable of base-pairing with the "'sense^'" (coding) strand of a specific nucleic acid sequence Antisense compositions may include DNA, RNA, peptide nucleic acid (PNA), oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates, oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars, or oligonucleotides having modified bases such as 5-methyl cytosine. 2'-deoxyuracιl, or 7-deaza-2'-deoxyguanosιne Antisense molecules may be produced by any method including chemical synthesis or transcription Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation "negative^"' or "minus" can refer to the antisense strand, and the designation "positive^" or "plus^"" can refer to the sense strand of a reference DNA molecule.

The term "biologically active'^" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active^'" or "immunogenic^" refers to the capability of the natural, recombinant, or synthetic DETX, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

'"Complementary^"' describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.

A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence^"' refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding DETX or fragments of DETX may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA. etc.).

"'Consensus sequence^*' refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG. Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.

'"Conservative amino acid substitutions^" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.

Original Residue Conservative Substitution

Ala Gly, Ser

Arg His, Lys

Asn Asp, Gin, His Asp Asn. Glu

Cys Ala. Ser Gin Asn, Glu, His

Gly Ala He Leu, Val

Leu He, Val

Lys Arg, Gin, Glu

Met Leu, He

Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr

Thr Ser, Val

Trp Phe, Tyr Val He, Leu. Thr

Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation,

(b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain A "deletion^" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more am o acid residues or nucleotides

The term "'derivative^** refers to a chemically modified polynucleotide or polypeptide Chemical modifications of a polynucleotide sequence can include, for example, replacement ot hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation. or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived

A '"detectable label^" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently pined to a polynucleotide or polypeptide A "fragment" is a umque portion of DETX or the polynucleotide encoding DETX which is identical in sequence to but shorter in length than the parent sequence A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5. 10. 15. 16, 20. 25, 30, 40, 50, 60. 75, 100. 150. 250 or at least 500 contiguous nucleotides or amino acid residues in length Fragments may be preferentially selected from certain regions of a molecule For example, a polypeptide fragment may comprise a certain length of contiguous ammo acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments

A fragment of SEQ ID NO.3-4 comprises a region of umque polynucleotide sequence that specifically identifies SEQ ID NO 3-4, for example, as distinct from any other sequence in the genome from which the fragment was obtained A fragment of SEQ ID NO 3-4 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO.3-4 from related polynucleotide sequences The precise length of a fragment of SEQ ID NO.3-4 and the region of SEQ ID NO 3-4 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment A fragment of SEQ ID NO 1-2 is encoded by a fragment of SEQ ID NO.3-4 A fragment of

SEQ ID NO 1-2 comprises a region of umque amino acid sequence that specifically identifies SEQ ID NO.1-2 For example, a fragment of SEQ ID NO.1-2 is useful as an immunogemc peptide for the development of antibodies that specifically recognize SEQ ID NO 1-2 The precise length of a fragment of SEQ ID NO 1-2 and the region of SEQ ID NO 1-2 to which the fragment coπesponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment

A "full-length^" polynucleotide sequence is one contaimng at least a translation initiation codon (e.g , methionine) followed by an open reading frame and a translation termination codon A '"lull- length" polynucleotide sequence encodes a "full-length^"" polypeptide sequence "Homology^" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences

The terms ""percent identity^'" and "% identity,^" as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm Such an algorithm may insert, in a standardized and reproducible way. gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences

Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEG ALIGN version 3 12e sequence alignment program This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI) CLUSTAL V is described in Higgins. D G and P M Sharp (1989) CABIOS 5 151 -153 and in Higgins, D G et al (1992) CABIOS 8 189-191 For pairwise alignments of polynucleotide sequences, the default parameters are set as follows Ktuple=2. gap penalty=5. wιndow=4, and "diagonals saved^"=4 The "weighted^"" residue weight table is selected as the default Percent identity is reported by CLUSTAL V as the " percent similarity^" between aligned polynucleotide sequences Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences^"" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences^" can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences^" tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences^" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:

Matrix: BLOSUM62 Reward for match: 1

Penalty for mismatch: -2

Open Gap: 5 and Extension Gap: 2 penalties

Gap x drop-off: 50

Expect: 10 Word Size: 11

Filter: on

Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

The phrases "'percent identity^" and "% identity.^" as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm Methods of polypeptide sequence alignment are well-known Some alignment methods take into account conservative amino acid substitutions Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEG ALIGN version 3 12e sequence alignment program (described and referenced above) For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows Ktuple=l, gap penalty=3, wmdow=5, and "diagonals saved^"=5 The PAM250 matrix is selected as the default residue weight table As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity^" between aligned polypeptide sequence pairs

Alternatively the NCBI BLAST software suite may be used For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2 0 12 (Apr-21-2000) with blastp set at default parameters Such default parameters may be, for example Matrix- BLOSUM62

Open Gap- 11 and Extension Gap 1 penalties

Gap x drop-off- 50

Expect- 10

Filter: on

Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured

"'Human artificial chromosomes^" (HACs) are linear rmcrochromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance

The term "humanized antibody^" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability

""Hybridization^" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 μg/ l sheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating T_m and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, 2^nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY; specifically see volume 2, chapter 9. High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.

The term "'hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C_nt or R_nt analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

The words "^■insertion^"" and "addition^" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively "Immune response^" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc These conditions can be characterized by expression of various factors, e g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems

An "immunogenic fragment^" is a polypeptide or oligopeptide fragment of DETX which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal The term "immunogenic fragment^"' also includes any polypeptide or oligopeptide fragment of DETX which is useful in any of the antibody production methods disclosed herein or known in the art

The term "microarray^" refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate The terms '"element^" and "aπay element^"* refer to a polynucleotide, polypeptide, or other chemical compound having a umque and defined position on a microarray

The term ""modulate^"' refers to a change in the activity of DETX For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of DETX The phrases '"nucleic acid" and "nucleic acid sequence^*" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material

"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression ot the coding sequence Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to ιoιn two protein coding regions, in the same reading frame

"Peptide nucleic acid^*" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine The terminal lysine confers solubility to the composition PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell

"Post-translational modification^"" of an DETX may involve lipidation, glycosylation. phosphorylation. acetylation. racemization proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of DETX

"Probe" refers to nucleic acid sequences encoding DETX, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes "Primers^"* are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing The primer may then be extended along the target DNA strand by a DNA polymerase enzyme Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e g., by the polymerase chain reaction (PCR)

Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used

Methods for preparing and using probes and primers are described in the reterences, for example Sambrook, J et al , 1989, Molecular Clomng A Laboratory Manual, 2^nd ed , vol 1-3, Cold Spring Harbor Press, Plamview NY; Ausubel, F M et al ,1987, Current Protocols in Molecular Biology, Greene Publ. Assoc & Wiley-Intersciences, New York NY, Innis, M. et al , 1990, PCR

Protocols. A Guide to Methods and Applications, Academic Press, San Diego CA PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0 5, 1991 , Whitehead Institute for Biomedical Research, Cambridge MA) Oligonucleotides for use as primers are selected using software known in the art for such purpose For example, OLIGO 4 06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases Similar primer selection programs have incorporated additional features for expanded capabilities For example, the PπmOU primer selection program (available to the public from the Genome Center at Umversity of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for desigmng primers on a genome-wide scope The Pπmer3 primer selection program (available to the public trom the Whitehead Institute/MIT Center for Genome Research. Cambridge MA) allows the user to input a "mispπming library,^'" in which sequences to avoid as primer binding sites are user-specified Pπmer3 is useful, in particular, for the selection of oligonucleotides for rmcroarrays (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user^'s specific needs ) The PπmeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences Hence, this program is useful for identification of both umque and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above

A "recombinant nucleic acid^" is a sequence that is not naturally occumng or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e g., by genetic engineering techniques such as those described in Sambrook, supra The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell Alternatively, such recombinant nucleic acids may be part of a viral vector, e g , based on a vaccima virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal

A "regulatory element^" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, mtrons, and 5' and 3' untranslated regions (UTRs) Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability

"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody Reporter molecules include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, substrates, cot actors, inhibitors, magnetic particles, and other moieties known in the art

An ""RNA equivalent,^" in reference to a DNA sequence, is composed of the same linear sequence ot nucleotides as the reterence DNA sequence with the exception that all occurrences of the mtrogenous base thymine are replaced with uracil. and the sugar backbone is composed of πbose instead of deoxyπbose

The term "sample^" is used in its broadest sense. A sample suspected of contaimng nucleic acids encoding DETX, or tragments thereof, or DETX itself, may comprise a bodily fluid, an extract from a cell, chromosome, organelle. or membrane isolated from a cell, a cell, genomic DNA, RNA, or cDNA, in solution or bound to a substrate, a tissue, a tissue print, etc

The terms ""specific binding^" and "specifically binding^"* refer to that interaction between a protein or peptide and an agomst, an antibody, an antagomst, a small molecule, or any natural or synthetic binding composition The interaction is dependent upon the presence of a particular structure of the protein, e g , the antigemc determinant or epitope, recognized by the binding molecule For example, if an antibody is specific for epitope "A,^"' the presence of a polypeptide comprising the epitope A, or the presence of tree unlabeled A, in a reaction contaimng free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody

The term '"substantially purified^"' refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated

A ""substitution^'* refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively

"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound

A "transcript image^" refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time "Transformation" describes a process by which exogenous DNA is introduced into a recipient cell Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteπophage or viral infection. electroporation, heat shock, lipotection, 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 transtormed cells which express the inserted DNA or RNA for limited periods of time

A "transgemc organism ' as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the orgamsm contains heterologous nucleic acid introduced by way of human intervention, such as by transgemc techmques well known in the art The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgemc organisms contemplated in accordance with the present invention include bacteria, cyanobacteπa, fungi, plants, and ammals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation Techmques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al (1989), supra

A "variant^"' of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences^"' tool Version 2.0.9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length A variant may be described as, for example, an "allelic^" (as defined above), "splice," '"species," or ""polymorphic^" variant. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule Species variants are polynucleotide sequences that vary trom one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "'single nucleotide polymorphisms^" (SNPs) in which the polynucleotide sequence varies by one nucleotide base The presence of SNPs may be indicative of. for example, a certain population, a disease state, or a propensity for a disease state

A "variant^" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length ol one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0 9 (May-07-1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50% . at least 60% , at least 70%. at least 80% , at least 90% . at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides THE INVENTION The invention is based on the discovery of new human detoxification proteins (DETX), the polynucleotides encoding DETX, and the use of these compositions for the diagnosis, treatment, or prevention of autoimmune inflammatory disorders, and cell proliferative disorders, including cancer Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding DETX Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each DETX were identified, and column 4 shows the cDNA libraries from which these clones were isolated Column 5 shows Incyte clones and their corresponding cDNA libraries Clones tor which cDNA libraries are not indicated were derived from pooled cDNA libraries The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each DETX and are useful as fragments in hybridization technologies

The columns of Table 2 show various properties of each of the polypeptides of the invention column 1 references the SEQ ID NO, column 2 shows the number of amino acid residues in each polypeptide, column 3 shows potential phosphorylation sites, column 4 shows potential glycosylation sites, column 5 shows the amino acid residues comprising signature sequences and motifs, column 6 shows homologous sequences as identified by BLAST analysis, and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding DETX The first column of Table 3 lists the nucleotide SEQ ID NOs Column 2 lists fragments of the nucleotide sequences of column 1 These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO 3-4 and to distinguish between SEQ ID NO 3-4 and related polynucleotide sequences The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides Column 3 lists tissue categories which express DETX as a fraction of total tissues expressing DETX Column 4 lists diseases disorders, or conditions associated with those tissues expressing DETX as a traction of total tissues expressing DETX Column 5 lists the vectors used to subclone each cDNA library

The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding DETX were isolated Column 1 references the nucleotide SEQ ID NOs, column 2 shows the clone IDs of the Incyte clones in which nucleic acids encoding each DETX were identified, column 3 shows the cDNA libraries from which these clones were isolated, and column 4 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 3 SEQ ID NO 4 maps to chromosome 16 within the interval from 88 80 to 90 20 centiMorgans The invention also encompasses DETX variants A preferred DETX variant is one which has at least about 80%. or alternatively at least about 90%, or even at least about 95% ammo acid sequence identity to the DETX amino acid sequence, and which contains at least one functional or structural characteristic of DETX

The invention also encompasses polynucleotides which encode DETX In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO.3-4, which encodes DETX The polynucleotide sequences of SEQ ID NO 3-4, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the mtrogenous base thymine are replaced with uracil, and the sugar backbone is composed of πbose instead of deoxyπbose The invention also encompasses a variant of a polynucleotide sequence encoding DETX In particular, such a variant polynucleotide sequence will have at least about 70% , or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding DETX A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO 3-4 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO 3-4 Any one of the polynucleotide variants descπbed above can encode an amino acid sequence which contains at least one functional or structural characteristic of DETX

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 DETX, some bearing mimmal similarity to the polynucleotide sequences of any known and naturally occumng 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 occumng DETX, and all such variations are to be considered as being specifically disclosed

Although nucleotide sequences which encode DETX and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring DETX under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding DETX 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 trequency with which particular codons are utilized by the host Other reasons for substantially altering the nucleotide sequence encoding DETX 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 DETX and DETX 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 DETX 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 NO 3-4 and fragments thereof under various conditions of stringency (See, e g , Wahl, G M and S L Berger (1987) Methods Enzymol 152 399-407, Kimmel, A R (1987) Methods Enzymol 152 507- 511 ) Hybridization conditions, including annealing and wash conditions, are described in "Definitions

Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD) Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosystems) Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEG AB ACE 1000 DNA sequencing system (Molecular Dynamics. Sunnyvale CA), or other systems known in the art The resulting sequences are analyzed using a variety ol algorithms which are well known in the art (See, e g , Ausubel, F M (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, umt 7 7, Meyers, R A (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp 856-853 )

The nucleic acid sequences encoding DETX may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements For example, one method which may be employed. restriction-site PCR, uses umversal and nested primers to amplify unknown sequence trom genomic DNA within a cloning vector (See. e g , Sarkar, G (1993) PCR Methods Applic 2 318-322 ) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (See, e g . Tπglia, T et al (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments ad]acent to known sequences in human and yeast artificial chromosome DNA (See, e.g., Lagerstrom, M. et al. ( 1991 ) PCR Methods Applic 1.111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art (See, e g , Parker, J.D et al. (1991) Nucleic Acids Res. 19.3055-3060) Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech. Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron exon junctions For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences. Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68 °C to 72°C.

When screemng for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs In addition, random-primed libraries, which often include sequences contaimng the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions

Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths Output light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems). and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample

In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode DETX may be cloned in recombinant DNA molecules that direct expression of DETX. 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 DETX

The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter DETX-encoding sequences lor a variety of purposes including, but not limited to, modification of the clomng, 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, ohgonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth

The nucleotides of the present invention may be subjected to DNA shuffling techmques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA, described in U.S Patent Number 5,837,458, Chang, C -C. et al. (1999) Nat Biotechnol 17.793-797, Chπstians, F C et al. (1999) Nat Biotechnol 17 259-264, and Crameπ, A et al (1996) Nat. Biotechnol 14.315-319) to alter or improve the biological properties of DETX, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds DNA shuffling is a process by which a library of gene vaπants is produced using PCR-mediated recombination of gene fragments The library is then subjected to selection or screemng procedures that identify those gene variants with the desired properties These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution For example, fragments of a single gene contaimng random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner

In another embodiment, sequences encoding DETX may be synthesized, in whole or in part, using chemical methods well known in the art (See, e g , Caruthers, M H et al (1980) Nucleic Acids Symp. Ser 7 215-223, and Horn, T et al (1980) Nucleic Acids Symp Ser. 7.225-232 ) Alternatively. DETX itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques (See, e g , Creighton, T. (1984) Proteins, Structures and Molecular Properties. WH Freeman, New York NY, pp 55-60, and Roberge, J Y. et al (1995) Science 269 202-204 ) Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (PE Biosystems) Additionally, the amino acid sequence of DETX, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence ol a naturally occumng polypeptide

The peptide may be substantially purified by preparative high performance liquid chromatography (See, e g , Chiez, R.M an F Z Regmer (1990) Methods Enzymol 182 392-421 ) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (See, e.g., Creighton, supra, pp. 28-53.)

In order to express a biologically active DETX, the nucleotide sequences encoding DETX or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3^" untranslated regions in the vector and in polynucleotide sequences encoding DETX. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding DETX. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding DETX and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding DETX and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology, 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 DETX. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke. G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer. CA. et al. (1994) Bio/Technology 12:181-184: Engelhard. E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3224-3227; Sandig. V. et al. (1996) Hum. Gene Ther. 7: 1937-1945; Takamatsu. N. (1987) EMBO J. 6:307-311 ; Coruzzi. G. et al. (1984) EMBO J. 3:1671-1680: Broglie, R. et al. (1984) Science 224:838-843: Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105: The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81 :3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di

Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al., (1993) Proc. Natl. Acad. Sci. USA 90(13): 6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, I.M. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed. In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding DETX. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding DETX can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding DETX into the vector's multiple cloning site disrupts the lacL 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 DETX are needed, e.g. for the production of antibodies, vectors which direct high level expression of DETX may be used. For example, vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of DETX. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter, supra; and Scorer, supra.)

Plant systems may also be used for expression of DETX. Transcription of sequences encoding DETX may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, supra; Broglie, supra; and Winter, supra.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.) In mammalian cells, a number of viral-based expression systems may be utilized In cases where an adenovirus is used as an expression vector, sequences encoding DETX may be ligated into an adenovirus transcription translation complex consisting of the late promoter and tripartite leader sequence Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses DETX in host cells. (See, e g , Logan, J and T Shenk (1984) Proc Natl Acad Sci USA 81 3655-3659 ) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells SV40 or EBV- based vectors may also be used for high-level protein expression

Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (lφosomes, polycatioruc amino polymers or vesicles) for therapeutic purposes (See, e g , Harrington, J J et al (1997) Nat Genet 15 345-355 ) For long term production of recombinant proteins in mammalian systems, stable expression of DETX in cell lines is preferred For example, sequences encoding DETX can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences Resistant clones of stably transformed cells may be propagated using tissue culture techmques 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 aderune phosphoπbosyltransferase genes, tor use in tk and apr cells, respectively (See, e g , Wigler, M et al (1977) Cell 11 223-232, Lowy, I et al (1980) Cell 22 817-823 ) Also, antimetabohte, antibiotic, or herbicide resistance can be used as the basis for selection For example, dhfr confers resistance to methotrexate, neo confers resistance to the aminoglycosides neomycin and G-418. and als and pat confer resistance to chlorsulfuron and phosphinotπcin acetyltransf erase, respectively (See, e g , Wigler, M et al (1980) Proc Natl Acad Sci USA 77 3567-3570. Colbere-Garapin, F et al (1981) J Mol Biol 150 1 -14 ) Additional selectable genes have been described, e g , trpB and hisD which alter cellular requirements for metabolites (See. e g , Hartman. S C and R C Mulligan (1988) Proc Natl Acad Sci USA 85 8047-8051 ) Visible markers, e g , anthocyanins green fluorescent proteins (GFP. Clontech). β glucuromdase and its substrate β-glucuronide or luciterase and its substrate lucifeπn may be used These markers can be used not only to identify transformants, but also to quantity the amount ol transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding DETX is inserted within a marker gene sequence, transformed cells containing sequences encoding DETX can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding DETX under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well. In general, host cells that contain the nucleic acid sequence encoding DETX and that express

DETX 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 DETX using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on DETX is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et al. (1997) Cuπent Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols. Humana Press, Totowa NJ.)

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding DETX include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding DETX, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like

Host cells transformed with nucleotide sequences encoding DETX may be cultured under conditions suitable for the expression and recovery of the protein from cell culture The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used As will be understood by those of skill in the art, expression vectors contaimng polynucleotides which encode DETX may be designed to contain signal sequences which direct secretion of DETX through a prokaryotic or eukaryotic cell membrane

In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation. and acylation Post-translational processing which cleaves a "prepro^" or "pro^"' form of the protein may also be used to specify protein targeting, folding, and/or activity Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e g , CHO, HeLa, MDCK, HEK293, and WI38) are available trom the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding DETX may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems For example, a chimeπc DETX protein contaimng a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screemng of peptide libraries for inhibitors of DETX activity Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affimty matrices Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP). thioredoxin (Trx), calmodulin binding peptide (CBP), 6-Hιs, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-Hιs enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively FLAG, c-myc, and hemagglutinin (HA) enable lmmunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags A fusion protein may also be engineered to contain a proteolytic cleavage site located between the DETX encoding sequence and the heterologous protein sequence, so that DETX may be cleaved away from the heterologous moiety following purification Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10) A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins

In a further embodiment of the invention, synthesis of radiolabeled DETX may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters Translation takes place in the presence of a radiolabeled amino acid precursor, for example, DETX of the present invention or fragments thereof may be used to screen for compounds that specifically bind to DETX At least one and up to a plurality of test compounds may be screened for specific binding to DETX. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

In one embodiment, the compound thus identified is closely related to the natural ligand of DETX, e.g , a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, Cohgan, J.E. et al. (1991) Current Protocols in Immunology 1(2). Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which DETX binds, or to at least a fragment of the receptor, e.g., the ligand binding site In either case, the compound can be rationally designed using known techmques. In one embodiment, screemng for these compounds involves producing appropriate cells which express DETX, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosopfula, or E_ coh. Cells expressing DETX or cell membrane fractions which contain DETX are then contacted with a test compound and binding, stimulation, or inhibition ot activity of either DETX or the compound is analyzed. An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combimng at least one test compound with DETX, either in solution or affixed to a solid support, and detecting the binding of DETX to the compound Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical hbraπes, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.

DETX of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of DETX Such compounds may include agomsts, antagomsts, or partial or inverse agomsts. In one embodiment, an assay is performed under conditions permissive for DETX activity, wherein DETX is combined with at least one test compound, and the activity of DETX in the presence ot a test compound is compared with the activity of DETX in the absence of the test compound. A change in the activity of DETX in the presence of the test compound is indicative ol a compound that modulates the activity of DETX Alternatively, a test compound is combined with an in vitro or cell-free system compπsing DETX under conditions suitable for DETX activity, and the assay is performed In either of these assays, a test compound which modulates the activity of DETX may do so indirectly and need not come in direct contact with the test compound At least one and up to a plurality of test compounds may be screened

In another embodiment, polynucleotides encoding DETX or their mammalian homologs may be "knocked out" in an ammal model system using homologous recombination in embryonic stem (ES) cells Such techmques are well known in the art and are useful for the generation of ammal models of human disease. (See, e g., U S Patent No. 5,175,383 and U.S Patent No. 5,767.337 ) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture The ES cells are transformed with a vector contaimng the gene of interest disrupted by a marker gene, e g , the neomycin phosphotransferase gene (neo, Capecchi, M R (1989) Science 244 1288-1292) The vector integrates into the corresponding region of the host genome by homologous recombination Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D (1996) Chn Invest 97.1999-2002, Wagner, K U. et al (1997) Nucleic Acids Res. 25 4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeπc progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgemc animals thus generated may be tested with potential therapeutic or toxic agents. Polynucleotides encoding DETX may also be manipulated in vitro in ES cells derived trom human blastocysts Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J A et al (1998) Science 282 1145-1 147) Polynucleotides encoding DETX can also be used to create "knockin" humamzed animals

(pigs) or transgemc animals (mice or rats) to model human disease With knockin technology, a region of a polynucleotide encoding DETX is ιn)ected into ammal ES cells, and the injected sequence integrates into the ammal cell genome Transformed cells are injected into blastulae. and the blastulae are implanted as described above Transgemc progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease Alternatively, a mammal inbred to overexpress DETX, e g , by secreting DETX in its milk, may also serve as a convement source of that protein (Janne, J et al (1998) Biotechnol Annu Rev 4 55-74) THERAPEUTICS

Chemical and structural similarity, e g . in the context of sequences and motifs, exists between regions of DETX and human detoxification proteins In addition the expression ot DETX is closely associated with nervous, reproductive, gastrointestinal, and musculoskeletal tissue Therefore, DETX appears to play a role in autoimmune/inflammatory disorders, and cell proliferative disorders, including cancer In the treatment of disorders associated with increased DETX expression or activity, it is desirable to decrease the expression or activity of DETX In the treatment of disorders associated with decreased DETX expression or activity, it is desirable to increase the expression or activity of DETX

Therefore, in one embodiment, DETX or a fragment or derivative thereof may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of DETX Examples of such disorders include, but are not limited to, an autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome (AIDS),

Addison^'s disease, adult respiratory distress syndrome, allergies, ankylosing spondyhtis. amyloidosis. anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocπnopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes melhtus, emphysema, episodic lymphopema with lymphocytotoxins, erythroblastosis fetahs, erythema nodosum, atrophic gastπtis, glomerulonephrius, Goodpasture^'s syndrome, gout, Graves^' disease, Hashimoto^'s thyroiditis, hypereosinophiha, irritable bowel syndrome, multiple sclerosis, myasthema gravis, myocardial or peπcardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter^'s syndrome, rheumatoid arthπtis, scleroderma, Sjogren^'s syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopemc purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma, and a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuπa, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma. and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, perns, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus In another embodiment, a vector capable of expressing DETX or a fragment or derivative thereof may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of DETX including, but not limited to, those described above

In a further embodiment, a pharmaceutical composition comprising a substantially purified DETX in conjunction with a suitable pharmaceutical carrier may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of DETX including, but not limited to, those provided above

In still another embodiment, an agomst which modulates the activity of DETX may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of DETX including, but not limited to, those listed above In a further embodiment, an antagomst of DETX may be admimstered to a subject to treat or prevent a disorder associated with increased expression or activity of DETX Examples of such disorders include, but are not limited to, those autoimmune/inflammatory disorders and cell proliferative disorders, including cancer, described above In one aspect, an antibody which specifically binds DETX may be used directly as an antagomst or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express DETX

In an additional embodiment, a vector expressing the complement of the polynucleotide encoding DETX may be admimstered to a subject to treat or prevent a disorder associated with increased expression or activity of DETX including, but not limited to, those described above

In other embodiments, any of the proteins, antagonists, antibodies, agomsts, complementary sequences, or vectors of the invention may be admimstered in combination with other appropriate therapeutic agents Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects

An antagomst of DETX may be produced using methods which are generally known in the art In particular, purified DETX may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind DETX Antibodies to DETX may also be generated using methods that are well known in the art Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeπc. and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use

For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with DETX or with any fragment or oligopeptide thereof which has immunogemc properties Depending on the host species, various adjuvants may be used to increase immunological response Such adjuvants include, but are not limited to, Freund^'s, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluromc polyols. polyamons, peptides, oil emulsions, KLH, and αimtrophenol Among adjuvants used in humans, BCG (bacilli Calmette-Guerm) and Corynebacteπum parvum are especially preferable It is prefeπed that the oligopeptides, peptides, or fragments used to induce antibodies to DETX have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of DETX 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 DETX may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.

Immunol. Methods 81 :31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimeric antibodies," such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Moπison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce DETX-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for DETX may also be generated. For example, such fragments include, but are not limited to, F(ab')₂ fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such lmmunoassays typically involve the measurement of complex formation between DETX and its specific antibody A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering DETX epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra) Various methods such as Scatchard analysis in conjunction with radioimmunoassay techmques may be used to assess the affimty of antibodies for DETX Affimty is expressed as an association constant, K,, which is defined as the molar concentration of DETX-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions The K_^ determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple DETX epitopes, represents the average affimty, or avidity, of the antibodies for DETX The K, determined tor a preparation of monoclonal antibodies, which are monospecific for a particular DETX epitope, represents a true measure ot affimty High-affimty antibody preparations with K_> ranging from about 10⁹ to 10¹² L/mole are preferred for use in lmmunoassays in which the DETX-antibody complex must withstand rigorous manipulations Low-affinity antibody preparations with K_j ranging trom about 10⁶ to 10⁷ L/mole are prefeπed for use in lmmunopuπfication and similar procedures which ultimately require dissociation of DETX, preferably in active form, from the antibody (Catty, D (1988) Antibodies, Volume I A Practical Approach, IRL Press, Washington DC, Liddell. J E and A Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY)

The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications For example, a polyclonal antibody preparation contaimng at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml. is generally employed in procedures requiring precipitation of DETX-antibody complexes Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available (See, e g , Catty, supra, and Coligan et al , supra )

In another embodiment of the invention, the polynucleotides encoding DETX, or any fragment or complement thereof, may be used for therapeutic purposes In one aspect, modifications of gene expression can be achieved by desigmng complementary sequences or antisense molecules (DNA. RNA. PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding DETX Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed trom various locations along the coding or control regions ot sequences encoding DETX (See, e g , Agrawal, S . ed (1996) Antisense Therapeutics. Humana Press Inc . Totawa NJ )

In therapeutic use. any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used Antisense sequences can be delivered lntracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g , Slater, J.E et al. (1998) J. Allergy Chn. Immunol 102(3).469-475; and Scanlon, K.J. et al. (1995) 9(13).1288- 1296 ) Antisense sequences can also be introduced lntracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (See, e.g., Miller, A.D. (1990) Blood 76.271 , Ausubel, supra; Uckert, W. and W Walther (1994) Pharmacol. Ther. 63(3).323-347 ) Other gene delivery mechanisms include liposome-deπved systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br Med. Bull. 51(1).217-225, Boado, R.J. et al. (1998) J Pharm Sci. 87(11).1308-1315; and Moms, M C et al. (1997) Nucleic Acids Res 25(14).2730-2736.)

In another embodiment of the invention, polynucleotides encoding DETX may be used for somatic or germline gene therapy. Gene therapy may be performed to (l) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-hnked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288.669-672), severe combined immunodeficiency syndrome associated with an inherited adenosme deaminase (ADA) deficiency

(Blaese, R.M et al. (1995) Science 270 475-480; Bordignon, C et al. (1995) Science 270 470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75.207-216, Crystal, R.G. et al. (1995) Hum. Gene Therapy 6.643-666; Crystal, R.G et al. (1995) Hum. Gene Therapy 6.667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R.G. (1995) Science 270.404-410; Verma, I.M. and Somia, N. (1997) Nature 389.239-242)), (n) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (in) express a protein which affords protection against intracellular parasites (e.g , against human retroviruses, such as human immunodeficiency virus (HIV) (Baltimore, D (1988) Nature 335.395-396, Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA 93.11395-11399), hepatitis B or C virus (HBV, HCV), fungal parasites, such as Candida albicans and Paracoccidioides brasihensis, and protozoan parasites such as Plasmodium falciparum and Trvpanosoma cruzi). In the case where a genetic deficiency in DETX expression or regulation causes disease, the expression of DETX from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency. In a further embodiment ot the invention, diseases or disorders caused by deficiencies in DETX are treated by constructing mammalian expression vectors encoding DETX and introducing these vectors by mechanical means into DETX-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (l) direct DNA microinjection into individual cells, (n) ballistic gold particle delivery, (in) liposome-mediated transtection. (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R A and W F Anderson (1993) Annu. Rev Biochem 62 191-217. Ivies, Z. (1997) Cell 91 :501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445- 450).

Expression vectors that may be effective for the expression of DETX include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,

PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). DETX may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 ; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding DETX from a normal individual.

Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to DETX expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding DETX under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus -acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61 :1647-1650: Bender, M.A. et al. (1987) J. Virol. 61 :1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806: Dull, T. et al. (1998) J. Virol. 72:8463-8471 ; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Patent Number 5,910,434 to Rigg ("Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant") discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4⁺ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71 :7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J. Virol. 71 :4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding DETX to cells which have one or more genetic abnormalities with respect to the expression of DETX. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544; and Verma, I.M. an N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.

In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding DETX to target cells which have one or more genetic abnormalities with respect to the expression of DETX. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing DETX to cells of the central nervous system, for which HS V has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye

Res.169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("Herpes simplex virus strains for gene transfer"), which is hereby incorporated by reference. U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161 , hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids contaimng different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techmques well known to those of ordinary skill in the art

In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding DETX to target cells The biology of the prototypic alphavirus,

Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H and K -J Li (1998) Curr Opin Biotech 9 464-469) During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e g , protease and polymerase) Similarly, inserting the coding sequence for DETX into the alphavirus genome in place of the capsid-coding region results in the production of a large number of DETX-coding RNAs and the synthesis of high levels of DETX in vector transduced cells While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S A et al. (1997) Virology 228 74-83) The wide host range of alphaviruses will allow the introduction of DETX into a variety of cell types The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art

Oligonucleotides derived from the transcription initiation site, e g , between about positions - 10 and +10 from the start site, may also be employed to inhibit gene expression Similarly, inhibition can be achieved using triple helix base-pairing methodology Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases. transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been descπbed in the literature (See, e g , Gee, J E et al (1994) in Huber, B E and B I. Carr. Molecular and Immunologic Approaches. Futura Publishing, Mt Kisco NY, pp. 163-177 ) A complementary sequence or antisense molecule may also be designed to block translation of mRNA b> preventing the transcript trom binding to πbosomes

Ribozymes. enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA The mechanism ot πbozyme action involves sequence-specific hybridization of the πbozyme molecule to complementary target RNA. followed by endonucleolytic cleavage For example engineered hammerhead motif πbozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding DETX

Specific πbozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for πbozyme cleavage sites, including the following sequences GUA, GUU, and GUC Once identified, short RNA sequences of between 15 and 20 πbonucleotides, corresponding to the region of the target gene contaimng 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 πbonuclease protection assays

Complementary πbonucleic acid molecules and πbozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules These include techmques tor chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding DETX Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6 Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues

RNA molecules may be modified to increase intracellular stability and half-life Possible modifications include, but are not limited to, the addition of flanking sequences at the 5^' and or 3' ends of the molecule, or the use of phosphorofhioate 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 ot nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of ademne, cytidine, guamne, thymine, and undine which are not as easily recognized by endogenous endonucleases An additional embodiment of the invention encompasses a method for screemng for a compound which is effective in altering expression of a polynucleotide encoding DETX Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcπption factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters ot polynucleotide expression Thus, in the treatment of disorders associated with increased DETX expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding DETX may be therapeutically useful, and in the treament ot disorders associated with decreased DETX expression or activity, a compound which specifically promotes expression of the polynucleotide encoding DETX may be therapeutically useful At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatoπally or randomly. A sample comprising a polynucleotide encoding DETX is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding DETX are assayed by any method commonly known in the art Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding DETX. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in alteπng the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomvces pombe gene expression system (Atkins, D. et al (1999) U.S. Patent No. 5.932,435, Arndt, CM. et al. (2000) Nucleic Acids Res. 28.E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys Res Commun. 268.8-13) A particular embodiment of the present invention involves screemng a combinatorial library of oligonucleotides (such as deoxyπbonucleotides. πbonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al (1997) U.S Patent No 5.686,242: Bruice, T W. et al. (2000) U.S Patent No. 6,022,691)

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (See, e g , Goldman, C.K et al (1997) Nat. Biotechnol. 15 462-466 )

Any of the therapeutic methods described above may be applied to any subject in need ot such therapy, including, tor example, mammals such as humans, dogs. cats, cows, horses, rabbits, and monkeys An additional embodiment of the invention relates to the administration ot a pharmaceutical composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient Excipients may include, for example, sugars, starches, celluloses, gums, and proteins Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington^'s Pharmaceutical Sciences (Maack Publishing, Easton PA) Such pharmaceutical compositions may consist of DETX, antibodies to DETX, and mimetics. agomsts, antagomsts, or inhibitors of DETX

The pharmaceutical compositions utilized in this invention may be admimstered by any number of routes including, but not limited to, oral, intravenous, intramuscular, lntra-arteπal, lntramedullary, lntrathecal, lntraventπcular, pulmonary, transdermal. subcutaneous, lntrapeπtoneal, intranasal, enteral, topical, sublingual, or rectal means

Pharmaceutical compositions for pulmonary administration may be prepared in liquid or dry powder form These compositions are generally aerosolized immediately prior to inhalation by the patient In the case of small molecules (e g traditional low molecular weight orgamc drugs), aerosol delivery of fast-acting formulations is well-known in the art In the case of macromolecules (e g larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e g , Patton, J S et al , U S Patent No 5,997,848) Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose The determination of an effective dose is well within the capability of those skilled in the art

Specialized forms of pharmaceutical compositions may be prepared for direct intracellular delivery of macromolecules comprising DETX or fragments thereof For example, liposome preparations contaimng a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule Alternatively, DETX or a fragment thereof may be joined to a short catiomc N-terminal portion from the HIV Tat-1 protein Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S R et al (1999) Science 285 1569-1572)

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e g , of neoplastic cells, or in ammal models such as mice, rats, rabbits, dogs, monkeys or pigs An ammal model may also be used to determine the appropriate concentration range and route of administration Such information can then be used to determine useful doses and routes for administration in humans

A therapeutically effective dose refers to that amount of active ingredient, for example DETX or fragments thereof, antibodies of DETX, and agomsts, antagomsts or inhibitors of DETX, which ameliorates the symptoms or condition Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental ammals. such as by calculating the ED,₀ (the dose therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD₅₀ ED,₀ ratio Pharmaceutical compositions which exhibit large therapeutic indices are preferred The data obtained from cell culture assays and ammal 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 ED_Λθ with little or no toxicity The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combιnatιon(s), reaction sensitivities, and response to therapy Long-acting pharmaceutical compositions may be admimstered 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 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc DIAGNOSTICS

In another embodiment, antibodies which specifically bind DETX may be used for the diagnosis of disorders characterized by expression of DETX, or in assays to momtor patients being treated with DETX or agomsts, antagomsts, or inhibitors of DETX Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics Diagnostic assays for DETX include methods which utilize the antibody and a label to detect DETX 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 ol a reporter molecule A wide variety of reporter molecules, several of which are described above, are known in the art and may be used

A variety ot protocols tor measuring DETX. including ELISAs. RIAs. and FACS, are known in the art and provide a basis tor diagnosing altered or abnormal levels of DETX expression Normal or standard values for DETX expression are established by combimng body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to DETX under conditions suitable for complex formation The amount of standard complex formation may be quantitated by various methods, such as photometric means Quantities of DETX 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 DETX may be used for diagnostic purposes The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of DETX may be coπelated with disease The diagnostic assay may be used to determine absence, presence, and excess expression of DETX, and to momtor regulation of DETX levels during therapeutic intervention

In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding DETX or closely related molecules may be used to identify nucleic acid sequences which encode DETX The specificity of the probe, whether it is made from a highly specific region, e.g , the 5^* regulatory region, or from a less specific region, e g , a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding DETX, allelic variants, or related sequences

Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the DETX encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO 3-4 or from genomic sequences including promoters, enhancers, and mtrons of the DETX gene Means for producing specific hybridization probes for DNAs encoding DETX include the cloning of polynucleotide sequences encoding DETX or DETX derivatives into vectors for the production of mRNA probes Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ^, 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 DETX may be used for the diagnosis of disorders associated with expression of DETX Examples of such disorders include, but are not limited to. an autoimmune/inflammatory disorder, such as acquired immunodeficiency syndrome (AIDS). Addison^'s disease, adult respiratory distress syndrome, allergies, ankylosing spondyhtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocπnopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes melhtus, emphysema, episodic lymphopema with lymphocytotoxins, erythroblastosis fetahs, erythema nodosum, atrophic gastritis, glomerulonephπtis, Goodpasture^' s syndrome, gout, Graves^' disease, Hashimoto's thyroiditis, hypereosinophiha, irritable bowel syndrome, multiple sclerosis, myasthema gravis, myocardial or pericardial inflammation, osteoarthπtis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter^'s syndrome, rheumatoid arthritis, scleroderma. Sjogren^'s syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopemc purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma, and a cell proliferative disorder, such as actimc keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuπa, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, perns, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus The polynucleotide sequences encoding DETX may be used in Southern or northern analysis, dot blot, or other membrane-based technologies, in PCR technologies, in dipstick, pin, and multiformat ELISA-hke assays, and in microarrays utilizing fluids or tissues from patients to detect altered DETX expression Such qualitative or quantitative methods are well known in the art

In a particular aspect, the nucleotide sequences encoding DETX may be useful in assays that detect the presence of associated disorders, particularly those mentioned above The nucleotide sequences encoding DETX may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the tormation of hybridization complexes After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding DETX 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 ammal studies, in clinical trials, or to momtor the treatment ot an individual patient

In order to provide a basis for the diagnosis of a disorder associated with expression of DETX. a normal or standard profile for expression is established This may be accomplished by combimng body fluids or cell extracts taken trom normal subjects, either ammal or human, with a sequence, or a fragment thereof, encoding DETX, under conditions suitable for hybridization or amplification Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder Deviation from standard values is used to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months

With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer

Additional diagnostic uses for oligonucleotides designed trom the sequences encoding DETX 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 DETX, or a fragment ot a polynucleotide complementary to the polynucleotide encoding DETX, and will be employed under optimized conditions for identification of a specific gene or condition Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences

In a particular aspect, oligonucleotide pπmers derived from the polynucleotide sequences encoding DETX may be used to detect single nucleotide polymorphisms (SNPs) SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods In SSCP. oligonucleotide primers derived from the polynucleotide sequences encoding DETX are used to amplify DNA using the polymerase chain reaction (PCR) The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single- stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels In fSCCP. the oligonucleotide pπmers are fluorescently labeled, which allows detection of the amphmers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence These computer- based methods filter out sequence variations due to laboratory preparation of DNA and sequencing eπors using statistical models and automated analyses of DNA sequence chromatograms In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc , San Diego CA) Methods which may also be used to quantify the expression of DETX include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e g , Melby, P.C. et al (1993) J Immunol Methods 159 235-244, Duplaa, C et al (1993) Anal Biochem 212 229-236 ) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometπc or coloπmetπc response gives rapid quantitation

In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray The microarray can be used in transcript imaging techniques which momtor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al , "Comparative Gene Transcript Analysis." U S. Patent No. 5,840.484, incorporated herein by reference The microarray may also be used to identify genetic variants, mutations, and polymorphisms This information may be used to determine gene function, to understand the genetic basis ot a disorder, to diagnose a disorder, to momtor progression/regression of disease as a function of gene expression, and to develop and momtor the activities of therapeutic agents in the treatment of disease In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile

In another embodiment, antibodies specific for DETX, or DETX or fragments thereof may be used as elements on a microarray The microarray may be used to momtor or measure protein-prote interactions, drug-target interactions, and gene expression profiles, as described above

A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type A transcript image represents the global pattern of gene expression by a particular tissue or cell type Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (See Seilhamer et al , "Comparative Gene Transcript Analysis.^" U S Patent Number 5,840,484, expressly incorporated by reference herein ) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity

Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies. or other biological samples The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line

Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E F. et al. (1999) Mol. Carcinog 24 153-159. Sterner, S. and N.L. Anderson (2000) Toxicol Lett 112-113 467-471 , expressly incorporated by reference herein) If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families Ideally, a genome-wide measurement of expression provides the highest quality signature Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data The normalization procedure is useful for comparison of expression data after treatment with different compounds While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released February 29. 2000, available at http.//www.mehs run gov/oc/news/toxchip htm ) Therefore, it is important and desirable in toxicological screemng using toxicant signatures to include all expressed gene sequences

In one embodiment, the toxicity of a test compound is assessed by treating a biological sample contaimng nucleic acids with the test compound Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcπpt levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g ,

Brennan, T.M et al. (1995) U S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci USA 93.10614-10619, Baldeschweiler et al. (1995) PCT application W095/251116, Shalon, D. et al (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94.2150- 2155, and Heller, M.J. et al. (1997) U.S. Patent No 5,605,662 ) Various types of microarrays are well known and thoroughly described in DNA Microarrays. A Practical Approach, M Schena, ed (1999) Oxford Umversity Press, London, hereby expressly incorporated by reference

In another embodiment of the invention, nucleic acid sequences encoding DETX may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries. (See, e.g , Harrington, J.J. et al. (1997) Nat Genet. 15.345-355, Pπce, CM. (1993) Blood Rev. 7.127-134, and Trask, B.J (1991) Trends Genet. 7.149-154 ) Once mapped, the nucleic acid sequences ot the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, e.g , Lander, E S. and D. Botstein (1986) Proc. Natl. Acad Sci. USA 83.7353-7357.)

Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e g , Heinz-Ulπch, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site Correlation between the location of the gene encoding DETX on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional clomng efforts

In situ hybridization of chromosomal preparations and physical mapping techmques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps Often the placement ot a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known This information is valuable to investigators searching for disease genes using positional clomng or other gene discovery techmques Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e g , ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation (See, e g , Gatti, R A et al (1988) Nature 336 577-580 ) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc , among normal, carrier, or affected individuals

In another embodiment of the invention, DETX, its catalytic or immunogemc fragments, or oligopeptides thereof can be used for screemng libraries of compounds in any of a variety of drug screemng techmques The fragment employed in such screemng may be free in solution, affixed to a solid support, borne on a cell surface, or located lntracellularly The formation of binding complexes between DETX and the agent being tested may be measured

Another technique for drug screemng provides for high throughput screemng of compounds having suitable binding affimty to the protein of interest (See, e g , Geysen, et al (1984) PCT application WO84/03564 ) In this method, large numbers of different small test compounds are synthesized on a solid substrate The test compounds are reacted with DETX, or fragments thereof, and washed Bound DETX is then detected by methods well known in the art Purified DETX can also be coated directly onto plates for use in the aforementioned drug screemng techmques 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 screemng assays in which neutralizing antibodies capable of binding DETX specifically compete with a test compound for binding DETX In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigemc determinants with DETX

In additional embodiments, the nucleotide sequences which encode DETX may be used in any molecular biology techniques that have yet to be developed, provided the new techmques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever

The disclosures ot all patents, applications and publications, mentioned above and below, in particular U S Ser No 60/142.678. are hereby expressly incorporated by reference

EXAMPLES I. Construction of cDNA Libraries

RNA was purchased from Clontech or isolated from tissues described in Table 4 Some tissues were homogenized and lysed in guanidinium lsothiocyanate, while others were homogemzed and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guamdine lsothiocyanate The resulting lysates were centπfuged over CsCl cushions or extracted with chloroform RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN) Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion. Austin TX)

In some cases, Stratagene was provided with RNA and constructed the coπesponding cDNA libraries Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, umts 5.1 -6.6 ) Reverse transcription was initiated using oligo d(T) or random primers Synthetic oligonucleotide adapters were hgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e g . PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA) Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue. XLl-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered trom host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis Plasmids were purified using at least one of the following a Magic or WIZARD Mimpreps DNA purification system (Promega), an AGTC Mimprep purification kit (Edge Biosystems, Gaithersburg MD). and QIAWELL 8 Plasmid. QIAWELL 8 Plus Plasmid. QIAWELL 8 Ultra Plasmid purification systems or the R E A.L PREP 96 plasmid purification kit trom QIAGEN Following precipitation, plasmids were resuspended in 0 1 ml ol

s distilled water and stored, with or without lyophilization, at 4°C

Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal Biochem 216 1-14) Host cell lysis and thermal cycling steps were carried out in a single reaction mixture Samples were processed and stored in 384- well plates, and the concentration of amplified plasmid DNA was quantified fluorometπcally using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland) III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems) Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics), the ABI PRISM 373 or 377 sequencing system (PE Biosystems) m conjunction with standard ABI protocols and base calling software, or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, u t 7.7) Some of the cDNA sequences were selected for extension using the techmques disclosed in Example VI

The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions. references, and threshold parameters The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences) Sequences were analyzed using MACDNASIS PRO software (Hitachi Software

Engineering, South San Francisco CA) and LASER GENE software (DNASTAR) Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEG ALIGN multisequence alignment program (DNASTAR). which also calculates the percent identity between aligned sequences The polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed. and were screened tor open reading frames using programs based on GeneMark, BLAST, and FASTA The full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM HMM is a probabilistic approach which analyzes consensus primary structures of gene families (See, e g , Eddy, S R (1996) Curr Opin Struct Biol 6 361-365 ) The programs described above for the assembly and analysis of full length polynucleotide and amino acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO 3-4 Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were descπbed in The Invention section above IV. Analysis of Polynucleotide Expression Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e g , Sambrook, supra, ch 7, Ausubel, 1995, supra, ch 4 and 16 )

Analogous computer techmques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics) 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

BLAST Score x Percent Identity 5 x minimum {length(Seq 1). length(Seq 2)}

The product score takes into account both the degree of similarity between two sequences and the length of the sequence match The product score is a normalized value between 0 and 100, and is calculated as follows the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences) The BLAST score is calculated by assigmng a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding DETX occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal. nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3. V. Chromosomal Mapping of DETX Encoding Polynucleotides The cDNA sequences which were used to assemble SEQ ID NO.3-4 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algoπthm. Sequences from these databases that matched SEQ ID NO.3-4 were assembled into clusters of contiguous and overlapping sequences using assembly algoπthms such as Phrap (Table 5). Radiation hybπd and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO , to that map location

The genetic map location of SEQ ID NO 4 is described in The Invention as ranges, or intervals, of human chromosomes The map position ot an interval, in centiMorgans, is measured relative to the terminus of the chromosome^'s p-arm. (The centiMorgan (cM) is a umt of measurement based on recombination trequencies between chromosomal markers On average, 1 cM is roughly equivalent to 1 megabase (Mb) ot DNA in humans, although this can vary widely due to hot and cold spots of recombination ) The cM distances are based on genetic markers mapped by Genethon which provide boundaries tor radiation hybrid markers whose sequences were included in each of the clusters.

VI. Extension of DETX Encoding Polynucleotides

The full length nucleic acid sequences of SEQ ID NO:3-4 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5 ' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides which would result in hairpin structures and primer -primer dimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.

High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂S0₄, and β-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1 : 94 °C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C.

The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence. The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E coh cells Transformed cells were selected on antibiotic-containing media, and individual colomes were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media

The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters Step 1 94°C, 3 m , Step 2 94°C, 15 sec, Step 3 60°C, 1 mm, Step 4 72°C 2 mm. Step 5 steps 2, 3, and 4 repeated 29 times, Step 6 72°C, 5 mm, Step 7 storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above Samples with low DNA recoveries were reamplified using the same conditions as described above Samples were diluted with 20% dimethysulfoxide (1 2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems) In like manner, the polynucleotide sequences of SEQ ID NO 3-4 are used to obtain 5 ' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library VII. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO 3-4 are employed to screen cDNAs, genomic DNAs, or mRNAs Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments Oligonucleotides are designed using state-of-the-art software such as OLIGO 4 06 software (National Biosciences) and labeled by combimng 50 pmol of each oligomer, 250 μCi of [γ-¹²P] adenosine tπphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA) The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech) An aliquot contaimng 10⁷ counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN) The DNA from each digest is tractionated on a 0 7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH) Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0 1 x saline sodium citrate and 0 5% sodium dodecyl sulfate Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared

VIII. Microarrays

The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e g , Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof The substrate in each of the aforementioned technologies should be umform and solid with a non-porous surface (Schena (1999), supra) Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface ot a substrate using thermal, UV, chemical, or mechanical bonding procedures A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (See, e.g , Schena, M et al (1995) Science 270 467-470. Shalon, D et al. (1996) Genome Res. 6.639-645, Marshall, A and J. Hodgson (1998) Nat Biotechnol 16 27-31 )

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR) The aπay elements are hybridized with polynucleotides in a biological sample The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection After hybridization, nonhybπdized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microaπay may be assessed In one embodiment, microarray preparation and usage is described in detail below Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guarudinium thiocyanate method and poly(A)⁺ RNA is purified using the ohgo-(dT) cellulose method Each poly(A)⁺ RNA sample is reverse transcribed using MMLV reverse-transcπptase, 0 05 pg/μl olιgo-(dT) pπmer (21mer), IX first strand buffer, 0 03 umts/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP. 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech) The reverse transcription reaction is performed in a 25 ml volume contaimng 200 ng poly(A)⁺ RNA with GEMB RIGHT kits (Incyte) Specific control poly(A)⁺ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA After incubation at 37 °C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2 5 ml of 0 5M sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc (CLONTECH), Palo Alto CA) and after combimng, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc , Holbrook NY) and resuspended in 14 μl 5X SSC/0 2% SDS Microarray Preparation

Sequences of the present invention are used to generate array elements Each array element is amplified from bacterial cells contaimng vectors with cloned cDNA inserts PCR amplification uses pπmers complementary to the vector sequences flanking the cDNA insert Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech)

Purified array elements are immobilized on polymer-coated glass slides Glass microscope slides (Coming) are cleaned by ultrasound in 0 1 % SDS and acetone, with extensive distilled water washes between and after treatments Glass slides are etched in 4% hydrofluoric acid (VWR

Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven

Array elements are applied to the coated glass substrate using a procedure descπbed in US Patent No. 5,807,522, incorporated herein by reference. 1 μl of the array element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus The apparatus then deposits about 5 nl of array element sample per slide

Microarrays are UV-crosshnked using a STRATALINKER UV-crosshnker (Stratagene) Microarrays are washed at room temperature once in 0 2% SDS and three times in distilled water Non-specific binding sites are blocked by incubation of microarrays in 0 2% casein in phosphate buffered saline (PBS) (Tropix, Inc , Bedford MA) for 30 minutes at 60 °C followed by washes in 0 2% SDS and distilled water as before Hybndization

Hybridization reactions contain 9 μl ot sample mixture consisting of 0 2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0 2% SDS hybridization buffer. The sample mixture is heated to 65 °C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm² covershp The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide The chamber is kept at 100% humidity internally by the addition of 140 μl of 5X SSC in a corner of the chamber. The chamber contaimng the arrays is incubated for about 6 5 hours at 60°C The arrays are washed for 10 mm at 45 °C in a first wash buffer (IX SSC 0.1 % SDS), three times for 10 minutes each at 45 °C in a second wash buffer (0.1X SSC), and dried Detection

Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent. Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY). The slide contaimng the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective The 1 8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers. In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially

Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photomcs Systems, Bridgewater NJ) corresponding to the two fluorophores Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1.100,000. When two samples from different sources (e g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital

(A/D) conversion board (Analog Devices, Inc , Norwood MA) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging trom blue (low signal) to red (high signal) The data is also analyzed quantitatively Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore' s emission spectrum

A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte) IX. Complementary Polynucleotides

Sequences complementary to the DETX-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring DETX Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4 06 software (National Biosciences) and the coding sequence of DETX To inhibit transcription, a complementary oligonucleotide is designed from the most umque 5 ^* sequence and used to prevent promoter binding to the coding sequence To inhibit translation, a complementary oligonucleotide is designed to prevent πbosomal binding to the DETX-encoding transcript X. Expression of DETX

Expression and purification of DETX is achieved using bacterial or virus-based expression systems For expression of DETX in bacteria, cDNA is subcloned into an appropriate vector contaimng an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element Recombinant vectors are transformed into suitable bacterial hosts, e g., BL21(DE3) Antibiotic resistant bacteria express DETX upon induction with lsopropyl beta-D- thiogalactopyranoside (IPTG). Expression of DETX in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica califorruca nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus The nonessential polyhedπn gene of baculovirus is replaced with cDNA encoding DETX by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates Viral lnfectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases Infection of the latter requires additional genetic modifications to baculovirus (See Engelhard, E K et al (1994) Proc Natl Acad Sci USA 91.3224-3227, Sandig, V et al (1996) Hum Gene Ther 7 1937-1945 )

In most expression systems, DETX is synthesized as a fusion protein with, e g , glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-Hιs, permitting rapid, single-step. affimty-based purification of recombinant fusion protein from crude cell lysates GST, a 26-kιlodalton enzyme from Schistosoma laporucum. enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech) Following purification, the GST moiety can be proteolytically cleaved from DETX at specifically engineered sites FLAG, an 8-amιno acid peptide, enables lmmunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak) 6- His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN) Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch 10 and 16) Purified DETX obtained by these methods can be used directly in the assays shown in Examples XI and XV

XL Demonstration of DETX Activity

For purposes of example, an assay measuring the β-galactosidase activity of a DETX molecule is described Varying amounts of DETX are incubated with 5-bromo-4-chloro-3-ιndoyl β-D- glycopyranoside solution (lmg ml 5-bromo-4-chloro-3-ιndoyl β-D-glycopyranoside, 2 mM magnesium chloride, 0 02% Nomdet P-40, 0.01%sodιum deoxycholate, 5 mM potassium femcyamde, and 5 mM potassium feπicyanate in PBS) at 37°C on a microtiter plate The sample^'s absorbance is measured spectrophotometπcally at 600 nm at hourly intervals, and is proportional to the activity of DETX in the sample (Palmer, C N A et al (1998) J Biol Chem 273(29) 18109-18116) XII. Functional Assays DETX function is assessed by expressing the sequences encoding DETX at physiologically elevated levels in mammalian cell culture systems cDNA is subcloned into a mammalian expression vector contaimng a strong promoter that drives high levels of cDNA expression. Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter 5-10 μg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation 1-2 μg of an additional plasmid contaimng sequences encoding a marker protein are co-transfected Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector Marker proteins of choice include, e g., Green Fluorescent Protein (GFP, Clontech), CD64, or a CD64-GFP fusion protein Flow cytometry (FCM), an automated, laser optics- based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death These events include changes in nuclear DNA content as measured by staimng 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 ot DNA synthesis as measured by decrease in bromodeoxyuπdine uptake, alterations expression ot cell surface and intracellular proteins as measured by reactivity with specific antibodies, and alterations in plasma membrane composition as measured by the binding of fiuorescem-conjugated Annexin V protein to the cell surface Methods in flow cytometry are discussed in Ormerod, M G (1994) Flow Cytometry, Oxford, New York NY

The influence of DETX on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding DETX and either CD64 or CD64-GFP CD64 and CD64- GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY) mRNA can be purified from the cells using methods well known by those of skill in the art Expression of mRNA encoding DETX and other genes of interest can be analyzed by northern analysis or microarray techmques XIII. Production of DETX Specific Antibodies

DETX substantially purified using polyacrylamide gel electrophoresis (PAGE, see, e g , Hamngton. M G (1990) Methods Enzymol 182 488-495), or other purification techmques, is used to immunize rabbits and to produce antibodies using standard protocols

Alternatively, the DETX amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high lmmunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (See, e.g , Ausubel, 1995, supra, ch 11.)

Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccimmide ester (MBS) to increase lmmunogenicity. (See, e g , Ausubel, 1995, supra ) Rabbits are immunized with the oligopeptide- KLH complex in complete Freund^'s adjuvant Resulting antisera are tested for antipeptide and anti-DETX activity by, for example, binding the peptide or DETX to a substrate, blocking with 1 % BS A, reacting with rabbit antisera. washing, and reacting with radio-iodinated goat anti-rabbit IgG XIV. Purification of Naturally Occurring DETX Using Specific Antibodies

Naturally occurring or recombinant DETX is substantially purified by lmmunoaffinity chromatography using antibodies specific for DETX An lmmunoaffinity column is constructed by covalently coupling anti-DETX antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) After the coupling, the resin is blocked and washed according to the manufacturer^'s instructions

Media contaimng DETX are passed over the lmmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of DETX (e g , high lomc strength buffers in the presence of detergent) The column is eluted under conditions that disrupt antibody/DETX 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 DETX is collected

XV. Identification of Molecules Which Interact with DETX

DETX, or biologically active fragments thereof, are labeled with ¹²T Bolton-Hunter reagent (See, e.g., Bolton A E. and W M Hunter (1973) Biochem. J 133 529-539 ) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled DETX, washed, and any wells with labeled DETX complex are assayed Data obtained using different concentrations of DETX are used to calculate values for the number, affimty, and association of DETX with the candidate molecules Alternatively, molecules interacting with DETX are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989, Nature 340 245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech)

DETX may also be used in the PATHCALLING process (CuraGen Corp , New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al (2000) U S Patent No. 6,057,101)

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 descπbed in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope ot the following claims

Table 1

Table 2

Table 3

Table 4

Table 5

Program Description Reference Parameter Threshold

ABI FACTURA A program that removes vectoi sequences and PE Biosystems, Foster City, CA masks ambiguous bases in nucleic acid sequences

ABI/PARACEL FDF A Fast Data Finder useful in comparing and PE Biosystems, Foster City, CA, Mismatch <50% annotating amino acid or nucleic acid sequences Paracel Inc , Pasadena, CA

ABI AutoAssemblei A program that assembles nucleic acid sequences PE Biosystems, Foster City CA

BLAST A Basic Local Alignment Search Tool useful in Altschul, S F et al (1990) J Mol Biol ESTs Probability value= 1 0E-8 sequence similarity seaich tor amino acid and 215 403 410, Altschul, S F et al (1997) or less nucleic acid sequences BLAST includes five Nucleic Acids Res 25 3389-3402 Full Length sequences Probabili functions blastp, blastn, blastx, tblastn, and tblastx value= 1 0E 10 or less

FASTA A Peaison and Lipman algorithm that searches foi Peaison, W R and D J Lipman (1988) Proc ESTs fasta E value=l 06E-6 similarity between a query sequence and a gioup of Natl Acad Sci USA 85 2444-2448, Peaison, Assembled ESTs fasta Identιty= sequences of the same type FASTA comprises as W R (1990) Methods Enzymol 183 63-98, 95% or greater and least five functions fasta, ttasta, fastx, tfastx, and and Smith, T F and M S Waterman (1981) Match length=200 bases or great ssearch Adv Appl Math 2 482-489 fastx E value=l 0E 8 or less

Full Length sequences fastx scores 100 or greater

BLIMPS A BLocks IMProved Seaicher that matches a Henikotf, S and J G Henikoff (1991) Nucleic Score=1000 or greater, sequence against those in BLOCKS, PRINTS, Acids Res 19 6565-6572, Henikoff, J G and Ratio of Score/Stiength = 0 75 or DOMO, PRODOM, and PFAM databases to search S Henikoff (1996) Methods Enzymol larger, and, if applicable, foi gene families, sequence homology, and structuial 266 88-105, and Attwood, T K et al (1997) J Probability value= 1 0E-3 or less fingerprint regions Chem Inf Co put Sci 37 417-424

HMMER An algoπthm toi searching a queiy sequence against Kiogh, A et al (1994) J Mol Biol Score=10-50 bits tor PFAM hits, hidden Markov model (HMM) based databases of 235 1501-1531 , Sonnhammer, E L L et al depending on individual protein protein family consensus sequences, such as PFAM (1988) Nucleic Acids Res 26 320 322 families

Table 5 (cont.)

Program Description Reference Parameter Threshold

ProfileScan An algorithm that seaiches foi structural and Gnbskov, M et al (1988) CABIOS 4 61 66, Normalized quality score≥GCG sequence motifs in piotein sequences that match Gnbskov, M et al (1989) Methods Enzymol specified "HIGH" value for that sequence patterns defined in Prosite 183 146-159, Bairoch, A et al (1997) particular Prosite motif Nucleic Acids Res 25 217-221 Generally, score=l 4-2 1

Phred A base-calling algorithm that examines automated Ewing, B et al (1998) Genome Res sequencer traces with high sensitivity and 8 175-185, Ewιng, B and P Green probability (1998) Genome Res 8 186 194

Phrap A Phils Revised Assembly Program including Smith, T F and M S Waterman (1981) Ad v Score= 120 or greater, SWAT and CrossMatch, programs based on Appl Math 2 482 489, Smith, T F and M S Match length= 56 or greater efficient implementation of the Smith Waterman Waterman (1981) J Mol Biol 147 195-197, algonthm, useful in searching sequence homology and Green, P , University of Washington, and assembling DNA sequences Seattle, WA

Consed A giaphical tool foi viewing and editing Phiap Goidon, D et al (1998) Genome assemblies Res 8 195 202

SPScan A weight matrix analysis program that scans protein Nielson, H et al (1997) Protein Engineering Score=3 5 or greater sequences for the piesence of secietory signal 10 1-6, Claveπe, J M and S Audic (1997) peptides CABIOS 12 431-439

Motifs A program that searches amino acid sequences for Bairoch, A et al (1997) Nucleic Acids Res patterns that matched those defined in Prosite 25 217-221 , Wisconsin Package Program Manual, version 9, page M51-59, Genetics Computei Group, Madison, WI

Claims

What is claimed is

1 An isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consishng of SEQ ID NO 1-2, h) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consishng of SEQ ID NO 1-2, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-2

2 An isolated polypeptide of claim 1 selected from the group consishng of SEQ ID NO 1-2

3 An isolated polynucleotide encoding a polypeptide of claim 1

4 An isolated polynucleotide encoding a polypeptide of claim 2

5 An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID NO 3-4

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

7 A cell transformed with a recombinant polynucleotide of claim 6

8 A transgemc orgamsm comprising a recombinant polynucleohde of claim 6

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

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

11 An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consishng of SEQ ID NO 3-4, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consishng of SEQ ID NO.3-4, c) a polynucleohde sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d)

12 An isolated polynucleohde compπsing at least 60 contiguous nucleotides of a polynucleotide of claim 11

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

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

15 A method for detechng a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising a) amplifying said target polynucleohde or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof

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

17 A pharmaceutical composition of claim 16, wherein the polypephde comprises an amino acid sequence selected from the group consisting of SEQ ID NO 1-2.

18 A method for treating a disease or condition associated with decreased expression of funchonal DETX, comprising admimsteπng to a patient in need of such treatment the pharmaceuhcal composihon of claim 16

19 A method for screemng a compound for effectiveness as an agomst of a polypeptide of claim 1, the method comprising a) exposing a sample comprising a polypephde of claim 1 to a compound, and b) detecting agomst activity in the sample

20 A pharmaceutical composihon comprising an agomst compound identified by a method of claim 19 and a pharmaceutically acceptable excipient

21 A method for treating a disease or condition associated with decreased expression of funchonal DETX, compπsing admimsteπng to a pahent in need of such treatment a pharmaceuhcal composihon of claim 20

22 A method for screemng a compound for effectiveness as an antagomst of a polypephde of claim 1, the method comprising a) exposing a sample compπsing a polypephde of claim 1 to a compound, and b) detecting antagomst activity in the sample

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

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

25 A method of screemng for a compound that specifically binds to the polypeptide of claim 1 , said method comprising the steps of a) combimng the polypephde ot claim 1 with at least one test compound under suitable conditions, and WO 01/04305 PCT/USOO/l 8509 b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypephde of claim 1

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

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

28 A method for assessing toxicity of a test compound, said method comprising a) treating a biological sample contaimng nucleic acids with the test compound, h) hybridizing the nucleic acids of the heated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 11 under conditions whereby a specific hybridizahon complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide compπsing a polynucleotide sequence of a polynucleohde of claim

11 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound