JP2003528567A - Human hydrolase protein - Google Patents

Abstract

(57) Abstract The present invention provides human hydrolase proteins (HYDRLs) and polynucleotides that identify and encode HYDRLs. The invention also provides expression vectors and host cells, antibodies, agonists, antagonists. Furthermore, the present invention provides a method for diagnosing, treating, or preventing a disease associated with the expression of HYDRL.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a nucleic acid sequence and an amino acid sequence of a hydrolase protein, and abnormal cell proliferation, autoimmune disorder / inflammatory disease, neuropathy, and the like using these sequences.
The present invention relates to diagnosis, treatment and prevention of renal disorders, adrenal insufficiency, and genetic disorders.

BACKGROUND OF THE INVENTION Hydrolysis is the disruption of covalent bonds within a substrate by the introduction of water molecules. The hydrolysis reaction is involved in the nucleophilic action of the oxygen atom of the water molecule in the target binding on the substrate. Water molecules split across target binding, breaking target binding and producing two product molecules. Hydrolases are used for cell signaling, cell proliferation,
It is involved in reactions essential for functions such as inflammation, apoptosis, differentiation and excretion. Hydrolases are involved in key steps in disease processes such as those involved in these functions. Hydrolases are hydrolases and are grouped according to their substrate properties into classes including aminohydrolases, phospholipases, carboxylesterases, phosphodiesterases, glycosidases, glyoxalase, sulfatases, phosphohydrolases, serine hydrolases, lysozymes. Let's do it.

NG, NG-dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes the endogenous nitric oxide synthase (NOS) inhibitor, NG-monomethylarginine, and NG, NG-dimethyl-L-arginine into L-citrulline. It is an enzyme that decomposes. By inhibiting DDAH, intracellular concentrations of NOS inhibitors can be increased to levels sufficient to inhibit NOS. Therefore, inhibiting DDAH is a means of inhibiting NOS,
Changes in H activity can play a role in abnormal physiological mutations in nitric oxide production (MacAllister, RJ et al. (1996) Br. J. Pharmacol.
119: 1533-1540). DDAH is found in neurons that exhibit cytoskeletal abnormalities and oxidative stress in Alzheimer's disease. Neurons did not show DDAH in age-matched management. This suggests that oxidative stress and nitric oxide-mediated events play a role in the pathogenesis of Alzheimer's disease (Smith, MA et al. (1998) Free Rad. Biol. Med. 25:89.
8-902).

Phosphodiesterase is one of two ester bonds in phosphodiester.
It catalyzes the hydrolysis of one ester bond. Therefore, phosphodiesterases are very important in various cellular processes. Phosphodiesterases include DNA and RNA endonucleases and exonucleases that are essential for cell growth and replication as well as protein synthesis.

Pancreatic lipase and co-lipase form a complex. This complex is likely to play a role in digesting dietary fat by converting insoluble long-chain triacylglycerols into more polar molecules that can cross the brush border of intestinal cells. . Complement lipase binds to the C-terminal domain of lipase. This interaction is involved in the formation of the ion pair formed in solution between the glutamate residue of co-lipase and the lysine residue of lipase. These residues are strictly converted between species (Ayvazian, L. et al. (1998) .J. Biol. Chem. 273: 33604-336.
09). Complement lipase appears to overcome the inhibitory effect of bile salts on pancreatic lipase (OMIM 246600, April 28, 1999).

[0006] Carboxylesterase is a protein that hydrolyzes carboxylic acid esters and is classified into three categories, A, B, and C. Most B-type carboxylesterases are evolutionarily related and are thought to constitute a protein family. The B-type carboxylesterase family of proteins includes vertebrate acetylcholinesterase, mammalian liver microsomal carboxylesterase, mammalian bile salt active lipase, duck fatty acyl-CoA hydrolase and the like. Some members of this protein family include domains that are not catalytically active but evolutionarily related to other B-type carboxylesterases, such as the thyroglobulin and Drosophila protein neuronal actin. The active site of carboxylesterase contains three residues of serine, an ester of glutamic acid or aspartic acid, and histidine. The sequence surrounding this catalytic site is well conserved and can be used as a signature pattern (see PROSITE, www.expasy.ch/cgi-bin/get-prodoc-entry, May 1999 11).
Documentation input as of day PDOC00112).

Acyl-CoA thioesterase is another member of the carboxylesterase family (Alexson, SE et al. (1993) Eur. J. Biochem. 214: 719-727).
. There is evidence suggesting that acyl-CoA thioesterases have a regulatory role in steroidogenic tissues (Finkielstein, C. et al. (1998) Eur. .J
Biochem. 256: 60-66).

Phospholipase A ₂ inhibitors are associated with human multileucine α ₂ glycoprotein and 3
It has been identified as having 3% sequence homology (Okumura, K. et al. (1998) J. B.
iol. Chem. 273: 19469-19475). The multileucine repeat (LRR) consensus sequence is
It is also found in the primary structure of many proteins, including proteins that are involved in biologically important processes such as hormone receptors, enzymes, enzyme inhibitors, proteins for cell adhesion, ribosome binding proteins and the like. All protein domains containing LRR are thought to be involved in protein-protein interactions.

The glyoxalase system consists of glyoxalase I, which catalyzes the formation of SD-lactoylglutathione from methiglyoxal, a by-product of triosephosphate energy metabolism, and SD-lactoylglutathione. Is hydrolyzed to D-lactic acid to reduce glutathione, and consists of glyoxalase II. Metglioxal levels are elevated during hyperglycemia, presumably by increasing triose phosphate energy metabolism. Elevated levels of glyoxalase II activity are found in human non-insulin dependent diabetes and rat models of the same. The glyoxalase system is involved in the detoxification of bacterial toxins and the regulation of cell growth and microtubule assembly. Elevated levels of SD-lactoyl glutathione are substrates for glyoxalase II and reduce growth arrest and toxicity in HL60 cells. Thus, the glyoxalase system and especially glyoxalase II
It may be associated with cell proliferation and autoimmune diseases such as diabetes.

[0010] The α / β hydrolase protein fold is common to several hydrolases of differing phylogenetic origin and catalytic function. Enzymes associated with the α / β hydrolase fold have a common nuclear structure consisting of eight β-sheets linked by α-helices. The most conserved structural function of this fold is the loop of the catalytic triad of nucleophilic histidine acids. The nucleophilic and acid loops accommodate more than one type of amino acid, but histidine in the catalytic triad is completely conserved (Olli
s, DL et al. (1992) Protein Eng. 5: 197-211).

Sulfatase is a member of a highly conserved gene family that shares extensive sequence homology and a high degree of structural similarity. Sulfatase catalyzes the cleavage of sulfates. To exert this function, sulfatase undergoes a unique post-translational mutation in the endoplasmic reticulum, which is involved in the oxidation of conserved cysteine residues. What is referred to as multiple sulfatase deficiency in human disease is due to a defect in this posttranslational mutational stage leading to inactive sulfatase (Recksiek, M. et al. (1998) J. Biol. Chem. .
273: 6096-6103).

Phosphohydrolase is an enzyme that hydrolyzes phosphate esters. Some phosphohydrolases contain a mutT domain signature sequence. mutT is DNA
GO responsible for removing oxidatively damaged forms of guanine from
It is a protein involved in the system. The region of about 40 amino acid residues found at the N-terminal of mutT is also found in other proteins including phosphohydrolase (
Documentation input for PROSITE at www.expasy.ch/cgi-bin/get-prodoc-entry as of April 27, 1999 PDOC00695).

Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides, such as compounds containing one or more sugars. Mammalian β-galactosidase removes terminal galactose from gangliosides, glycoproteins, and glycosaminoglycans. β-galactosidase belongs to family 35, which is classified as a glycosyl hydrolase. Deficiency of this enzyme is associated with GM1-gangliosidosis, a genetic disease known as type B Morquio disease (www.expasy.ch/cgi-
Documentation input PDOC00910 of PROSITE in bin / get-prodoc-entry as of May 12, 1999).

Serine hydrolases are a functional class of hydrolases that contain a serine residue in their active site. The biological role of this class is different because it includes proteinases, esterases, and lipases that hydrolyze various substrates. Proteins in this superfamily can be further grouped into subfamilies based on substrate properties or amino acid similarities (
Puente, XS and C. Lopez-Ont (1995) J. Biol. Chem. 270: 12926-12932).
One member of the serine hydrolase superfamily is Kraken, a Drosophila gene isolated from a Drosophila embryo cDNA library. Krakens belong to a subfamily with members that catalyze the resolution of substrates with groups containing carbonyl groups (Chan, E.
(1998) Gene 222: 195-201).

The lysozyme c superfamily consists of normal lysozyme c, calcium-binding lysozyme c, and alpha lactalbumin (Prager, EM and P. Jolles.
(1996) EXS 75: 9-31). 35 proteins of the lysozyme c superfamily
It has ~ 40% sequence homology and a common three-dimensional fold, but may have different functions. Lysozyme c is ubiquitous in various tissues and secretions and can lyse the cell wall of certain bacteria (McKenzie, HA (1996) EXS 75: 365-409.
). Alpha-lactalbumin is a metalloprotein that binds calcium and is involved in lactose synthesis (Iyer, LK and PK Qasba (1999) Protein).
Eng. 12: 129-139). Alpha-lactalbumin is present in mammalian milk and colostrum (McKenzie, supra).

Lysozyme catalyzes the hydrolysis of β (1-4) glycosidic bonds between certain mucopolysaccharides of the bacterial cell wall, especially N-acetylmuramic acid and N-acetylglucosamine, Causes bacterial lysis. Lysozyme occurs in a variety of organisms including viruses, birds and mammals. In humans, lysozyme is found in the spleen, liver, kidneys, white blood cells, plasma, saliva, milk, tears, and cartilage (Online Mendeli
an Inheritance in Man (OMIM) # 153450 Lysozyme, Weaver, LH et al. (1985) J.
Mol. Blot. 184: 739-741). Lysozyme c functions as a digestive enzyme that releases proteins from ingested bacterial cells in ruminants and may exert similar functions in human neonates (Braun, OH et al. (1995) Klin. Pediatr. 207). : 4-7
).

Two types of lysozyme are well known, a chicken type and a goose type, and they are originally isolated from chicken egg white and goose egg white, respectively. Chicken lysozyme and goose lysozyme have similar three-dimensional structures but different amino acid sequences (Nakano, T. and T. Graf (1991) Biochim. Biophys. Acta 1090: 2).
73-276). Both chicken-type and goose-type lysozymes are found in the neutral-affinity granulocytes of chickens (metachromatic), but only chicken-type lysozyme is found in the egg white of chickens. Usually, chicken lysozyme mRNA is found not only in cells of bone marrow, spleen, sac, and oviduct, but also in adherent monocytes and macrophages, and in nonadherent promyelocytes and granulocytes. Goose-type lysozyme mRNA is found in non-adherent cells of bone marrow and lung. Some isozymes are found in rabbits, including leukocyte, gastrointestinal, and possibly lymphoid (OMIM # 153450, supra, Nakano & Graf, supra, and GenBank GI 1310929). The human lysozyme gene, which encodes a protein similar to chicken lysozyme, has been cloned and replicated (Yoshimura, K.
(1988) Biochem. Biophys. Res. Commun. 150: 794-801). A consensus motif that features regularly spaced cysteine residues is derived from lysozyme C enzymes of diverse species (Swiss Institute of Bloinformatics http: // exp.
Prosite documentation input provided by asy.hcuge.ch P500128). Lysozyme C is 40% identical in amino acid sequence to α-lactalbumin.

Lysozyme is associated with several diseases. Lysozyme urine includes diabetic neuropathy (Shima, M. et al. (1986) Clin. Chem. 32: 1818-1822), epidemic neuropathy (Bruckner, I et al. (1978) Med. Interne. 16: 117-125), Observed in urinary tract dysfunction (Heidegger, H. (1990) Minerva Ginecol. 42: 243-250) and acute monocytic leukemia (Shaw, MT (1978) Am. J. Hematol. 4: 97-103). It Nakano (
The above) suggests the role of lysozyme in the host defense system.
Older rabbits lacking hereditary lysozyme are more susceptible to infections such as subcutaneous abscesses (OMIM # 153450, supra). Mutations in the human lysozyme gene cause genetic phylogenetic amyloidosis. Genetic systematic amyloidosis is a rare autosomal dominant genetic disease in which amyloid deposits in internal organs including kidney, adrenal gland, spleen, and liver, and usually causes fatal symptoms by the age of 50 years. Amyloid deposits include various forms of lysozyme. Renal amyloidosis is associated with the most common and potentially the most severe organ morphology (Pepys, MB et al. (1993) Nature
362: 553-557, OMIM # 105200 Familial Visceral Amyloidosis, Cotran, RS
Et al (1994) Robbins Pathologic Basis of Disease , WB Saunders Company, Ph
iladelphia PA, pp. 231-238). Increased levels of lysozyme and lactate are observed in the cerebrospinal fluid of patients with bacterial meningitis. (Ponka, A. et al. (1983) In
infection 11: 129-131). Acute monocytic leukemia is characterized by massive lysozyme urine (Den Tandt, WR (1988) Int. J. Biochem. 20: 713-719).

The discovery of new hydrolase proteins and polynucleotides encoding them has led to the diagnosis, prevention and treatment of cell proliferative disorders, autoimmune / inflammatory disorders, neurological disorders, renal illness, adrenal disorders, and hereditary disorders. To meet the demands of those skilled in the art by providing new compounds such as

With the discovery of novel hydrolase proteins and polynucleotides encoding them, diagnosis and treatment of cell proliferative disorders, autoimmune disorders / inflammatory diseases, neuropathy, renal disorders, adrenal insufficiency, and genetic disorders , Can provide novel compositions useful in prevention to meet the needs of the art.

(Summary of the Invention) [0021] The present invention refers to "HYDRL" as a general term, and individually "HYDRL-1", "HYDRL-2", and "HYDRL".
-3 "," HYDRL-4 "," HYDRL-5 "," HYDRL-6 "," HYDRL-7 "," HYDRL-8 ",
"HYDRL-9", "HYDRL-10", "HYDRL-11", "HYDRL-12", "HYDRL-13", "H
Provided are hydrolase proteins that are substantially purified polypeptides designated YDRL-14 "," HYDRL-15 ", and" HYDRL-16 ". In one embodiment of the invention there is provided a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. The present invention also provides SEQ ID NO:
There is provided a polypeptide comprising an amino acid sequence in which one or more conserved amino acids of an amino acid sequence selected from the group consisting of 1-16 are substituted.

The present invention further provides a substantially purified variant having 90% or more amino acid identity with at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. provide. The invention also provides an isolated and substantially purified polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. The present invention also provides SEQ ID
An isolated and purified polynucleotide variant sequence having 90% or more polynucleotide sequence identity with a polynucleotide encoding a polypeptide containing an amino acid sequence selected from the group consisting of NO: 1-16 and fragments thereof. provide.

Furthermore, the present invention provides an isolated hybridisation under stringent conditions to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. To provide a purified polynucleotide.
The present invention also provides an isolated and purified polynucleotide containing a sequence complementary to a polynucleotide encoding a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. provide.

The present invention is also a method of detecting a polynucleotide in a sample containing nucleic acids, the method comprising: (a) hybridizing a complement of a polynucleotide sequence with a polynucleotide of at least one sample, and performing hybridization. A detection method comprising the step of forming a complex and (b) the step of detecting the hybridization complex, wherein the presence of the hybridization complex and the presence of the polynucleotide in the sample are correlated. . In one embodiment of the invention, the step of amplifying the polynucleotide prior to hybridization is included.

The present invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32 and fragments thereof. The present invention also provides an isolated and purified polynucleotide variant sequence having 90% or more polynucleotide sequence identity with a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32 and fragments thereof. provide. Further, the present invention provides SEQ
There is provided an isolated and purified polynucleotide having a sequence complementary to a polynucleotide comprising a polynucleotide sequence selected from the group consisting of ID NO: 17-32 and fragments thereof.

The invention further provides an expression vector comprising at least one fragment of a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16. In another embodiment, the expression vector is contained within a host cell.

The present invention also provides a method for producing a polypeptide, which comprises (a) culturing a host cell containing an expression vector having the polynucleotide of the present invention under conditions suitable for expression of the polypeptide. , (B) recovering the polypeptide from the medium of the host cell.

The present invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof, together with a suitable pharmaceutical carrier. provide.

The present invention further provides a purified antibody that binds to a polypeptide selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. The invention also provides purified agonists and antagonists of the polypeptide.

The present invention also provides a method of treating or preventing a disease associated with decreased expression or activity of HYDRL, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. There is provided a method of treatment or prophylaxis comprising administering to a patient an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide together with a suitable pharmaceutical carrier.

The present invention also provides a method of treating or preventing a disease associated with increased HYDRL expression or activity, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-16 and fragments thereof. Methods of treatment or prophylaxis are provided that include administering to the patient an effective amount of a polypeptide antagonist.

Regarding the Description of the Present Invention Before explaining the protein and nucleic acid sequences and methods of the present invention, the present invention is not limited to the specific devices and materials and methods disclosed herein, and its embodiments are modified. Please understand what you can do. Also, the terminology used herein is for the purpose of describing particular embodiments only and is limited only by the scope of the following claims,
It should also be understood that it is not intended to limit the scope of the invention.

In the present specification and claims, “a”, “the (the same),” etc. represent the singular.
Note that includes plural forms unless the context clearly indicates otherwise. Thus, for example, “a host cell” includes a plurality of host cells, “antibody” includes a plurality of antibodies, and equivalents well known to those skilled in the art.

All technical and scientific terms used herein, unless defined otherwise,
It has the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.
Although any apparatus and materials and methods similar or equivalent to those described herein can be used in the practice and testing of the present invention, suitable apparatus and materials and methods are described herein.
All references mentioned herein are cited to describe and disclose the cell lines, protocols, reagents, vectors described in the references that may be used in connection with the present invention. The mere fact that the conventional invention is cited does not mean that the novelty of the present invention is impaired.

Definitions The term "HYDRL" is substantially purified obtained from all species including natural, synthetic, semi-synthetic or recombinant (especially mammals including bovine, ovine, porcine, murine, equine and human). HYDRL amino acid sequence.

The term “agonist” refers to a molecule that enhances or mimics the biological activity of HYDRL. This agonist either interacts directly with HYDRL or acts with components of the biological pathway in which HYDRL is involved to regulate the activity of HYDRL such as proteins, nucleic acids, carbohydrates, small molecules, any other compound or The composition may be included.

The term “allelic variant sequence” refers to another form of the gene that encodes HYDRL. The allelic variant sequence results from at least one mutation in the nucleic acid sequence,
It may be NA or a mutated polypeptide, and its structure and function may or may not change. Some genes include those in which no naturally-occurring allelic variant is present, or one gene or many genes are present. Generally, mutations that give rise to allelic variant sequences are due to natural deletions, additions, or substitutions of nucleotides. Each of these mutations, either alone or concurrently with other mutations, occurs one or more times within a given sequence.

“Mutant” nucleic acid sequences encoding HYDRL include various nucleotide deletions, insertions,
Alternatively, it refers to a polypeptide which, even if substitutions occur, has the same polypeptide as HYDRL or which has at least one functional property of HYDRL. This definition includes improper or unexpected hybridization of a HYDRL-encoding polynucleotide sequence with an allelic variant at a position other than the normal chromosomal locus, as well as specific oligonucleotide probes of the HYDRL-encoding polynucleotide. Includes polymorphisms that are easily or difficult to detect using. The encoded protein can also be mutated,
Deletion, insertion of amino acid residues that cause silent changes and are functionally equivalent to HYDRL,
Alternatively, it may include substitution. Intentional amino acid substitution is a biological or immunological HY
To the extent that the activity of DRL is retained, the polarity, charge, solubility, hydrophobicity, hydrophilicity of the residue,
And / or based on similarities for amphipathicity. For example, negatively charged amino acids can include aspartic acid and glutamic acid, and positively charged amino acids can include lysine and arginine. Amino acids with similar hydrophilicity values and having polar uncharged side chains can include asparagine, glutamine, serine, threonine. Amino acids with similar hydrophilicity values and having uncharged side chains can include leucine, isoleucine, valine, glycine, alanine, phenylalanine and tyrosine.

The terms “amino acid” and “amino acid sequence” refer to oligopeptide, peptide, polypeptide, protein sequences, or any fragment thereof, including naturally occurring and synthetic molecules. When the "amino acid sequence" is a naturally occurring protein molecule, "
Amino acid sequence "and like terms are not intended to limit the amino acid sequence to the complete, unaltered amino acid sequence associated with the protein molecules described.

The term “amplification” refers to making copies of nucleic acid sequences. Amplification is generally performed by the polymerase chain reaction (PCR) technique well known in the art.

The term “antagonist” is a molecule that inhibits or attenuates the biological activity of HYDRL. Antagonists are antibodies, nucleic acids, carbohydrates, that interact directly with HYDRL or act with components of the biological pathways in which HYDRL is involved to regulate HYDRL activity.
It may include proteins such as small molecules, any other compound or composition.

The term “antibody” refers to intact molecules such as Fab and F (ab ′) ₂ and fragments thereof, Fv fragments, which are capable of binding antigenic determinants. Antibodies that bind HYDRL polypeptides can be produced using intact molecules, or fragments thereof, containing small peptides that immunize the antibody. The polypeptide or oligopeptide used to immunize an animal (eg, mouse, rat, or rabbit) can be synthesized from the translation of RNA or chemically and is optionally conjugated with a carrier protein. It is also possible. Commonly used carriers that are chemically linked to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemonean (KLH). The conjugated peptide is then used to immunize the animal.

The term “antigenic determinant” refers to the region (ie, epitope) of a molecule that makes contact with a particular antibody.
Refers to. When a protein or a fragment of a protein is used to immunize a host animal, various regions of this protein are antibodies that specifically bind to an antigenic determinant (a specific region on the protein or a three-dimensional structure). Can induce the production of The antigenic determinant may compete with the intact antigen (ie, the immunogen used to elicit an immune response) to bind the antibody.

The term “antisense” refers to any composition containing a nucleic acid sequence complementary to the sense strand of a particular nucleic acid sequence. Antisense molecules can be produced by any method including synthesis or transcription. Once introduced into a cell, complementary nucleotides combine with the natural sequences made by the cell to form a duplex, which inhibits transcription or translation.
The expression "minus (-)" refers to the antisense strand, and the expression "plus (+)" refers to the sense strand.

The term “biological activity” refers to a protein having the structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, the term "immunologically active" means that a natural or recombinant HYDRL, synthetic HYDRL or any oligopeptide thereof induces a specific immune response in a suitable animal or cell to induce a specific antibody. Refers to the ability to combine with.

The terms “complementary” and “complementarity” refer to the natural binding of polynucleotides to form base pairs. For example, the sequence "5'AG-T3 '" binds to the complementary sequence "3'TC-A5'". The complementarity between two single-stranded molecules may be partial, where only some of the nucleic acids bind, or there may be complete complementarity between the single strands resulting in perfect complementarity. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on binding between nucleic acid strands, as well as in the design or use of peptide nucleic acid (PNA) molecules.

“Composition comprising a given polynucleotide sequence” or “composition comprising a given amino acid sequence” refers broadly to any composition that comprises a given nucleotide or amino acid sequence. The composition may include a dry formulation or an aqueous solution.
A composition comprising a polynucleotide sequence encoding HYDRL or a fragment of HYDRL,
It can be used as a hybridization probe. This probe can be stored in a freeze-dried state and can be bound to a stabilizer such as sugar.
In hybridization, the probe is developed in an aqueous solution containing salts (eg, NaCl) and detergents (eg, SDS: sodium dodecyl sulfate) and other substances (eg, Denhardt's solution, dried milk, salmon sperm DNA, etc.). obtain.

A “consensus sequence” is a nucleic acid sequence that has been sequenced to separate unwanted bases and is 5'and / or 3'using XL-PCR ^™ (Perkin Elmer, Norwalk, CT). Sequenced nucleic acid sequence or GELVI
EW fragment construction system (GCG, Madison, WI), etc. One or more Insight clones using computer programs for fragment construction,
And, in some cases, refers to nucleic acid sequences constructed by duplication of one or more public domain ESTs. Some consensus sequences are constructed by both extension and overlap.

The term “conservative amino acid substitution” refers to a substitution that does not significantly alter the properties of the original protein. That is, the substitution does not significantly change the structure or function of the protein,
The structure of the protein, in particular its function, is preserved. The following shows conservative amino acid substitutions in which the original amino acid of one protein is replaced with another amino acid. Original residue Conservative substitution Ala Gly, Set Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln , Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile . Leu, Thr In general, in the case of conservative amino acid substitutions, a) the backbone structure of the polypeptide in the substituted region, eg β sheet or α helix conformation, b) the charge of the molecule at the substituted site or Hydrophobic and / or c) most of the side chains are retained.

The term “deletion” refers to an amino acid sequence change that lacks one or more amino acid residues, or a nucleic acid sequence change that lacks one or more nucleotides.

The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of polynucleotide sequences include, for example, replacement of hydrogen with an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide has at least one biological or immunological function of a natural molecule (unmodified molecule).
Encodes a polypeptide that maintains one. A derivative polypeptide is a polypeptide modified by glycosylation, polyethylene glycolation, or any similar process that maintains at least one of the biological or immunological functions of the original polypeptide. That is.

The term “fragment” refers to a unique portion of HYDRL or a polynucleotide encoding HYDRL that is identical to its parent sequence but shorter than that sequence. The maximum length of a "fragment" is 1 nucleotide / from the parent sequence.
It is the length less amino acid residues. For example, a fragment contains 5 to 1000 consecutive nucleotides or amino acid residues. Probes, primers, antigens, therapeutic molecules, or fragments used for other purposes should be at least 5, 10, 15
, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250
Alternatively, the length is 500 consecutive nucleotides or amino acid residues. Fragments may also be preferentially selected from particular regions of the molecule. For example, a polypeptide fragment may comprise a predetermined length of contiguous amino acids selected from the first 250 or 500 amino acids shown in the given sequence (or the first 25% or 50% of the polypeptide). . These lengths are examples, and any length described in the specification including the sequence listing and the tables and drawings can be included in the embodiments of the present invention.

Certain fragments of SEQ ID NO: 17-32 include, for example, a region of unique polynucleotide sequence that uniquely identifies SEQ ID NO: 17-32 that differs from other sequences in the same genome. . Certain fragments of SEQ ID NO: 17-32 are useful, for example, in hybridization and amplification techniques, or similar methods of distinguishing SEQ ID NO: 17-32 from related polynucleotide sequences. The exact fragment length or region of SEQ ID NO: 17-32 that matches a fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

Certain fragments of SEQ ID NO: 1-16 are encoded by certain fragments of SEQ ID NO: 17-32. Certain fragments of SEQ ID NO: 1-16 contain regions of unique amino acid sequence that uniquely identify SEQ ID NO: 1-16. For example, certain fragments of SEQ ID NO: 1-16 are useful as immunogenic peptides for the production of antibodies that specifically recognize SEQ ID NO: 1-16. The exact fragment length and region of SEQ ID NO: 1-16 that matches a given fragment can be routinely determined by techniques common in the art based on the purpose of the fragment.

The term “similarity” refers to a degree of complementarity. This includes partial similarity and complete similarity. The term “identity” can also be called “similarity”. Partially complementary sequences in which hybridization of the same sequence to the target nucleic acid is at least partially blocked are said to be "substantially similar." Inhibition of hybridization between the perfectly complementary sequence and the target sequence is tested using a hybridization assay (Southern or Northern blotting, solution hybridization, etc.) under mildly stringent conditions. Substantially similar sequences or hybridization probes competitively suppress binding of the perfectly similar (identical) sequence to the target sequence under mildly stringent conditions. This does not mean that non-specific binding is allowed under mildly stringent conditions, but under mildly stringent conditions, the binding of two sequences to each other is specific (ie, selective). You have to interact. A second target sequence, which may not be said to be partially complementary (eg, less than 30% similarity or identity), can be used to test for the absence of non-specific binding. In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.

The term “percent identity” or “% identity” with respect to polynucleotide sequences refers to matching residues between two or more polynucleotide sequences that are aligned using a standardized algorithm. It is a percentage. Such algorithms can insert gaps in the sequences to make a more meaningful comparison between the two sequences in a standardized and reproducible manner to optimize the alignment between the two sequences.

The percent identity between polynucleotide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package and includes a suite of molecular biology analysis programs (DNASTAR, Madison W
I). This CLUSTAL V is for Higgins, DG and PM Sharp (1989) CABIOS
5: 151-153, Higgins, DG et al. (1992) CABIOS 8: 189-191. For pairwise alignments of polynucleotide sequences, the default parameter is Kt.
Set uple = 2, gap penalty = 5, window = 4, and “diagonals saved” = 4. A "weighted" residue weight table was selected as the default. Percentage identity is reported by CLUSTAL V as the "percentage similarity" of aligned polynucleotide sequence pairs.

Alternatively, a set of commonly used and freely available sequence comparison algorithms is
NCBI, Bethesda, MD, and Internet (http://WWW.ncbi.nlm.nih.gov/BLAS
National Center for Biotechnology Information (NCB)
I) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J
Mol. Biol. 215: 403-410). The suite of BLAST software includes various sequence analysis programs, including "blastn" used to align known polynucleotide sequences with other polynucleotide sequences in various databases. A tool called "BLAST 2 Sequences" is available,
Used to directly compare pairs of two nucleotide sequences. "BLAST 2 Sequen
"ces" can be used interactively by accessing http://WWW.ncbi.nlm.nih.gov/gorf/b12.html. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (described below). The BLAST program is commonly used with gaps and other parameters that set defaults. For example,
When comparing two nucleotide sequences, one may blast with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set to default parameters.
will use n. Such default parameters are, 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 May be measured for the entire length of a given sequence, as determined by the sequence number of, or for shorter lengths, for example, fragments obtained from a given given sequence, for example at least contiguous , 20 or 30
, 40, 50, 70, 100, 200 nucleotide fragments. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

Nucleic acid sequences that do not show high identity can encode similar amino acid sequences due to the degeneracy of the genetic code. Degeneracy can be used to alter nucleic acid sequences to create multiple nucleic acid sequences each encoding substantially the same protein.

The term “percentage of identity” or “% of identity” as used for polypeptide sequences
"Is the percentage of matching residues between two or more polypeptide sequences that are aligned using a standardized algorithm." Methods of polypeptide sequence alignment are well known. Some alignment methods consider conservative amino acid substitutions. Such conservative substitutions, described in detail above, generally conserve charge and hydrophobicity at the site of substitution, and result in polypeptide structure (and thus function) as well.
Is saved.

The percent identity between polypeptide sequences can be determined using the default parameters of the CLUSTAL V algorithm incorporated into the MEGALIGN version 3.12e sequence alignment program (supra). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are Ktuple = 1, gap
Set penalty = 3, window = 5, and “diagonals saved” = 5. The PAM250 matrix is selected as the default residue weight table. Similar to polynucleotide alignments, the percent identity of paired aligned polypeptide sequences is reported by CLUSTAL V as the "percentage of similarity."

Alternatively, the NCBI BLAST software suite is used. For example, when making a pairwise comparison of two polypeptide sequences, one would use blastp with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set with default parameters. . Such default parameters are, for example: Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50 Expect: 10 Word Size: 3 Filter: on It may be measured over the entire length of the peptide sequence, or for shorter lengths, eg fragments derived from one large given polypeptide sequence, eg at least 15, 20, or 30, 40 contiguous. , 50, 70, 150 residues may be measured against the length of the fragment. Such lengths are merely exemplary, and fragments of any length of the sequences set forth in the specification including sequence listing and tables and figures may be used to indicate the length at which percent identity is measured. it can.

A “human artificial chromosome (HAC)” can contain a DNA sequence of about 6 kb to 10 Mb in size and is a linear, small, linear unit containing all the elements necessary for stable segregation and maintenance of mitotic chromosomes. It is a chromosome.

The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence of the non-antigen binding region has been altered in order to resemble a more human antibody while retaining its original binding ability.

“Hybridization” is the process of annealing in which a single-stranded polynucleotide forms a base pair with a complementary single strand under defined hybridization conditions. Specific hybridization means that two nucleic acid sequences have high identity. Under conditions that allow annealing, specific hybridization complexes are formed and remain hybridized after the washing process. The washing process is particularly important in determining the stringency or stringency of the hybridization process, and under more stringent conditions, non-specific binding, ie binding of pairs of nucleic acid strands that are not perfectly matched. Is reduced. The conditions under which annealing between nucleic acid sequences is permissible are routinely determined by those of skill in the art and are constant during hybridization, but the washing process involves changing the conditions during which to achieve the desired stringency. Is possible and hybridization specificity is obtained. Annealing conditions are, for example, at a temperature of 68 ° C., about 6 × SSC, about 1% (w / v) SDS, and about 100.
Includes μg / ml shear denatured salmon sperm DNA.

In general, hybridization stringency can be expressed in part by the temperature at which the wash process occurs. The wash temperature is usually selected to be about 5-20 ° C below the thermal melting point (Tm) of the particular sequence at a given ionic strength and pH. This Tm is
It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The formula for calculating the Tm and the conditions for nucleic acid hybridization are well known, Sambrook, J. et al., 1989, Molecular Cloning : A Laboratory Manual , Volume 2 1-3, Cold Spring Harbor Press, Plainvi.
ew NY; Especially described in Chapter 2 of Volume 2.

High stringency hybridization between the polynucleotides of the present invention involves a wash step at about 68 ° C. for 1 hour in the presence of about 0.2 × SSC and about 1% SDS. Alternatively, it is carried out at temperatures of 65 ° C, 60 ° C, 55 ° C, 42 ° C. The concentration of SSC is in the range of about 0.1-2xSSC in the presence of about 0.1% SDS. Normally,
Blocking agents are used to prevent non-specific hybridization. Such blocking agents include, for example, about 100-200 μg / ml denatured salmon sperm DNA. Organic solvents such as formamide at a concentration of about 35-50% v / v, for example RNA and
It can be used in specific cases such as hybridization of DNA. Useful modifications of these wash conditions are well known to those of skill in the art. Hybridization, particularly under highly stringent conditions, may suggest evolutionary similarities between the nucleotides. Such similarities strongly suggest that the nucleotides and encoded polypeptides play a similar role.

The term “hybridization complex” refers to a complex of two nucleic acid sequences formed by hydrogen bonding between complementary bases. Hybridization complexes may be formed in solution (eg, C ₀ t or R ₀ t analysis), or one nucleic acid sequence in solution and a solid support (eg, paper, membrane, filter, chip, pin). ,
Or a slide glass, or any suitable substrate for immobilizing cells and their nucleic acids)
Can be formed with another nucleic acid sequence fixed to.

The term “insertion” or “addition” refers to a change in an amino acid sequence or nucleic acid sequence in which one or more amino acid residues or nucleotides have been added, respectively.

“Immune response” refers to conditions associated with inflammatory diseases and trauma, immune disorders, infectious diseases, genetic diseases and the like. These conditions are characterized by the expression of various factors such as cytokines and chemokines, other signaling molecules that affect the cell and systemic defense systems.

The term “microarray” refers to an array of different polynucleotides arranged on a substrate.

The term “element” or “array element” as used in the context of microarray refers to hybridizable polynucleotides arranged on the surface of a substrate.

The term “modulation” refers to a change in the activity of HYDRL. For example, modulation results in a change in protein activity or binding properties of HYDRL, or other biological, functional or immunological properties.

The term “nucleic acid” or “nucleic acid sequence” refers to nucleotides, oligonucleotides, polynucleotides, or fragments thereof. In addition, DN of genomic or synthetic origin, which is a single-strand or double-strand and is a sense or antisense strand
A or RNA, peptide nucleic acid (PNA), any DNA-like substance, and RNA-like substance.

“Operably linked” refers to the condition in which a first nucleic acid sequence and a second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are very contiguous or contiguous if the regions encoding the two proteins must be joined in the same reading frame.

“Peptide nucleic acid (PNA)” refers to an antisense molecule or anti-gene agent that comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. This terminal lysine renders the composition soluble. PNAs can bind preferentially to complementary single-stranded DNA or RNA to stop elongation of transcripts and polyethylene glycol to prolong life in cells.

The “probe” is a nucleic acid sequence encoding the same sequence or allele nucleic acid sequence, HYDRL, a complementary sequence thereof, or a fragment thereof used for detecting a related nucleic acid sequence. A probe is an oligonucleotide or polynucleotide that has been isolated with a detectable label or reporter molecule attached. Typical signs include
There are radioactive isotopes and ligands, chemiluminescent reagents, enzymes. "Primer"
Is a short nucleic acid, usually a DNA oligonucleotide, capable of forming complementary base pairs and annealing to a target polynucleotide. After the primer anneals to the polynucleotide, the target DNA is
It is extended along a single chain. The primer set can be used, for example, for amplification (and identification) of a nucleic acid sequence in a PCR method.

The probes and primers used in the present invention include at least 15 of known sequences.
Of contiguous nucleotides. Longer probes and primers can be used to increase specificity. For example, at least 20 or 25, 30, 30, 40, 50, 60, 70, 80, 90, 100 contiguous of the disclosed nucleic acid sequences.
, 150 nucleotides. It should be understood that probes and primers that are considerably longer than the above examples can be used, and nucleotides of any length shown in the tables and drawings of the present specification and the sequence listing can also be used.

For the preparation and use of the probe and the primer, see, for example, Sambrook,
J. et al., 1989, Name "Molecular Cloning: A Laboratory Manual", 2nd
Editions 1-3 (Cold Spring Harbor Press, Plainview NY), or Ausubel, FM
In 1987, the name "Current Protocols in Molecular Biology" (Greene
Pubi. Assoc. & Wiley-Intersciences, New York NY), and Innis et al.,
In 1990, the name `` PCR Protocols, A Guide to Methods and Applications '' (Acade
mic Press, San Diego CA.). The set of primers for PCR is, for example, Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research
, Cambridge MA), and the like, and can be derived from one known sequence using a computer program for such purpose.

Oligonucleotides used as primers are selected by computer programs well known in the art for primer selection. For example, OLIGO 4.06 software selects primer pairs for PCR, each up to 100 nucleotides,
And is useful for the analysis of large polynucleotides and oligonucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32,000 bases. Similar primer selection programs include additional features that expand capacity. For example, the PrimOU primer selection program (Genome Center at Uni
versity of Texas South West Medical Center, available from Dallas TX)
Since a specific primer can be selected from the megabase sequence, it is useful for designing a primer in a genome-wide scope. Primer3 primer selection program (Whitehead Institute / MIT Center for Genome Research,
(Available from Cambridge MA1) allows users to specify sequences that they want to avoid as primer binding sites.
) ”Can be entered. In addition, Primer3 is especially useful for the selection of oligonucleotides for microarrays (the source code of the latter two primer selection programs can be obtained from each source and modified to meet the needs of users). Can also). PrimerGen Program (UK Human Genome Mapping Pro
(available from the ject Resource Center, Cambridge UK), which designs primers based on multiple sequence alignments, so that they will hybridize to either the most conserved or the least conserved regions of the aligned nucleic acid sequences. Can be selected. Therefore, this program is useful for identifying unique and conserved oligonucleotide or polynucleotide fragments. The oligonucleotide or polynucleotide fragment identified by any of the selection methods described above is, for example, a PCR method or a sequencing primer, a microarray element, or a specific polynucleotide that identifies a polynucleotide that is completely or partially complementary to a sample nucleic acid. It is useful for the hybridization technique such as the probe. The method for selecting the oligonucleotide is not limited to the above method.

As used herein, “recombinant nucleic acid” is a sequence that is not a naturally occurring sequence, but is an artificial combination of two or more distant segments of a sequence. Often, this artificial combination is made by chemical synthesis, but it is more common to artificially manipulate distant segments of nucleic acid using genetic engineering techniques such as those described in Sambrook, supra. is there. The "recombinant nucleic acid" also includes a nucleic acid modified by simply adding or substituting a part of the nucleic acid. The recombinant nucleic acid may also include a nucleic acid sequence operably linked to a promoter sequence. Such recombinant nucleic acid can be, for example, part of a vector used to transform a cell.

Alternatively, such a recombinant nucleic acid is a vaccinia virus-based virus that, when used to vaccinate a mammal expressing the recombinant nucleic acid, elicits a defensive immune response in the mammal. It can be part of the vector.

The term “sample” is used in its broadest sense. Samples suspected of containing HYDRL-encoding nucleic acid or a fragment thereof, and HYDRL itself include body fluids, extracts from cells, chromosomes or intracellular organelles isolated from cells, membranes, cells, and solutions. Genomic DNA, RNA, cDNA present in or immobilized on a substrate, tissues or tissue prints and the like may also be included.

The terms “specific binding” and “specifically bind” refer to the interaction between a protein or peptide and an agonist, antibody, antagonist, small molecule, or any natural or synthetic binding composition. Point to. This interaction depends on the presence of a particular structure of the protein recognized by the binding molecule, eg, an antigenic determinant or epitope. For example, when the antibody is specific to the epitope "A", the reaction solution containing the unbound labeled "A" and the antibody is bound to the polypeptide containing the epitope A or the unbound unlabeled "A". Is present, the amount of labeled A bound to the antibody is reduced.

The term “substantially purified” refers to a nucleic acid sequence or amino acid sequence that has been isolated or separated after being removed from its natural environment, such that the composition to which it is naturally associated has at least about 60 amino acids. % Or more, preferably about 75% or more, and most preferably about 90% or more.

“Substitution” is the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides.

The term “substrate” refers to any suitable solid or semi-solid support, including membranes and filters, chips, slides, wafers, fibers, magnetic or non-magnetic beads, gels, tubes, plates, polymers, microspheres. Includes particles and capillaries. The substrate has various surface morphologies such as walls or trenches, pins, channels, pores, to which polynucleotides and polypeptides bind.

“Transformation” is the process by which exogenous DNA enters and changes the recipient cell. Transformation can occur under natural or artificial conditions by a variety of methods well known in the art and can be performed by any well known method of inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell. be able to. This transformation method is
The choice depends on the type of host cell to be transformed. This method includes, but is not limited to, viral infection, electroporation, lipofection, and particulate irradiation. "Transformed" cells include stably transformed cells in which the introduced DNA is capable of replicating autonomously, either as a plasmid or as part of a host chromosome. Further, a cell that transiently expresses the introduced DNA or the introduced RNA within a limited time is also included.

A “variant sequence” of a particular nucleic acid sequence refers to the default parameter setting “BLAST
2 Sequences "tool Version 2.0.9 (May-07-1999) shows that the specific nucleic acid sequence identity for a given length of a given nucleic acid sequence is at least 40%.
The nucleic acid sequence determined to be. Such nucleic acid pairs may, for example, be at least 50% or 60%, 70%, 80%, 85%, 90%, over a length.
It may exhibit 95%, 98%, or more identity. A variant sequence can be represented, for example, as an "allelic" variant sequence (described above) or a "splice" variant sequence, a "species" variant sequence, a "polymorphism" variant sequence. Splice variant sequences may have very high identities to the reference molecule, but alternative splicing of exons during mRNA processing may result in more or less polynucleotides. The corresponding polypeptide may have additional functional domains present in the reference molecule or may lack domains present in the reference molecule. Species variant sequences are polynucleotide sequences that vary from species to species. The resulting polypeptides have a high degree of amino acid identity with each other. Polymorphic variant sequences differ in the polynucleotide sequence of a given gene from a given species and the species. The polymorphic variant sequence also includes one of the polynucleotide sequences
A "single nucleotide polymorphism" (SNP) that differs by two nucleotides may also be included. The presence of SNPs may be indicative of, for example, a population, a pathological condition, a characteristic of the pathological condition.

A “variant” of a particular polypeptide sequence refers to the default parameter setting “
Blast 2 Sequences "Tool Version 2.0.9 (May-07-1999) by blastp, the identity of the particular polypeptide sequence to a certain length of a certain nucleic acid sequence is determined to be at least 40% of the nucleic acid sequence That is. Such polypeptide pairs may, for example, be at least 50% or 60%, 70%, 8 in a length.
It may exhibit 0%, 85%, 90%, 95%, 98%, or more identity.

(Invention) The present invention is based on the discovery of a novel human hydrolase protein (HYDRL) and a polynucleotide encoding HYDRL. It relates to the use of these compositions in the diagnosis, treatment and prevention of adrenal insufficiency and genetic disorders.

Table 1 shows the Incyte clones used to construct the full length nucleotide sequence encoding HYDRL. Columns 1 and 2 show the SEQ ID NOs for the polypeptide and polynucleotide, respectively. Column 3 shows Incy in which the nucleic acid encoding each HYDRL was identified.
The clone IDs of the te clones are shown and column 4 shows the cDNA library from which those clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. The Incyte clones for which the cDNA library is not shown are derived from the pooled cDNA library. The clone in row 5 was used to construct the consensus nucleotide sequence for each HYDRL and is useful as a fragment in hybridization techniques.

Each column of Table 2 shows various properties of each polypeptide of the invention, column 1 is the SEQ ID NO (SEQ ID NO), column 2 is the number of amino acid residues in each polypeptide, column 3 is Potential phosphorylation site, row 4 potential glycosylation site, row 5 signature
ature) amino acid residues having a sequence and a motif, column 6 shows a homologous sequence identified by BLAST analysis, column 7 shows an analytical method, and in some cases, a searchable database in which the analytical method can be used. The analysis method in column 7 can be used to characterize each polypeptide from sequence homology and protein motifs.

As shown in FIG. 1, HYDRL-1 is a Colobus guereza lysozyme c precursor
(GI 1790927; SEQ ID NO: 33), Colobus angolensis lysozyme c precursor (GI 17
90967; SEQ ID NO: 34), and Nasalis larvatis lysozyme c precursor (GI 1790984;
It has chemical and structural similarities to SEQ ID NO: 35). Specifically, HYDRL-1 is, Colo bus guereza lysozyme c 40% identity with the precursor, 40% identity to the Colobus Angolensis lysozyme c precursor, the same 41% and Nasalis Larvatis lysozyme c precursor Have sex.

The columns of Table 3 show the tissue specificity and diseases or disorders associated with the nucleotide sequence encoding HYDRL. Column 1 of Table 3 shows the nucleotide sequence number (SEQ ID NO). Column 2 shows a fragment of the nucleotide sequence of column 1. For example, these fragments are useful in hybridization or amplification techniques that identify SEQ ID NO: 17-32 and distinguish SEQ ID NO: 17-32 from related polynucleotide sequences. Polynucleotides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 shows the tissue category expressing HYDRL and its proportion in total tissues expressing HYDRL. Column 4 is a disease or disorder associated with tissues expressing HYDRL, symptoms, and HYD
Their proportions in the total tissues expressing RL are shown. Column 5 shows the vector used to subclon each cDNA library.

Northern analysis of SEQ ID NO: 17 showed expression of this sequence in tissues associated with cancer.

SEQ ID NO: 18 represents the range of chromosome 6 from 42.30 cmorgan to 45.40 cmorgan, the range of chromosome 9 from 130.40 cmorgan to 166.50 cmorgan, and the chromosome 16 of 88.10 cmorgan to 92.60 cmorgan. Mapped to a range up to.

SEQ ID NO: 25 has been mapped to the range from 22.90 cmorgan to 39.90 cmorgan on chromosome 1 and the range from 30.90 cmorgan to 43.00 cmorgan on chromosome 3. In addition, the range from 30.90 centimorgans to 43.00 centimorgans on chromosome 3 also includes expressed gene sequence fragments associated with von Hippel-Lindau syndrome.

SEQ ID NO: 28 has been mapped to chromosome 10 in the range from 137.60 centimorgans to 139.20 centimorgans.

The columns of Table 4 provide a description of the tissues used to generate the cDNA library in which clones of cDNA encoding HYDRL were isolated. Row 1 shows the SEQ ID NO of the nucleotides, row 2 shows the cDNA library from which those clones were isolated, row 3 shows the cDNA library of row 2.
The origin and details of the tissue corresponding to the cDNA library are shown.

The present invention also includes variants of HYDRL. Preferred variants of HYDRL have at least about 80%, or at least about 90%, or at least about 95% amino acid sequence identity with the HYDRL amino acid sequence and possess at least one of the functional or structural characteristics of HYDRL. Have.

The present invention also includes polynucleotides that encode HYDRL. In a particular embodiment, the invention comprises a polynucleotide sequence having a sequence selected from the group consisting of SEQ ID NO: 17-32 encoding HYDRL.

The invention also includes variant sequences of the polynucleotide sequence encoding HYDRL.
In particular, such polynucleotide variants have at least about 80%, or at least about 90%, or even at least about 95% polynucleotide sequence identity with the polynucleotide sequence encoding HYDRL. A particular embodiment of this invention is a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17-32 and having at least about 80%, more preferably at least about 90%, and most preferably at least about 95% poly nucleotide sequence. A variant sequence of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 17-32, having nucleotide sequence identity, is included. Variant sequences of any of the above polynucleotides may encode an amino acid sequence having at least one functional or structural characteristic of HYDRL.

Various polynucleotide sequences encoding HYDRL that may be created by the degeneracy of the genetic code include those that have minimal similarity to the polynucleotide sequences of any known naturally occurring gene. Those of ordinary skill in the art will understand. Thus, the present invention may include all of the possible polynucleotide sequence variations that may be created by the selection of combinations based on possible codon choices. These combinations are made on the basis of the standard triplet genetic code applied to the naturally occurring HYDRL polynucleotide sequences, and are considered to be the clear disclosure of all mutations.

It is desirable that the nucleotide sequence encoding HYDRL and its mutated sequences be capable of hybridizing to naturally occurring HYDRL nucleotides under appropriately selected stringent conditions, but with non-naturally occurring codons. It may be beneficial to create a nucleotide sequence that encodes HYDRL or a derivative thereof with substantially different codon usage, such as inclusion. Codons can be selected based on the frequency with which a particular codon is utilized by the host to increase the rate at which peptide expression occurs in a particular eukaryotic or prokaryotic host. Another reason for substantially altering the nucleotide sequence encoding HYDRL and its derivatives without changing the encoded amino acid sequence is that RNA with favorable properties, such as a longer half-life, than transcripts made from naturally occurring sequences. To make a transcript.

The present invention also includes the production of DNA sequences encoding HYDRL and its derivatives or fragments thereof, entirely by synthetic chemistry. After construction, the synthetic sequence can be inserted into any of the various available expression vectors and cell lines using reagents well known in the art. In addition, synthetic chemistry may be used to introduce mutations into the sequence encoding HYDRL or any fragment thereof.

The present invention further provides a polynucleotide sequence capable of hybridizing to the claimed polynucleotide sequences, in particular SEQ ID NO: 17-32 and fragments thereof, under various stringent conditions. Included (eg Wahl, GM and SL Berger
(1987) Methods Enzymol. 152: 399-407; and Kimmel. AR (1987) Methods En
zymol. 152: 507-511.). Hybridization conditions, including annealing and wash conditions, are described in the "Definitions".

Any of the embodiments of the present invention can be carried out using DNA sequencing methods well known in the art. This method includes, for example, the Klenow fragment of DNA polymerase I, SE
QUENASE (US Biochemical, Cleveland OH), Taq Polymerase (Perkin Elmer)
, Thermostable T7 polymerase (Amersham, Pharmacia Biotech Piscataway NJ),
Alternatively, enzymes such as a combination of proofreading exonuclease and polymerase as found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD) are used. Preferably, MICROLAB 2200 Liquid Transfer System (Hamilton, Reno, NV),
PTC200 Thermal Cycler200 (MJ Research, Watertown MA) and ABI CATALYST 80
Automate sequence preparation using a device such as 0 (Perkin-Elmer). Then ABI 373
Alternatively, sequencing is performed using the 377 DNA Sequencing System (Perkin-Elmer), MEGABACE 1000 DNA Sequencing System (Molecular Dynamics. Sunnyvale CA) or other methods known in the art. The resulting sequences are analyzed using various algorithms well known in the art (e.g., Ausubel, FM (1997) Short Protocols in Molecular Biology , John Wiley & Sons, New York NY, unit 7.7;
Meyers, RA (1995) Molecular Biology and Biotechnology , Wiley VCH, New
York NY, pp. 856-853.).

A variety of PCR-based methods well known in the art can be used to extend the nucleic acid sequence encoding HYDRL using partial nucleotide sequences to extract upstream sequences such as promoters and regulatory elements. To detect. For example, one method utilizing restriction site PCR is to amplify an unknown sequence from genomic DNA in a cloning vector using common and nested primers (eg, Sarkar, G. (1993) PCR M).
See ethods Applic 2: 318-322). An alternative method using the inverse PCR method uses primers that extend in a wide range of directions and amplify unknown sequences from a circularized template. This template is derived from a restriction fragment containing known genomic loci and sequences surrounding it (see, eg, Triglia, T. et al. (1988) Nucleic Acids Res 16: 8186). Capture pc
A third method using the R method involves PCR amplification of DNA fragments flanking known sequences of human and yeast artificial chromosome DNA (eg, Lagerstrom, M. et al. (1991) PCR Methods Ap.
See plic 1: 111-119). This method allows digestion and ligation with multiple restriction enzymes to insert the recombinant double-stranded sequence into a region of unknown sequence prior to PCR. It is also possible to obtain the unknown sequence using other methods well known in the art. (For example, Parker, JD et al. (1991) Nucleic Acids Res
. 19: 3055-3060). Furthermore, by using PCR, nested primers, and PROMOTER FINDER library (Clontech, Palo Alto CA), walking within genomic DNA is possible. This method does not require screening the library and is useful for looking for intron / exon junctions. In all PCR-based methods, the primers are commercially available OLIGO 4.06 Primer Analysis software (National Bioscien
ces, Plymouth MN) or another suitable program etc.
It is designed to anneal to the template at a temperature of about 68 ° C to 72 ° C with 0 nucleotides, a GC content of 50% or more.

When screening for full-length cDNAs, it is preferable to use libraries that are size-selected to contain large cDNAs. Furthermore, if the oligo d (T) library cannot produce a full-length cDNA, a randomly primed library, which often contains a sequence having the 5'region of the gene, is useful. Genomic libraries will be useful for extension of sequences into the 5'non-transcribed regulatory regions.

Sequencing or PCR using a commercially available capillary electrophoresis system
It is possible to analyze or confirm the size of the nucleotide sequence of the product. For more information,
Capillary sequencing uses a flowable polymer for electrophoretic separation, and a laser-activated fluorochrome specific for four different nucleotides and a CCD camera used to detect emitted wavelengths. It is possible.
The output / light intensity is determined by the appropriate software (eg GENOTYPER and SEQUENCE NAVIGA
TOR, Perkin-Elmer) is used to convert to an electric signal, and the process from sample loading to computer analysis and electronic data display can be controlled by computer. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA, which may be present in small amounts in a particular sample.

In another embodiment of the invention, the polynucleotide sequence encoding HYDRL or a fragment thereof is used in a recombinant DNA molecule to express HYDRL, a fragment or functional equivalent thereof in a suitable host cell. Is possible. Due to the unique duplication of the genetic code, alternative DNA sequences may be produced which encode substantially the same or functionally equivalent amino acid sequences, which sequences are available for cloning and expression of HYDRL.

The nucleotide sequences of the present invention can be recombined using methods generally known in the art to alter the HYDRL coding sequence for a variety of purposes. This purpose includes, but is not limited to, cloning, processing and / or regulating expression of the gene product. Nucleotide sequences can be recombined using DNA mixing with random fragments or PCR reassembly of gene fragments and synthetic oligonucleotides. For example, oligonucleotide-mediated directed mutagenesis can be used to introduce mutations that create new restriction sites, alter glycosylation patterns, alter codon preferences, generate splice variants, and the like.

According to another embodiment, the sequences encoding HYDRL can be wholly or partially synthesized using chemical methods well known in the art (eg, Carthers. MH et al. (198).
0) Nucl. Acids Res. Symp. Ser 7: 215-223; and Horn, T. et al. (1980) Nucl. Aci.
See ds Res. Symp. Ser. 225-232). Alternatively, chemical methods can be used to synthesize HYDRL itself or fragments thereof. For example, peptide synthesis can be performed using a variety of solid phase techniques (eg, Roberge, JY et al. (1995) Science 26.
9: 202-204). Alternatively, automation of synthesis may be achieved using, for example, the ABI 431A Peptide Synthesizer (Perkin Elmer). Furthermore, the amino acid sequence of HYDRL, or any part thereof, may be altered by direct synthetic alterations and / or combinations with sequences from other proteins or any part thereof using chemical methods. It is possible to make a polypeptide.

This peptide is used for preparative high performance liquid chromatography (eg, Chiez, RM).
And FZ Regnier (1990) Methods Enzymol. 182: 392-421)). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (see, for example, Creighton. T. (1983) Proteins , Structures and Molecular Properties , WH Freeman, New York, NY).
.

For expression of biologically active HYDRL, the nucleotide sequence encoding HYDRL or a derivative thereof is inserted into a suitable expression vector. This expression vector
It contains the elements necessary to regulate the transcription and translation of the coding sequences inserted into a suitable host. These elements include regulatory sequences such as enhancers, constitutive and expression-inducible promoters, 5'and 3'untranslated regions in the vector and polynucleotide sequences encoding HYDRL. Such elements vary in length and specificity. With particular initiation signals, it is possible to achieve more efficient translation of the HYDRL coding sequence. Such signals include the ATG initiation codon and nearby sequences such as the Kozak sequence. If the HYDRL coding sequence and its initiation codon, upstream regulatory sequences are inserted into a suitable expression vector, no additional transcriptional or translational regulatory signals will be required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal containing an in-frame ATG initiation codon must be included in the expression vector. Exogenous translational elements and initiation codons can be obtained from a variety of natural and synthetic sources. The efficiency of expression can be increased by the inclusion of enhancers suitable for the particular host cell system used. (For example, Scharf, D
Et al. (1994) Results Probl. Cell Differ. 201-18-162.).

[0117]   Sequences encoding HYDRL, suitable transcription and translation, using methods well known to those of skill in the art.
It is possible to make expression vectors that include regulatory elements. These methods include in vitro Recombinant DNA technology, synthetic technology, andin vivoIncludes gene recombination technology.
(For example, Sambrook, J. et al. (1989)Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Press, Plainview NY, Chapters 4 & 8, and 16-17; and
 Ausubel, F.M. et al. (1995)Current Protocols in Molecular Biology, John W
iley & Sons, New York NY, ch. 9 and 13-1-4).

A variety of expression vector / host systems may be utilized to retain and express sequences encoding HYDRL. These include, but are not limited to, microorganisms such as recombinant bacteriophage, plasmids, or bacteria transformed with cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression vectors. Insect cell lines infected with (eg, baculovirus) or plants transformed with viral expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg, Ti or pBR322 plasmids) Cell lines and animal cell lines are included. The present invention is not limited by the host cell used.

In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for the polynucleotide sequence encoding HYDRL. For example, HYDR
For routine cloning, subcloning, and propagation of L-encoding polynucleotide sequences, multifunctional E. coli vectors such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORT1 plasmid (GIBCO BRL) can be used. Ligation of the HYDRL coding sequence into multiple cloning sites of the vector resulted in lac.
The Z gene is disrupted, allowing a colorimetric screening method for identification of transformed bacteria containing recombinant molecules. In addition, these vectors can be used to generate in vitro transcription of cloned sequences, dideoxin screening, single chain rescue by helper phage, and nested deletions. (For example, Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509.
.). For example, if a large amount of HYDRL is needed for antibody production,
Vectors that induce high levels of expression can be used. For example, a vector containing a strongly inducible T5 or T7 bacteriophage promoter can be used.

It is possible to use yeast expression systems for the expression of HYDRL. Various vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH promoter can be used for yeast Saccharomyces cerevisiae or Pichia pastoris . Furthermore, such vectors induce either the secretion of the expressed protein or its retention within the cell, integrating foreign sequences into the host genome for stable growth. (For example, Ausubel .; and Bitter, GA et al. (1987) Method above.
s Enzymol. 153: 51-794: Scorer. CA et al. (1994) Bio / Technology 121-181-18
Plant lines can also be used to express HYDRL. Transcription of the sequence encoding HYDRL is, for example, a viral promoter such as the 35S and 19S promoters derived from CaMV alone or an omega leader sequence derived from TMV (Takamatsu, N. et al. (1987) EMBO J 6: 307-311). Is promoted in combination with. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (For example, The McGraw Hill Yearbook of Science and Technology (1992)
See McGraw Hill NY, pp.191-196).

In mammalian cells, a variety of virus-based expression systems may be utilized. When adenovirus is used as an expression vector, the HYDRL coding sequence may be linked to an adenovirus transcript / translation complex consisting of a late promoter and a triple leader sequence. By insertion into the nonessential E1 or E3 region of the viral genome,
It is possible to obtain live virus expressing HYDRL in infected host cells (Logan
, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659). In addition, transcription enhancers, such as the Rous Sarcoma Virus (RSV) enhancer, can be used to increase expression in mammalian host cells. For high level expression of proteins, SV40 or EBV based vectors can be used.

Human artificial chromosomes (HACs) can be used to supply fragments of DNA that are larger than those expressed in the plasmid and contained therein. HACs of about 6kb-10Mb for treatment
Are prepared and delivered by conventional delivery methods (liposomes, polycationic amino polymers, or vesicles). (For example, Harrington.JJ et al. (1997) Nat Genet. 15: 3.
45-355.).

For long-term production of recombinant proteins in mammalian systems, stable expression of HYDRL in cell lines is desirable. For example, the expression vector can be used to transform a cell line with a sequence encoding HYDRL. Such expression vectors contain replication and / or endogenous expression elements of viral origin and a selectable marker gene on the same or another vector. After the introduction of the vector, the cells are grown in enriched medium for about 1-2 days before being transferred to selective medium. The purpose of the selectable marker is to confer resistance to selective mediators, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells can be propagated using tissue culture techniques suitable for that cell type.

[0124] Any number of selection systems can be used to recover transformed cell lines. The selection systems include, but are not limited to, include herpes simplex virus thymidine kinase gene and adenine phosphoribosyltransferase genes, respectively tk ^- or apr ^- used in the cell. (For example, Wigler, M. et al. (1
977) Cell 11: 223-232; and Lowy, I. et al. (1980) Cell 22: 817-823). Also, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, dhfr confers resistance to methotrexate, neo confers resistance to aminoglycoside neomycin and G-418, and als or pat confers resistance to chlorsulfuron (cHYDRLsulfuron) and phosphinotricin acetyltransferase (eg, phosphinotricin acetyltransferase). , Wigl
er, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-3570; Colbere-Garapin,
F. et al. (1981) J. Mol. Biol. 150: 1-14). Further genes available for selection have been described in the literature, eg trpB and hisD which alter what the cell needs for metabolism (eg Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad.
. Sci. 85: 8047-51). Anitocyanin, green fluorescent protein (GFP; Clon
tech), β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin and other visible markers are used. Green fluorescent protein (GFP) (Clon
tech, Palo Alto, CA) can also be used. These markers can be used not only to identify transformants, but also to quantify transient or stable protein expression due to a particular vector system (eg, Rhodes, CA et al. (19
95) Methods Mol. Biol. 55: 121-791).

Even if the presence / absence of the expression of the marker gene indicates the presence of the gene of interest, it may be necessary to confirm the presence and expression of the gene. For example, if the sequence encoding HYDRL is inserted within a marker gene sequence, transformed cells containing the sequence encoding HYDRL can be identified by the lack of marker gene function. Alternatively, the marker gene can be placed in tandem with the sequence encoding HYDRL under the control of one promoter. Expression of the marker gene in response to induction or selection usually also indicates expression of the tandem gene.

In general, a host cell containing a nucleic acid sequence encoding HYDRL and expressing HYDRL can be identified using a variety of methods well known to those of skill in the art. These methods include DNA-DNA or DNA-RNA hybridizations, PCR methods, protein biological methods including membrane-based, solution-based, or chip-based techniques for the detection and / or quantification of nucleic acids or proteins. Includes, but is not limited to, test methods or immunological assays.

HY with either specific polyclonal or monoclonal antibodies
Immunological methods for detecting and measuring the expression of DRL are well known in the art. Such techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorter (FACS), and the like. A two-site, monoclonal-based immunoassay using a monoclonal antibody that reacts with two non-interfering epitopes on HYDRL.
) Is preferred, but competitive binding assays can also be used. These and other assays are well known in the art. (For example, Hampton.
R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press. St Pa
ul. MN, Sect. IV; Coligan, JE et al Current Protocols in Immunology , Gree
ne Pub. Associates and Wiley-Interscience, New York. NY; and Pound, JD
(1990) Immunochemical Protocols , Humans Press, Totowa NJ).

Various labeling and conjugation methods are well known to those of skill in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to HYDRL-encoding polynucleotides include oligo-labeling, nick-translation, end-labeling, or labeled nucleotides. Includes the PCR amplification used.
Alternatively, the HYDRL coding sequence, or any fragment thereof, can be cloned into a vector to generate an mRNA probe. Such vectors, which are well known in the art and are commercially available, can be prepared using suitable R7 such as T7, T3, or SP6.
RNA in vitro by the addition of NA polymerase and labeled nucleotides
It can be used for probe synthesis. These methods are described, for example, in Amersham Pha.
rmacia Biotech and Promega (Madison WI), US Biochemical Corp (Clevelan
d OH) can be carried out using various commercially available kits. Suitable reporter molecules or labels that can be used for easy detection include substrates, cofactors, inhibitors, magnetic particles, and radionuclides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents and the like.

Host cells transformed with a nucleotide sequence encoding HYDRL are cultured under conditions suitable for the expression and recovery of this protein in cell culture. Whether the protein produced by a transformed cell is secreted or remains intracellular depends on the sequence and / or the vector used. It will be appreciated by those of skill in the art that expression vectors containing a polynucleotide encoding HYDRL can be designed to include a signal sequence that directs secretion of HYDRL across prokaryotic and eukaryotic cell membranes.

In addition, a host cell strain may be chosen for its ability to regulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Modifications of such polypeptides include acetylation, carboxylation, glycosylation, phosphorylation, lipidation (
lipidation), and acylation, but is not limited thereto.
Post-translational processing which cleaves a "prepro" or "pro" form of the protein can be used to identify target proteins, folds and / or activities. A variety of host cells (eg, CHO, HeLa, MDCK, MEK293, WI38) with specific cellular machinery and characteristic mechanisms for post-translational activity are available in the American Type Culture C
Ollection (ATCC; Bethesda, MD) and is selected to ensure correct modification and processing of foreign proteins.

In another embodiment of the invention, the natural or altered or recombinant nucleic acid sequence encoding HYDRL is linked to a heterologous sequence which results in the translation of the fusion protein of any of the host systems described above. For example, a chimera containing a heterologous moiety that can be recognized by a commercially available antibody
HYDRL proteins may facilitate the screening of peptide libraries for inhibitors of HYDRL activity. Also, the heterologous protein portion and the heterologous peptide portion can facilitate purification of the fusion protein using commercially available affinity substrates. Such moieties include glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6
-His, FLAG, c-mc, hemagglutinin (HA), but are not limited to these. GST and MBP, Trx, CBP, 6-His allow purification of cognate fusion proteins with immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal chelate resins, respectively.
FLAG, c-mc, and hemagglutinin (HA) allow purification of immunoaffinity of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. Alternatively, HYDRL can be cleaved from the heterologous moiety after purification by genetically engineering the fusion protein to include a proteolytic cleavage site between the HYDRL coding sequence and the heterologous protein sequence. Methods for expressing and purifying fusion proteins are described in Ausubel. (1995, supra ch 10). A variety of commercially available kits can be used to facilitate expression and purification of the fusion protein.

In another embodiment of the invention, it is possible to synthesize radiolabeled HYDRL in vitro using the TNT rabbit reticulocyte lysate or wheat germ extraction system (Promega). These systems connect transcription and translation of sequences encoding proteins that are operably linked to the T7 or T3, SP6 promoters. Translation occurs in the presence of a radiolabeled amino acid precursor, which is, for example, ³⁵ S methionine.

Fragments of HYDRL can be produced by direct peptide synthesis using solid phase techniques as well as recombinant products (see, eg, Creighton, pp. 55-60. Supra). Protein synthesis can be performed manually or automatically. Automated synthesis can be performed using, for example, a 431A Peptide Synthesizer (Perkin Elmer). Various fragments of HYDRL are separately synthesized and then ligated to produce the full length molecule.

Treatment There are chemical and structural similarities between certain regions of HYDRL and certain hydrolase proteins, for example in the context of sequences and motifs. Furthermore, HYDRL
Is closely associated with proliferative tissue, inflamed tissue, and cancer. In some cases,
The sequence encoding HYDRL is located in a region of the chromosome associated with inherited disease.
Therefore, HYDRL is associated with cell proliferative disorders, autoimmune disorders / inflammatory diseases, neuropathy,
It is thought to play a role in renal disorders, adrenal insufficiency, and genetic disorders. In the treatment of diseases associated with increased HYDRL expression or activity, it is desirable to reduce HYDRL expression or activity. In addition, in the treatment of diseases associated with decreased HYDRL expression or activity, it is desirable to increase HYDRL expression or activity.

Accordingly, in one embodiment, HYDRL or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of disorders associated with decreased HYDRL expression or activity. Examples of such disorders include, but are not limited to, cell proliferative disorders, including actinic keratosis and atherosclerosis, bursitis,
Cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, bone marrow Tumor, sarcoma, and teratocarcinoma, specifically adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, accessory Includes thyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, uterine cancer, and also autoimmune disorders / inflammatory diseases, including inflammation, actinic keratosis, Acquired immunodeficiency syndrome (AIDS) and adrenal insufficiency, adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmunity Thyroiditis, autoimmune polyglandular endocrine candidiasis Mesodermal dystrophy (APECED), bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
Emphysema, lymphotoxin-induced transient lymphopenia, erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, Hepatitis, hypereosinophilia, irritable bowel syndrome, lymphotoxin-induced temporary lymphopenia, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, Myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Schögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic Sclerosis, primary thrombocythemia, thrombocytopenia, ulcerative colitis, Werner syndrome,
Cancer complications, hemodialysis, extracorporeal circulation and hematopoiesis, including viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminth infections, trauma, and lymphomas, leukemias, and myeloma Cancers of the system are also included, including neuropathy, including epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and others. Extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, inherited ataxia, multiple sclerosis and other demyelinating diseases, bacterial and Viral meningitis, cerebral abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, and Kuru and Creutzfeldt-Jakob disease , Gerstmann's syndrome , Gerstmann-Straussler-Scheinker syndrome prion containing mellitus (prion disease), fatal familial insomnia, nervous system trophic diseases and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma (cerebelloretinal hemangioblast
cerebral trigeminal vascular syndrome, central nervous system mental retardation and other developmental disorders,
Cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nerve diseases, dermatomyositis and polymyositis, and hereditary, metabolic, endocrine, and moderate Toxic myopathy, myasthenia gravis, periodic quadriplegia, mood and anxiety psychiatric disorders, and delusional psychosis, and seasonal affective disorders (SA
D), akathisia, amnesia, catatonia, diabetic neuropathy, extrapyramidal terminal defect syndrome, dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease; Including amyloid nephropathy, hypertension,
Primary aldosteronism, adrenal insufficiency, renal failure, nephritis, chronic nephritis, tubuloinserstitial nephritis, cystic disease of the kidney such as polycystic disease, dysplastic malformation such as renal dysplasia, cortex Congenital polycystic renal dysplasia (PD), including congenital or medullocystic disease, autosomal dominant and recessive congenital polycystic renal dysplasia (PRD)
RD), medullary cysts, cavernous and tubular dysplasia, Alport syndrome, non-renal cancers that have a physiological effect on the kidneys, such as lung and basilar tumors, multiple myeloma, renal adenocarcinoma , Metastatic renal cancer, and also adrenal insufficiency, including:
Orbital gland inflammation, anaphylaxis, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, pheochromocytoma, and also genetic disorders, including: GM1 gangliosidosis, Niemann-Pick disease, adrenoleukodystrophy, Alport syndrome, colloideremia, Duchenne-Becker muscular dystrophy, Down syndrome, cystic fibrosis, chronic granulomatosis, Gaucher disease, Huntington disease, Marfan syndrome, muscle Dystrophy, myotonic dystrophy,
Frequent pycnodysostosis, Lefsum syndrome, retinoblastoma, sickle cell anemia, thalassemia, Werner syndrome, von Willebrand disease, von Hippel-Lindau syndrome, Wilms tumor, Zellweger syndrome, peroxisome acyl-CoA oxidation Enzyme deficiency (peroxisomal acyl-CoA oxidase deficiency
), Peroxisome thiolase deficiency, peroxisome bifunctional protein deficiency, mitochondrial carnitine palmitoyl transferase and carnitine deficiency, mitochondrial ultralong chain acyl-CoA dehydrogenase deficiency, mitochondrial short chain acyl-CoA dehydrogenase deficiency, mitochondrial electron transfer flavoprotein And electron transfer flavoprotein: ubiquinone oxidoreductase deficiency,
Mitochondrial trifunctional protein deficiency, Mitochondrial short-chain 3-hydroxyacyl-C
Includes oA dehydrogenase deficiency.

In another embodiment, HYDRL or a fragment or derivative thereof is expressed for the treatment or prevention of diseases associated with decreased expression or activity of HYDRL, including but not limited to the diseases listed above. It is also possible to administer the resulting vector to a patient.

In yet another embodiment, HYDRL, including but not limited to the diseases listed above.
For the treatment or prevention of diseases associated with decreased expression or activity of A., it is possible to administer a pharmaceutical composition comprising substantially purified HYDRL to a patient together with a suitable pharmaceutical carrier.

In yet another embodiment, HYDRL, including but not limited to the diseases listed above.
It is also possible to administer to the patient an agonist that regulates the activity of HYDRL for the treatment or prevention of diseases associated with decreased expression or activity of HYDRL.

In a further embodiment, a patient may be administered an antagonist of HYDRL for the treatment or prevention of disorders associated with increased HYDRL expression or activity.
Examples of such diseases include, but are not limited to, the above described cell proliferative disorders,
Includes autoimmune disorders / inflammatory disorders, neuropathy, renal disorders, adrenal insufficiency, and genetic disorders. In one embodiment, an antibody that specifically binds to HYDRL can be used directly as an antagonist or indirectly as a targeting or delivery mechanism to deliver the drug to cells or tissues that express HYDRL.

In another embodiment, the complement of a polynucleotide encoding HYDRL for the treatment or prevention of diseases associated with increased expression or activity of HYDRL, including but not limited to the diseases listed above. It is also possible to administer a vector that expresses

In another example, any of the proteins, antagonists, antibodies, agonists, complementary sequences, vectors of the invention may be administered in combination with another suitable therapeutic agent. One of ordinary skill in the art can select suitable therapeutic agents for use in the combination therapy according to conventional pharmaceutical principles. The combination with therapeutic agents may bring about a synergistic effect in the treatment or prevention of the various diseases listed above. Using this method, it is possible to achieve a medicinal effect with a small amount of each drug, and it is possible to reduce the possibility of a wide range of side effects.

HYDRL antagonists can be prepared using methods conventional in the art. Specifically, purified HYDRL can be used to produce antibodies, and therapeutic drug libraries can be screened to identify those that specifically bind to HYDRL. The antibody of HYDRL can also be produced using a method generally used in this field.
Such antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies,
Included are single chains, Fab fragments, and fragments produced by Fab expression libraries. However, it is not limited to these. For therapeutic purposes, neutralizing antibodies (ie, those that inhibit dimer formation) are particularly preferred.

For the production of antibodies, various hosts, including goats, rabbits, rats, mice, humans and others, can be injected with HYDRL or any fragment or oligopeptide thereof with immunogenic properties. Can be immunized with. Depending on the host species, various adjuvants may be used to enhance the immune response. Such adjuvants include Freund's adjuvant, mineral gel adjuvants such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and surfactants such as dinitrophenol. However, it is not limited thereto. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parv um it is particularly preferred.

Oligopeptides, peptides used to induce antibodies to HYDRL,
Alternatively, fragments preferably consist of at least about 5 amino acids, generally about 10 or more amino acids. These oligopeptides or peptides, or fragments thereof, are preferably identical to a portion of the amino acid sequence of the native protein and also include the entire amino acid sequence of small naturally occurring molecules. Short stretches of HYDRL amino acids can be fused with sequences of another protein, such as KLH, to produce antibodies to the chimeric molecule.

Monoclonal antibodies against HYDRL can be produced by successive cell lines in culture, using any technique that produces antibody molecules. These techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV-hybridoma technology (eg, Kohler.
, G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) .J. Immunol. M.
ethods 81-8-42; Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-20.
30; Cole, SP et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, techniques such as splicing the mouse antibody gene onto the human antibody gene developed for the production of “chimeric antibodies” can be used to obtain molecules with suitable antigen specificity and biological activity. (For example, Morrison, SL et al. (1984) Proc.
Natl. Acad. Sci. 81-4851-4855; Neuberger, MS et al. (1984) Nature 312: 6.
04-608; Takeda, S. et al. (1985) Nature 314: 452,454). Alternatively, the techniques described for the production of single chain antibodies are applied using methods well known in the art to produce HYDRL specific single chain antibodies. Antibodies with related specificities but of different idiotype composition can also be generated by chain mixing from random combinatorial immunoglobulin libraries (eg, Burton DR (1991) Proc. Nat).
l. Acad. Sci. 88: 11120-3).

Antibodies can be induced by in vivo production in a population of lymphocytes, or by screening immunoglobulin libraries or a panel of highly specific binding reagents, as shown in the literature. (See, for example, Orlando, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833.
-3837; Winter, G. et al. (1991) Nature 349: 293-299).

Antibodies containing specific binding sites for HYDRL can also be produced. For example, such fragments include F (ab ') fragments produced by pepsin digestion of antibody molecules and Fa (ab') fragments produced by reducing the disulfide bridges of the fragments.
b fragments are included, but are not limited to. Alternatively, generating a Fab expression library allows for rapid and easy identification of monoclonal Fab fragments with the desired specificity (eg, Huse, WD et al. (1989) Science 254: 12.
See 75-1281).

Various immunoassays are used to screen to identify antibodies with the desired specificity. Numerous protocols for competitive binding, or immunoradioactivity, using either polyclonal or monoclonal antibodies with isolated specificity are well known in the art. Usually for such immunoassays, HYDR
Included is the measurement of complex regulation between L and its specific antibody. Two incoherent HYDRL
Two-site monoclonal-based immunoassays are generally used with monoclonal antibodies reactive to the epitope, but competitive binding assays can also be used (Pound, supra).

Using various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques, H 2
Assess the affinity of the YDRL antibody. Affinity is represented by the binding constant Ka, which is the molar concentration of HYDRL antibody complex divided by the molar concentration of free antibody and free antigen under equilibrium conditions. The measured value Ka of the polyclonal antibody reagent having a heterogeneous affinity for many HYDRL epitopes represents the average affinity or binding activity of the HYDRL antibody. The Ka of a monoclonal antibody reagent monospecific for a particular HYDRL epitope represents a true measure of affinity. The high affinity antibody reagent having a Ka value of 10 ⁹ to 10 ¹² L / mol is
It is preferably used in immunoassays in which the HYDRL antibody complex must withstand vigorous manipulation. Low affinity antibody reagents with Ka values of 10 ⁶ to 10 ⁷ L / mol are HYDRL
Is required to dissociate from the antibody in the final activated state (immunopurifica
tion) and similar treatments. (Catty, D. (1988) Antibodies, Volume I: A Practical Approach .IRL Press, Washington, DC; Liddell, J. E
. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies , John
Wiley & Sons, New York NY).

The antibody titer and avidity of the polyclonal antibody reagents are further evaluated to determine the quality and suitability of such reagents for certain downstream applications. For example, at least 1
Polyclonal antibody reagents containing 2 mg / ml of specific antibody, preferably 5-10 mg / ml of specific antibody, are generally used in processes in which the HYDRL antibody complex must be precipitated. Specificity of antibodies and antibody titer, binding activity, quality of antibodies and guidelines for usage in various applications are publicly available. (See, eg, Catty, supra, and Coligan et al., Supra).

In another embodiment of the invention, the polynucleotide encoding HYDRL, or any fragment or complementary sequence thereof, can be used for therapeutic purposes. In certain embodiments, if the complementary sequence of a polynucleotide encoding HYDRL is suitable for blocking the transcription of mRNA, it can be used. In particular, cells can also be transformed with sequences complementary to the polynucleotide encoding HYDRL. Thus, the complementary molecule or fragment can be used to regulate the activity of HYDRL or the regulation of gene function. Such techniques are well known in the art, and sense or antisense oligonucleotides or large fragments can be designed from the control region of the HYDRL coding sequence or at various positions along the coding region.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia, or various bacterial plasmids can also be used to deliver the nucleotide sequence to the target organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to generate vectors expressing nucleic acid sequences complementary to HYDRL-encoding polynucleotides (see, eg, Sambrook et al., Supra, and Ausubel et al., Supra). .

The gene encoding HYDRL can be stopped by transforming cells or tissues with an expression vector that expresses high levels of a polynucleotide encoding HYDRL or a fragment thereof. Such constructs can be used to introduce non-translatable sense or antisense sequences into cells. Even when not integrated into DNA, such vectors continue to transcribe mRNA molecules until impaired by endogenous nucleases. Non-replicating vectors also have transient expression that lasts for a month or more, and may last even longer if suitable replication elements are part of the vector system.

[0155] As described above, the expression of a gene is the sequence complementary to the regulatory 5'or regulatory region of the gene encoding HYDRL or an antisense molecule (DNA or RNA, PNA).
Can be adjusted by designing. For example, it is possible to use an oligonucleotide derived from the transcription start site from about -10 to about +10 from the start site. Similarly, it can be blocked using "triple helix" and base pairing methods. The triple helix structure is useful because it prevents the double helix from expanding sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances with triplex DNA have been described in the literature (eg Gee, JE et al. (1994) In: H
uber, BE and BI Carr, Molecular and Immunologic Approaches , Futura
Publishing Co., Mt. Kisco, NY). Complementary sequences or antisense molecules can also be designed to block translation of mRNA by blocking the binding of transcripts to ribosomes.

Ribozymes, enzymatic RNA molecules, can be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by nucleotide strand scission.
For example, recombinant hammerhead ribozyme molecules that specifically and effectively catalyze nucleotide scission of sequences encoding HYDRL are included.

A specific ribozyme cleavage site in any potential RNA target is identified by the subsequent sequence GU.
Initially identified by scanning the target molecule against ribozyme cleavage sites including A, GUU, and GUC. Once identified, a short RNA sequence of 15 to 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site is evaluated for secondary structural features that impair the function of the oligonucleotide. Is possible. The suitability of candidate targets can also be assessed by testing their ease of hybridization with complementary oligonucleotides using a ribonuclease protection assay.

Complementary ribonucleic acid molecules and ribozymes of the invention can be made for the synthesis of nucleic acid molecules using methods well known in the art. These methods include methods of chemically synthesizing oligonucleotides such as solid phase phosphoramidite compounds. Alternatively, RNA molecules can be generated in vitro and in vivo by transcription of the DNA sequence encoding HYDRL, such DNA sequences being produced using various RNA vector promoters such as T7 or SP6. Can be incorporated into. Alternatively, these cDNA constructs that synthesize complementary RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.

Modification of RNA molecules can increase intracellular stability and prolong half-life. Possible modifications include the addition of flanking sequences at the 5'and / or 3'ends of the molecule, or the use of phosphorothionate or 2'O methyl rather than phosphodiesterase linkages within the backbone of the molecule. , Are not limited to these. Unique to PNA production, this concept is that adenine, cytidine, guanine, thymine, which are not readily recognized by endogenous endonucleases,
And the acetyl-, methyl-, thio-, and similar modified forms of uridine, as well as the inclusion of non-conventional bases such as inosine, queosine, and wybutosine, in whole of these molecules. Can be expanded to.

[0160] vectors available are a number of ways of introducing into a cell or tissue, in vivo, are equally suitable for use in in vitr o, and ex vivo. For ex vivo treatment, the vector is introduced into hepatocytes taken from a patient and cloned and propagated for autologous transplant back into the same patient. Transfection, ribosome injection or delivery by polycationic aminopolymers can be performed using methods well known in the art (eg Goldman, CK et al. (1997) Nature Biotechnology 15: 462.
-66 :).

Any of the above methods of treatment can be applied to any subject in need of treatment including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits and monkeys.

Another embodiment of the present invention relates to the administration of a pharmaceutical or sterile composition in association with a pharmaceutically acceptable carrier for all of the above mentioned therapeutic effects. Such a pharmaceutical composition comprises HYDRL, an antibody against HYDRL, a mimetic, an agonist, an antagonist, or HYDR.
It consists of L inhibitors and so on. This composition can be used alone or as a stabilizer.
It may be administered in sterile biocompatible pharmaceutical carriers with more than one additional agent. Such pharmaceutical carriers include, but are not limited to, saline, buffered saline, glucose, water and the like. This composition can be administered alone or with another agent such as a drug or hormone.

The pharmaceutical composition used in the present invention can also be administered using any number of routes. This route includes oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, ectopic, sublingual, It also includes, but is not limited to, the rectum.

In addition to the active ingredients, these pharmaceutical compositions contain suitable pharmaceutically acceptable excipients and auxiliaries, which facilitate the formulation of the active compounds into pharmaceutically usable agents. The carrier may be included. Detailed techniques for formulation and administration are described in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, PA).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers allow tablets, pills, dragees, capsules, and pharmaceutical compositions to be ingested by patients.
It is formulated as a liquid, gel, syrup, mud, or suspension.

Medicaments for oral use include tablets or dragee cores (after grinding if desired) by mixing the active compound with solid drug additives and treating the resulting granule mixture. cores). Suitable excipients include lactose, sucrose, mannitol, or sugars including sorbitol, starch from corn, wheat, rice, potato, or other plants, methylcellulose, hydroxypropylmethylcellulose, or cellulose such as sodium carboxymethylcellulose, Gum, including gum arabic and gum tragacanth, carbohydrates or protein excipients such as proteins such as gelatin and collagen.
If desired, a disintegrant or solubilizer such as cross-linked polyvinylpyrrolidone, kanten, alginic acid, or its salt sodium alginate is added.

Dragee cores are used with a suitable coating such as a concentrated sugar solvent. Such concentrated sugar solvents may include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solvents, and suitable organic or mixed solvents. Dyestuffs or pigments are added to the tablets or dragee coatings for identification of the product or to indicate the quantity of active compound, ie the dose.

Pharmaceutical formulations for oral use include push-fit capsules made of gelatin, encapsulated capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules contain the active ingredient in admixture with excipients or binders such as lactose or starch, lubricants such as talc or magnesium stearate, and, if desired, stabilizers. In soft capsules, the active compound, with or without stabilizer,
It may be dissolved or suspended in a suitable solution such as a fatty oil, solution, or polyethylene glycol solution.

Pharmaceutical agents suitable for parenteral administration are formulated in aqueous solutions, but physiologically compatible buffers such as Hank's solution, Ringer's solution, physiological buffered saline are preferred. Aqueous suspension injections may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be formulated as suitable oily injection suspensions. Suitable hydrophilic solutions or vehicles include fatty oils such as sesame oil, ethyl oleate, triglycerides or synthetic fatty acids such as ribosomes. Non-lipid polycationic amino polymers are used for delivery purposes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for concentrated solutions.

For local or nasal administration, suitable penetrants that penetrate certain barriers are used in the formulation. Such penetrants are well known to those of ordinary skill in the art.

The pharmaceutical compositions of the present invention may be prepared using methods well known in the art, such as conventional mixing, dissolving, granulating, sugar coating, elevating, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Can be manufactured.

Pharmaceutical compositions are formulated as salts and can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid. Salts are more soluble in water or other protic solvents than are the corresponding free base systems. Another drug form comprises some or all of 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, with a pH range of 4.5-5.
5, a lyophilized powder that is buffered prior to use can be used.

After the pharmaceutical compositions have been prepared, they are packaged in suitable boxes and labeled as medication for the indicated symptoms. For the administration of HYDRL, such a label would include the amount,
Frequency and method of administration will be included.

Suitable pharmaceutical compositions for use in the present invention include compositions wherein the active ingredients in effective amounts to achieve the objectives are included. Those of ordinary skill in the art can determine a dose that is sufficiently effective based on their ability.

For any composition, the therapeutically effective dose can be estimated initially either in tumor cell assays, eg, of tumor cells, or in animal models. Usually, a mouse, a rabbit, a dog, a pig, or the like is used as the animal model. Animal models can also be used to determine the appropriate concentration range and route of administration. Based on such treatment, a beneficial dose and administration route for human can be determined.

A pharmaceutically effective dose is related to the amount of active ingredients, such as HYDRL or fragments thereof, HYDRL antibodies, HYDRL agonists or antagonists, inhibitors, etc. that ameliorates symptoms or conditions. Medicinal efficacy and toxicity can be calculated, for example, by calculating the ED ₅₀ (50% of the population has a medicinal effect on dose) or LD ₅₀ (50% of the population is fatal on dose) statistics. , Can be determined by standard drug techniques in cell culture or animal studies. Dosage ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD _{50 /} ED _50. Pharmaceutical compositions that exhibit high therapeutic indices are desirable. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human applications. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the patient sensitivity, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide effective levels of the active ingredient or to maintain the desired effect. Dosage factors considered include disease severity, general health of the patient, age, weight, and sex of the patient, time and frequency of administration, concomitant medications, response susceptibility, and treatment. Contains the response. Long-acting pharmaceutical compositions can be administered every three or four days, once a week, once every two weeks, depending on the half-life and clearance rate of the particular formulation.

The usual dosage varies depending on the route of administration, but is about 0.1-100,000 μg.
Up to about 1 gram. Guidance on specific dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. One of skill in the art will utilize formulations of nucleotides that are different than the protein or inhibitor. Similarly, delivery of a polynucleotide or polypeptide is
It will be specific to a particular cell, condition, location, etc.

(Diagnosis) In another embodiment, a patient whose antibody specifically binding to HYDRL is diagnosed with a disease characterized by the expression of HYDRL or treated with HYDRL or an agonist, antagonist or inhibitor of HYDRL. Used in an assay to monitor Antibodies useful for diagnosis are formulated in the same manner as described in treatment. HYDRL diagnostic assays include methods of detecting HYDRL from human body fluids or from cells and tissues using antibodies and labels. These antibodies, used with or without modification, can be labeled by covalent or non-covalent attachment of reporter molecules. Various reporter molecules known in the art are used, some of which have been mentioned above.

Various protocols for measuring HYDRL, including ELISA, RIA, and FACS, are well known in the art and provide the basis for diagnosing altered or abnormal levels of HYDRL expression. The value of normal or standard HYDRL expression is determined by binding an antibody against HYDRL to a body fluid or cell collected from a subject such as a normal mammal, for example, a human under conditions suitable for complex formation. To do. The amount of canonical complex formation can be quantified by various methods such as photometric. The amount of HYDRL expression, control and disease in the subject, samples from biopsy tissue are compared to standard values. The deviation between the standard value and the subject is the parameter for diagnosing the disease.

According to another embodiment, the polynucleotide encoding HYDRL can also be used for diagnosis. The polynucleotides used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. This polynucleotide is used to detect and quantify the expression of genes in biopsy tissues that express HYDRL that can be correlated with disease. This diagnostic assay is used to examine the absence, presence, and overexpression of HYDRL and monitor the regulation of HYDRL levels during treatment.

In certain embodiments, it is possible to identify a nucleic acid sequence encoding HYDRL by hybridization with a PCR probe capable of detecting a polynucleotide sequence comprising a gene sequence encoding HYDRL or a closely related molecule. is there. For example, 5 '
The specificity of the probe, whether it is a highly specific region that is a regulatory region or a region with a slightly low specificity that is a conserved motif, and the stringency of hybridization or amplification depends on the probe HYDRL. It will depend on whether to identify only the natural sequences that encode or whether to identify only the natural sequences that encode the allele or related sequence.

The probe may also be used for detection of related sequences and have at least 50% sequence identity with any sequence encoding HYDRL. The desired hybridization probe of the present invention can be DNA or RNA and can be derived from the sequence of SEQ ID NO: 17-32 or the genomic sequence of HYDRL gene including promoter, enhancer and intron.

As a method for producing a hybridization probe specific to HYDRL-encoding DNA, there is a method of cloning a polynucleotide sequence encoding HYDRL and a HYDRL derivative into a vector for producing an mRNA probe. Such vectors are commercially available, well known to those skilled in the art, and are used to synthesize RNA probes in vitro by adding a suitable RNA polymerase and a suitable labeled nucleotide. The hybridization probe is, for example, ³² P.
Alternatively, it may be labeled with a radionuclide such as ³⁵ S, or a population of various reporters such as an enzyme label such as alkaline phosphatase linked to the probe by an avidin / biotin binding system.

Polynucleotide sequences encoding HYDRL can be used to diagnose disorders associated with expression of HYDRL. Examples of such disorders include, but are not limited to, cell proliferative disorders, including actinic keratoses and atherosclerosis, bursitis, cirrhosis, hepatitis, mixed Type connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and adenocarcinoma and leukemia, lymphoma, melanoma, myeloma, sarcoma, and malformations Cancer, specifically, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate, It includes salivary glands, skin, spleen, testes, thymus, uterine cancer, and also autoimmune disorders / inflammatory diseases, including inflammation, actinic keratosis, and acquired immunodeficiency syndrome ( AIDS) and adrenal insufficiency,
Adult respiratory distress syndrome, allergy, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyglandular endocrine candidiasis Ectodermal dystrophy (APECED), bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes, emphysema, lymphotoxin-induced temporary lymphopenia , Erythroblastosis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, paroxysmal nocturnal hemoglobinuria, hepatitis, hypereosinophilia, irritable colon Syndrome, lymphotoxin-induced transient lymphopenia, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis , Polycythemia vera , Polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenia, ulcerative colon Inflammation, Werner syndrome, cancer complications, hemodialysis, extracorporeal circulation and viral and bacterial infections, fungal infections, parasitic infections, protozoal infections, helminthic infections, trauma and lymphoma, leukemia, and Includes hematopoietic cancers including myeloma, also includes neuropathy,
Among them are epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasm, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders,
Amyotrophic lateral sclerosis and other motor neuron disorders, progressive neuromuscular atrophy, retinitis pigmentosa, hereditary ataxia, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis , Brain abscess, subdural empyema, epidural abscess, purulent intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease and Kuru and Creutzfeldt-Jakob disease, Gerstmann syndrome, Gerstmann -Prion disease including Straussler-Scheinker syndrome and fatal familial insomnia, neurotrophic and metabolic diseases, neurofibromatosis, tuberous sclerosis, cerebellar retinal hemangioblastoma (cerebelloretin)
al hemangioblastomatosis), cerebral trigeminal neurovascular syndrome, central nervous system mental retardation and other developmental disorders, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, myelopathy, muscular dystrophy and other neuromuscular disorders, Peripheral neuropathy, dermatomyositis and polymyositis, hereditary, metabolic, endocrine, and toxic myopathy, myasthenia gravis, periodic quadriplegia, mood and anxiety psychosis, and delusional psychosis And seasonal affective disorder (SAD), akathisia, amnesia, catatonic disease, diabetic neuropathy,
Includes extrapyramidal terminal deficiency syndrome, dystonia, schizophrenia, postherpetic neuralgia, and Tourette's disease, and also includes renal impairment, including amyloid nephropathy, hypertension, primary Cystic disease of the kidney such as aldosteronism, adrenal insufficiency, renal failure, nephritis, chronic nephritis, tubuloinserstitial nephritis, polycystic disease, dysplastic malformations such as renal dysplasia, cortical or Congenital polycystic renal dysplasia (PRD), including medullary cystosis, autosomal dominant and recessive congenital polycystic renal dysplasia (PRD), medullary cystosis, cavernous and tubular dysplasia, Alport Non-renal cancers that have a physiological effect on the kidneys, such as the syndrome, tumors of the lungs and tumors of the fundus of the brain,
Includes multiple myeloma, adenocarcinoma of the kidney, metastatic renal cancer, and also adrenal insufficiency, including gut inflammation, anaphylaxis, arrhythmias, asthma, cardiovascular shock, Cushing's syndrome. , Hypertension, hypoglycemia, myocardial infarction, migraine headache, pheochromocytoma, and genetic disorders, including GM1 gangliosidosis, Niemann-Pick disease, adrenoleukodystrophy, Alport syndrome , Colloideremia, Duchenne-Becker muscular dystrophy, Down syndrome, cystic fibrosis, chronic granulomatosis, Gaucher disease, Huntington's disease, Marfan syndrome, muscular dystrophy, myotonic dystrophy, frequent bone dystrophy (pycnodysostosis), Refsum syndrome, retinoblastoma, sickle cell anemia, thalassemia, Werner syndrome, von Willebrand disease, von Hippel-li Dow syndrome, Wilms' tumor, Zellweger syndrome, peroxisomal acyl -CoA oxidase deficiency (peroxisomal acyl-CoA o
xidase deficiency), peroxisome thiolase deficiency, peroxisome bifunctional protein deficiency, mitochondrial carnitine palmitoyl transferase and carnitine deficiency, mitochondrial ultralong chain acyl-CoA dehydrogenase deficiency, mitochondrial short chain acyl-CoA dehydrogenase deficiency, mitochondrial electron transfer Flavoproteins and electron transfer flavoproteins: ubiquinone oxidoreductase deficiency, mitochondrial trifunctional protein deficiency, mitochondrial short chain 3-
Includes hydroxyacyl-CoA dehydrogenase deficiency. The polynucleotide sequence encoding HYDRL can be obtained by the Southern method, Northern method, dot blot method, or other membrane-based technology, PCR method, dipstick, pin,
It can be used in ELISA-based assays and microarrays that utilize body fluids or tissues collected from patients to detect the expression of mutant HYDRL. Such qualitative or quantitative methods are well known in the art.

In a particular form, the nucleotide sequences encoding HYDRL will be useful in assays to detect related disorders, in particular the disorders mentioned above. The nucleotide sequence encoding HYDRL could be labeled by standard methods and added to a body fluid or tissue sample taken from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the samples are washed and the signal is quantified and compared to standard values. If the amount of signal in the patient sample is significantly altered compared to the control sample, the level of mutation of the nucleotide sequence encoding HYDRL in the sample reveals the presence of the associated disease. Such assays can be used to estimate the effectiveness of a particular treatment in animal studies, clinical trials, or monitoring individual patient treatment.

Normal or canonical expression profiles are established to provide a basis for diagnosis of diseases associated with expression of HYDRL. This can be achieved by combining the HYDRL coding sequence or a fragment thereof with a body fluid or cell extracted from a normal subject, either animal or human, under conditions suitable for hybridization or amplification. . Standard hybridizations can be quantitated by comparing the values obtained from normal subjects to those from experiments in which known amounts of substantially purified polynucleotides are used. Standard values obtained from normal samples can be compared with those obtained from subjects who exhibit symptoms of the disease. Deviation between standard and subject values is used to establish the presence of disease.

Once the presence of the disease is established and the treatment protocol is initiated, the hybridization assay can be repeated on a regular basis to estimate whether the level of expression in the subject has begun to approach the values shown in normal patients. It is possible. The results of repeated assays can be used to see the effect of treatment over a period of days to months.

In cancer, abnormal amounts of transcripts in living tissue from an individual predispose to the development of the disease and may provide a method of detecting the disease before actual clinical symptoms develop. is there. This type of clearer diagnosis allows medical professionals to use preventative or aggressive therapies early to prevent the development or progression of cancer.

Further diagnostic uses for oligonucleotides designed from the HYDRL coding sequence may include the use of PCR. Such oligomers can be chemically synthesized, enzymatically produced, or produced in vitro . The oligomer preferably contains a fragment of a polynucleotide encoding HYDRL or a fragment of a polynucleotide complementary to the polynucleotide encoding HYDRL, and is used to identify a specific gene or condition under the optimum conditions. It Further, the oligomer can be used for detecting and / or quantifying a closely related DNA or RNA sequence under slightly mild stringent conditions.

Methods that can be used to quantify the expression of HYDRL include radiolabeling or biotin labeling of nucleotides, coamplification of regulatory nucleic acids, and standard curves with the results added. (For example, Melby, PC et al. (1993) J. I.
mmunol. Methods, 159: 235-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The quantitation rate of a large number of samples was accelerated by using a high throughput assay in which the oligomer of interest was included in different dilutions and quantified by spectrophotometry or non-color response.

In another example, oligonucleotides or longer fragments from any of the polynucleotide sequences described herein are used as targets in microarrays. Microarrays are used to monitor the expression levels of a large number of genes at the same time and identify gene mutations, mutations and polymorphisms. This information is useful in determining gene function, interpreting the genetic basis of disease, diagnosing disease, and monitoring and developing the activity of therapeutic agents.

Microarrays are prepared, used, and analyzed by methods well known in the art. (For example, Brennan, TM et al. (1995) US Pat. No. 5,474,796; Schena, M. et al. (1996) Pro.
c. Natl. Acad. Sci. 93: 10614-10619; Baldeschweiler et al. (1995) PCT application number W
O95 / 251116; Shalon, D. et al. (1995) PCT application number WO95 / 35505; Heller, RA et al. (1
997) Proc. Natl. Acad. Sci. 94: 2150-2155; and Heller, MJ et al. (1997) U.S. Pat.No. 5,605,662) Another embodiment of the invention also uses a nucleic acid sequence encoding HYDRL. Thus, it is possible to generate hybridization probes useful for mapping naturally occurring genomic sequences. This array maps to: It is a specific chromosome, a specific region of the chromosome or an artificially generated chromosome, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a bacterial P1 product or a single chromosome cDNA library. (Eg Harrington, 1.3. Etc.
(1997) Nat Genet. 15: 345-355; Price, CM (1993) Blood Rev. 5-87-134,
And Trask, BJ (1991) Trends Genet. 7: 149-154) in sit fluorescence hybridization (FISH) will correlate with other physical chromosome mapping techniques and genetic map data (eg Heinz- Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.). Examples of genetic map data can be found in various scientific journals or the World Wide Web site of the Online Mendelian Inheritance in Man (OMIM). On a physical chromosome map
The correlation between the location of the gene encoding HYDRL and a particular disease, or the predisposition to a particular disease, helps determine the region of DNA associated with such a disease. The nucleotide sequences of the present invention may also be used to detect differences in gene sequences between normal, carrier and infected individuals.

The genetic map can also be extended using physical mapping techniques such as in sit hybridization of chromosomal preparations and binding analysis using established chromosomal markers. By placing the gene on the chromosome of another mammal, such as a mouse,
Relevant markers are often revealed, even if the number or arm of a particular human chromosome is not known. The new array by physical mapping
It can also be assigned to a chromosome arm. This is valuable information for researchers studying genetic disorders using positional cloning or other gene discovery techniques. The location of the disease or syndrome is, for example, 11q22-
Once roughly determined by gene binding to a particular gene region such as 23, any sequence mapping for that region represents a relevant gene or regulatory gene for further investigation (eg, Gatti, RA et al. (1988). Nature 336: 577-580
See). Further, by using the nucleotide sequence of the present invention of interest, a normal person, a holder,
That is, a difference in chromosomal position due to translocation, inversion, etc. between infected persons may be detected.

In another embodiment of the invention, HYDRL, its catalytic or immunogenic fragments or oligopeptides thereof can be used to screen a library of compounds in any of a variety of drug screening techniques. Fragments used for such screening may be free in solution, immobilized on a solid support, retained on the surface of cells, or present intracellularly. Formation by binding the complex of HYDRL with the drug being tested may be measured.

Another method used for drug screening is used to increase the screening throughput of compounds with suitable binding affinities for the protein of interest (eg, Geysen, et al. (1984) PCT Application No. See WO84 / 03564). In this method, a significant number of different small test compounds are synthesized on plastic pins or other substrates. The test compound is reacted with HYDRL, or a fragment thereof, and then washed. Bound HYDRL is then detected by methods well known in the art.
Purified HYDRL can also be coated directly onto the plates used in the drug screening techniques described above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another example, competitive drug screening assays can be used in which neutralizing antibodies capable of binding HYDRL specifically compete with the test compound for binding HYDRL. In this method, the antibody detects the presence of any peptide that shares one or more antigenic determinants with HYDRL.

In another embodiment, nucleotide sequences encoding HYDRL are used in developing molecular biology techniques, including, but not limited to, currently known nucleotides such as the triplet code and specific base pair interactions. New technologies can be provided that depend on the characteristics of the sequence.

Those skilled in the art will be able to make full use of the present invention without further explanation given the preceding description. Therefore, the preferred specific embodiments described below are for purposes of illustration and not limitation of the invention.

All patent applications, patents, publications, particularly US applications [1998] mentioned above and below.
No. PF-0643P filed on November 12, 2012 and US serial number 60 / 135,51
References to 9 are incorporated herein by reference.

Example 1 Preparation of cDNA Library RNA was purchased from Clontech or isolated from the tissues listed in Table 4. A tissue was homogenized and dissolved in a guanidinium isothiocyanate solution. On the one hand, another tissue is homogenized and dissolved in phenol, or TRIZOL (
Life Technologies), guanidinium isothiocyanate and a suitable modifier such as a single phase solution of phenol. The lysate was centrifuged in cesium chloride or extracted with chloroform. RNA was precipitated from this lysate with either isopropanol or sodium acetate and ethanol, or otherwise.

RNA extraction with phenol and precipitation were repeated as many times as necessary to increase RNA purity. In some cases, RNA is treated with a DNA degrading enzyme. Most libraries have oligo d (T) -linked paramagnetic particles (Promega) or OLIGOTEX latex particles (QIAGEN).
Valencia CA), OLIGOTEX mRNA purification kit (QIAGEN) was used to isolate poly (A +) RNA. Alternatively, another RNA isolation kit such as the POLY (A) PURE mRNA purification kit (Ambion, Austin TX) was used to isolate directly from tissue lysates.

In some cases, RNA was provided to Stratagene and a cDNA library corresponding to Stratagene was created. Otherwise, UNIZAP vector system (Stratagene)
Alternatively, a cDNA library was prepared by using the SUPERSCRIPT plasmid system (Life Technologies) to synthesize cDNA by a recommended method known in the art or a similar method.
(See, eg, Ausubel, 1997, supra, Units 5.1-6.6). Reverse transcription is oligo d (T)
Or started with random primers. A synthetic oligonucleotide adapter was attached to the double-stranded cDNA and the cDNA was digested with a suitable restriction enzyme or restriction enzymes. For most libraries, SEPHACRYL S 1000 or SEPHAROSE
CL2B, SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech
), And the size of the cDNA (300-1000 bp) was selected by agarose gel electrophoresis. PBLUESCRIPT plasmid (Stratagene) or pSPORT1 plasmid (Life Technolo
gies), plNCY (Incyte Pharmaceuticals, Palo Alto CA), etc., and the cDNA was ligated to the compatible restriction enzyme site of the polylinker of a suitable plasmid. Use this recombinant plasmid with Stratagene XL1-Blue, XL1-BIueMRF, SOLR, or Life Technologi.
The cells were transformed into competent E. coli cells containing es DH5α or DH10B and ELECTROMAX DH10B.

2 Isolation of cDNA clones Plasmids were recovered from host cells by in vivo excision utilizing the UNIZAP vector system (Stratagene) or cell lysis. Magic or WIZARD Minipreps
DNA purification system (Promega) and AGTC Miniprep purification kit (Edge Biosystems, G
aithersburg MD), QIAGEN's QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QI
Plasmids were purified using at least one of the AWELL 8 Ultra Plasmid Purification System, REAL Prep 96 Plasmid Kit. After precipitation, it was resuspended in 0.1 ml of distilled water and stored at 4 ° C. with or without freeze-drying.

Alternatively, plasmid DNA was amplified from host cell lysates by a high throughput direct ligation PCR method. (Rao, VB (1994) Anal. Biochem. 216: 1-14). Host cell lysis and thermal cycling processes were performed in a single reaction mixture. Samples are processed and transferred to 384-well plates for storage and the concentration of amplified plasmid DNA is quantified using PICOGREEN dye (Molecular Probes, Eugene OR) and Fluoroskan II fluorescence scanner (Labsystems Oy, Helsinki, Finland). Measured.

3 Sequencing and Analysis cDNA sequencing reactions can be performed using standard methods or ABI CATALYST 800 (
Perkin-Elmer) thermal cycler or PTC-200 thermal cycler (MJ Research)
HYDRA Microdispenser (Robbins Scientific) or MICROLAB 2200 (Ham
ilton) High throughput equipment such as combination with liquid transfer system. c
For the preparation of a DNA sequencing reaction, a reagent from Amersham Pharmacia Biotech or a reagent contained in an ABI sequencing kit such as ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer) was used. c
For electrophoretic separation of DNA sequencing reactions and detection of labeled polynucleotides, the MEGABACE 1000 DNA Sequencing System (Molecular Dynamics
), ABI PRISM 373 using standard ABI protocol and base pair calling software
Alternatively, the 377 Sequencing System (Perkin-Elmer), other sequence analysis systems well known in the art, was used. The open reading frame of the cDNA sequence is standard (Ausubel, 1997
, Unit 7.7), supra. Some of the cDNA sequences were selected and extended by the method described in 5 of this example.

The construction and analysis of the polynucleotide sequences obtained from the sequencing of the cDNA was carried out in combination with software utilizing algorithms well known to those skilled in the art.
Table 5 outlines the tools, software, algorithms utilized, their description, references, and threshold parameters used. Column 1 of Table 5 is the tools and programs used, algorithms, column 2 is a brief description thereof, column 3 is a reference cited as part of this specification by reference, column 4 is a sequence of two sequences. The score, probability value, and other parameters used in the evaluation of the degree of coincidence are shown (the higher the probability value, the higher the homology between sequences). MACDNASIS PRO software (Hitachi Softwa
re Engineering, S. San Francisco CA) and LASER GENE software (DNASTAR)
Was used.

Confirmation of polynucleotide sequences is accomplished by using BLAST and dynamic programming, programs and algorithms based on dinucleotide nearest neighbor analysis to remove vectors and linkers, poly A sequences and mask ambiguous base pairs. I went there. next,
Using databases based on BLAST, FASTA and BLIMPS, public databases such as GenBank primates and rodents, mammals, vertebrates, eukaryotic databases and BLOCKS, PRINTS, DOMO, PRODOM and PFAM databases. Annotations were obtained by querying these sequences against sequences selected from. These sequences were constructed into full-length polynucleotide sequences using programs based on Phred and Phrap, Consed and screened for open reading frames with programs based on BLAST and FASTA, BLIMPS. The full-length polynucleotide sequence is translated to derive the corresponding full-length amino acid sequence, and the GenBank database (above) and Swi
These full-length sequences were analyzed by querying databases such as ssProt, BLOCKS, PRINTS, DOMO, PRODOM and Prosite, or Hidden Markov Model (HMM) based protein family databases such as PFAM. HMMs use probabilities to analyze the consensus primary structure of gene families (eg, Eddy
, SR (1996) Curr. Opin. Struct. Biol. 6: 361-365).

The programs described above for the construction and analysis of full length polynucleotide and amino acid sequences can also be used to identify fragments of the polynucleotide sequence from SEQ ID NO: 17-32. Fragments of about 20 to 4000 nucleotides are useful for hybridization and amplification and have been described in the invention above.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of transcripts of a gene, labeled nucleotides on the membrane to which RNA from a particular cell type or tissue is bound. With sequence hybridization (see, for example, Sambrook, supra,
Chapter 7; and Ausubel. FM et al., Supra, Chapters 4 and 16).

Using similar computer technology for BLAST, GenBank or LIFESEQ (Inc
Search for identical or related molecules in nucleotide databases such as yte Pharmaceuticals). This assay is much faster than many membrane-based hybridizations. You can also change the sensitivity of the computer search so that any particular match
It can be established whether it is classified as an exact match or a homologous match. The search criterion is the product score defined as (% sequence identity x% maximum BLAST score) / 100. The product score takes into account both the similarity between the two sequences and the length of the sequence match. For example, if the product score is 40, the match is 1 to 2
Exact within% error, at 70 the match would be exact. Similar molecules are usually identified by selecting those molecules that show a product score in the range of 15-40, although lower scores may identify related molecules.

Results of Northern analysis are reported as the percent distribution of the library in which transcripts encoding HYDRL occurred. The analysis also includes classification of the cDNA library by organ / tissue and disease. Organ / tissue categories include cardiovascular, skin, development, endocrine, gastrointestinal, hematopoietic / immune, musculoskeletal, neural, reproductive, urological. Disease categories include cancer, inflammation / trauma, cell proliferation, nerves, pooled. By category, the number of libraries expressing the sequence of interest was counted and divided by the number of libraries in the entire range. Table 3 shows the ratio (percentage) of specific expression in each tissue and the ratio of expression in each disease.

Chromosomal Mapping of the Polynucleotide Encoding 5 HYDRL The cDNA sequence used to construct SEQ ID NO: 18-32 was prepared using BLAST or Smith-Wat.
The erman algorithm is used to compare sequences with the Insight LIFESEQ database and public databases. Sequences from these databases that match SEQ ID NO: 18-32 were incorporated into contiguous and overlapping cluster sequences using construction algorithms such as Phrap (Table 5). Stanford Human Genonse Center (SHGC), Wh
Public radiation hybrid and gene mapping data such as theitehead Institute for Genome Research (WIGR) and Ge'ne'thon will be used to determine if the clustered sequences have already been mapped. If the cluster contains mapped sequences, all sequences in that cluster (
A specific SEQ ID NO) was assigned to that map position.

The positions of the gene maps of SEQ ID NO: 18, SEQ ID NO: 25, and SEQ ID NO: 28 have been described herein as sections or ranges of the human chromosome. SEQ ID NO: 18 and SEQ ID N
For O: 25, more than one map position is listed, which is SEQ ID NO: 18.
And that the previously mapped sequence similar to SEQ ID NO: 25 but not completely identical is included in each cluster. The range of map positions indicated in centimorgan was measured from the end of the p-arm of the chromosome (centimorgan (cM) is a unit that represents the distance based on the transfer rate between genes on the same chromosome). On average, 1 cM is approximately equal to 1 megabase of human chromosomes, but can vary significantly due to the high recombination rate and the low recombination rate). cm
The distances are based on the genetic markers mapped by Ge'ne'thon which delineate the boundaries of the radiation hybrid markers containing sequences in each cluster. Where there are diseases associated with the public and insight sequences located within the indicated range, they are listed in the invention section.

6 Extending the Polynucleotide Encoding HYDRL The full-length nucleic acid sequence of SEQ ID NO: 17-32 is prepared using oligonucleotide primers designed from suitable fragments of the full-length molecule. The fragment was extended and produced. One primer was synthesized to initiate the 5'extension of the known fragment and the other primer was synthesized to initiate the 3'extension of the known fragment. The starting primer has a GC content of about 50% or more and a length of about 22 to about 30 nucleotides and a pH of about 68 using OLIGO 4.06 software (National Biosciences) or other suitable program. It was designed to anneal to the target sequence at a temperature of ~ 72 ° C. The nucleotides were extended so that hairpin structures and primer-primer dimers did not occur.

This sequence was extended with selected human cDNA libraries. If more than one extension is required or desired, additional or nested primer sets are designed.

Amplification was performed with high fidelity by the PCR method using a method known in the art. PCR is PTC-200
It was performed in a 96-well block plate using a thermal cycler (MJ Research, Inc.). The reaction mixture consisted of template DNA, 200 nmol of each primer, Mg ²⁺ and (NH ₄ ) ₂ SO ₄ and β.
− Buffer containing mercaptoethanol, Taq DNA polymerase (Amersham Pha
rmacia Biotech), ELONGASE enzyme (Life Technologies), Pfu DNA polymerase (St
ratagene). Amplification was performed on the primer set, PCI A and PCI B, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 60 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Storage at 4 ° C Alternatively, amplification was performed on the primer set, T7 and SK +, with the following parameters. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 57 ° C. for 1 minute Step 4 68 ° C. for 2 minutes Step 5 Repeat steps 2, 3, and 4 20 times Step 6 68 ° C. for 5 minutes Step 7 Store at 4 ° C.

The DNA concentration in each well was dissolved in 1 × TE and 0.5 μl of undiluted PCR product.
100 μl PICOGREEN quantification reagent (0.25% (v / v) PICOGREEN; Molecular Probes, Eugen
e OR) is distributed to each well of an opaque fluorometer plate (Coming Costar, Acton MA) to allow the DNA to bind to the reagent and be measured. Fluoroskan this plate
Scan with II (Labsystems Oy, Helsinki, Finland) and measure the fluorescence of the sample to quantify the concentration of DNA. A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresis on a 1% agarose minigel to determine which reaction succeeded in extending the sequence.

The extended nucleotides are desalted and concentrated before being transferred to a 384 well plate and Cvi
JI Cholera virus endonuclease (Molecular Biology Research, Madison W
Digested with I) and sonicated or sheared before religation to pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on a low concentration (0.6-0.8%) agarose gel to cut the fragments and the agar was digested with Agar ACE (Promega). T4 Ligase (New England B
clones extended with iolabs, Beverly MA) were cloned into pUC 18 vector (Amersham Pha
rmacia Biotech) and treated with Pfu DNA polymerase (Stratagene) for restriction site extension to transfect competent E. coli cells. The transfected cells were selected and transferred to a medium containing antibiotics, each colony was cut off, and cultured in a 384-well plate of LB / 2X carbenicillin culture solution at 37 ° C. overnight.

The cells were lysed and Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu
DNA was PCR amplified using DNA polymerase (Stratagene) according to the following procedure. Step 1 94 ° C. for 3 minutes Step 2 94 ° C. for 15 seconds Step 3 60 ° C. for 1 minute Step 4 72 ° C. for 2 minutes Step 5 Repeat Steps 2, 3 and 4 29 times Step 6 72 ° C. for 5 minutes Step 7 Store at 4 ° C. DNA was quantified with PICOGREEN reagent (Molecular Probes) as described above. Samples with poor DNA recovery were amplified again under the above conditions. Dilute the sample with 20% dimethysulphoxide (1-15, v / v) and use the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or ABI PRISM BIGDYE Terminator cycle.
Sequencing was performed using the sequencing ready reaction kit (Perkin-Elmer).

Similarly, following the procedure described above, utilizing the nucleotide sequence of SEQ ID NO: 17-32 and using the oligonucleotide designed for this extension and a suitable gene library, 5 '
The regulatory sequence was obtained.

7 Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID NO: 17-32 are used to screen cDNA, mRNA, or genomic DNA. Special mention is made of labeling of oligonucleotides of about 20 base pairs, but essentially the same procedure is used for larger cDNA fragments. Oligonucleotides were designed using state-of-the-art software, such as OLIGO 4.06 software (National Bioscience), with 50 pmol of each oligomer and 250 μCi of [γ- ³² P] adenosine triphosphate (A
mersham, Chicago, IL) and T4 polynucleotide kinase (DuPont NEN, Bost)
on MA) and used in combination for labeling. The labeled oligonucleotide was loaded onto a SEPHADEX G-25 ultrafine exclusion dextran bead column (Amersham Phar
Substantially purified using macia Biotech). An aliquot containing the labeled probe at 10 ⁷ counts per minute was added to the following endonucleases: Ase I, Bgl II, Eco R.
Human genome cleaved with one of I, Pst I, Xba1 or Pvu II (DuPont NEN)
Used in typical membrane-based hybridization analysis of DNA.

DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, the blots are sequentially washed at room temperature, eg, under conditions of max 0.1 × sodium citrate saline and 0.5% sodium dodecyl sulfate. The hybridization patterns are visualized and compared by autoradiography or another imaging means.

8 It is possible to synthesize array elements on the surface of a substrate using a microarray chemical bonding method and an inkjet device (see eg Baldeschweiler, supra). Using an array similar to dot blotting or slot blotting, heat, UV,
It is placed and bonded to the surface of the substrate using mechanical or chemical bonding methods. A typical array can be made manually or using available methods and machines and can include any suitable number of elements. After hybridization, unhybridized probe is removed and a scanner is used to determine the level and pattern of fluorescence. Scanned images can be analyzed to determine the degree of complementarity and the relative amount / expression level of each probe that hybridizes to the element on the microarray.

Full-length cDNAs, expressed gene sequence fragments (ESTs), or fragments thereof, may form part of the microarray. A fragment suitable for hybridization is LASERG
Selection can be made using software known in the art such as ENE software (DNASTAR). A full-length cDNA corresponding to one of the nucleic acid sequences of the invention, EST,
Alternatively, those fragments, or cDNA arbitrarily selected from the cDNA library related to the present invention, are aligned on a suitable substrate such as a glass slide. cDNA is, for example, UV
It is immobilized on slides using UV cross-linking, then heat and chemical treated and finally dried (eg Schena, M. et al. (1995) Science.
270: 467-470; and Shalon, D. et al. (1996) Genome Res. 6: 639-645). A fluorescent probe is prepared and used to hybridize to the element on the substrate. This substrate is analyzed as described above.

9 A sequence complementary to the sequence encoding the complementary polynucleotide HYDRL, or any portion thereof, is used to reduce or inhibit the expression of naturally occurring HYDRL. Although the use of oligonucleotides containing from about 15 to about 30 base pairs is described, essentially the same method can be used with smaller or larger sequence fragments. Oligo4.0
6 Use software (National Biosciences) and HYDRL coding sequences to design appropriate oligonucleotides. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5'sequence and used to prevent the promoter from binding to the coding sequence. To inhibit translation, complementary oligonucleotides are designed to prevent the ribosome from binding to the HYDRL-encoding transcript.

10 Expression of HYDRL Expression and purification of HYDRL can be performed using a bacterial or virus-based expression system. For HYDRL expression in bacteria, the cDNA is subcloned into a suitable vector containing an antibiotic resistance and an inducible promoter that enhances the transcription level of the cDNA. Such promoters include a T associated with the lac operator regulatory element.
5 or T7 bacteriophage promoters and trp-lac (tac) hybrid promoters, but are not limited thereto. Recombinant vector,
Transform into a suitable bacterial host such as BL21 (DE3). Bacteria with antibiotic resistance
It expresses HYDRL when induced with isopropyl β-D thiogalactopyranoside (IPTG). Expression of HYDRL in eukaryotic cells is accomplished by infecting insect or mammalian cell lines with the Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with the cDNA encoding HYDRL by either homologous recombination or bacterial-mediated gene transfer involving transfer plasmid mediated. Viral infectivity is maintained and the strong polyhedrin promoter results in high levels of cDNA transcription. Recombinant baculovirus is often used to infect Spodoptera frugiperda (Sf9) insect cells, but can also be used to infect human hepatocytes. In the case of the latter infection, further genetic modification of the baculovirus is required. (For example, Engelhard.EK et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3224-3227; S
andig, V. et al. (1996) Hum. Gene Ther. 7: 1937-1945.).

In most expression systems, HYDRL is used, for example glutathione S transferase (GST).
, Or a fusion protein synthesized with a peptide epitope tag such as FLAG or 6-His, the affinity-based purification of the recombinant fusion protein from crude cell lysate can be performed rapidly in one step. The 26-kilodalton enzyme GST from Schistosoma japonicum enables purification of fusion proteins with immobilized glutathione while maintaining protein activity and antigenicity (Amersham Pharma
cia Biotech). After purification, the GST moiety can be proteolytically cleaved from HYDRL at specific manipulation sites. FLAG, a peptide of 8 amino acids, allows immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His in which 6 histidine residues are continuously extended enables purification with a metal chelate resin (QIAGEN). The method of protein expression and purification is described by Ausu
bel (1995, supra, ch 10, 16). HYDRL purified by these methods
Can be used directly to perform the following assays.

11 Demonstration of HYDRL Activity For purposes of illustration, an assay for measuring β-glucosidase and lysozyme activity of the HYDRL molecule is described. Assays for β-glucosidase activity showed varying amounts of HYDR
L to 1 mM 4-nitrophenyl β-in 50 mM sodium acetate buffer (pH 5.0)
Incubate with D-glycopyranoside (substrate) at 37 ° C for various times (typically 1-5 minutes). The reaction is terminated by heating at 100 ° C for 2 minutes. The absorbance at 410 nm is measured with a spectrophotometer. This value is proportional to the β-glucosidase activity of HYDRL in the sample (see Hrmova, M et al. (1998) J. Biol. Chem. 273: 11134-11143).

The lysozyme activity of HYDRL can be demonstrated by its ability to lyse Micrococcus lysodeikticus bacterial cells (eg, Enzymatic Assay of Lysozyme 1 , Sigma Aldri.
ch, St. Louis MO). 66 mM potassium phosphate buffer (pH6.24) at 25 ℃
Lyophilized Micrococcus lysodeikticus cells (ATCC 4698 ) in (buffer A)
To 0.015% suspension. Pipet a 2.5 ml aliquot of the cell suspension into an optical cuvette and bring to 25 ° C. Monitor with a thermostat spectrophotometer until the absorbance at 450 nm is constant between 0.6 and 0.7. Prepare in a second cuvette containing 2.5 ml Buffer A to prevent reaction. H
Dissolve YDRL in chilled buffer A. Add a 0.1 ml aliquot of the HYDRL solution to the test cuvette and 0.1 ml Buffer A to the empty cuvette. The cuvette is inverted and mixed quickly, and the decrease in absorbance at 450 nm is recorded for about 5 minutes.
When bacteria lyse, the turbidity decreases and the absorbance at 450 nm also decreases. The rate of decrease in absorbance of the test cuvette at 450 nm is proportional to the HYDRL lysozyme activity in the original sample.

12 Functional assay function is HYDRL at physiologically elevated levels in mammalian cell culture systems.
Is evaluated by the expression of the sequence encoding The cDNA is subcloned into a mammalian expression vector containing a strong promoter that expresses the cDNA at high levels. Such vectors include pCMV SPORT ^™ (Life Technologies.) And pCR 3.1 (Invitr
ogen, Carlsbad, CA), both containing the cytomegalovirus promoter. For example, 5 to 10 μg of the recombinant vector is transiently transfected into a human cell line derived from endothelium or hematopoiesis by liposome preparation or electroporation. In addition, 1-2 μg of plasmid containing the sequence encoding the labeled protein is co-transfected. Expression of the labeled protein allows to distinguish between transfected and non-transfected cells. Moreover, the expression of the cDNA from the recombinant vector can be accurately predicted by the expression of the labeled protein. Such labeled proteins include
Includes green fluorescent protein (GFP; Clontech), and CD64 or CD64-GFP fusion protein. Automated flow cytometry (FCM) using technology based on laser optics
Are used to identify transfected cells expressing GFP or CD64-GFP and to assess their apoptotic status and other cellular characteristics. In addition, FCM detects and weighs uptake of fluorescent molecules that diagnoses the phenomenon of preceding or simultaneous cell death. These phenomena are measured by changes in nuclear DNA content as measured by DNA staining with propidium iodide and by a decrease in bromodeoxyuridine uptake.
Down-regulation of DNA synthesis, changes in cell surface and intracellular protein expression measured by reactivity with specific antibodies, and cytoplasm measured by binding of fluorescent complex annexin V protein to cell surface And changes in film composition. Flow cytometry by Ormerod, MG (1994) Flow Cytometry Oxford, New York, N
It is described in Y.

[0232] The effect of HYDRL on gene expression depends on the sequence encoding HYDRL and CD64 or CD64.
-A highly purified cell population transfected with either GFP can be evaluated. CD64 or CD64-GFP is expressed on the surface of transformed cells and binds to a conserved region of human immunoglobulin G (IgG). Transformed and non-transformed cells can be separated using magnetic beads coated with either human IgG or antibodies against CD64 (DYNAL. Lake Success. NY). mRNA can be purified from cells by methods known in the art. Expression of mRNA encoding HYDRL and other genes of interest can be analyzed by Northern analysis or microarray technology.

13 Production of Antibodies Specific for HYDRL Polyacrylamide gel electrophoresis (PAGE; eg, Harrington, MG (1990)
Methods Enzymol. 1816-3088-495) or substantially purified by other purification techniques, and HYDRL is used to immunize rabbits using standard protocols to generate antibodies.

Alternatively, the HYDRL amino acid sequence can be analyzed using LASERGENE software (DNASTAR) to determine highly immunogenic regions and the corresponding oligopeptides synthesized and used to determine which methods are well known to those of skill in the art. To produce antibodies. Selection of suitable epitopes, such as those near the C-terminus or within adjacent hydrophilic regions, is well known in the art (see, eg, Ausubel, 1995, supra, Chapter 11).

Generally, oligopeptides of about 15 residues in length were synthesized by fmoc chemistry using an Applied Biosystems peptide synthesizer ABI 431A Peptide Synthesizer (Perkin-Elmer), and N-maleimidobenzoyl-N-hydroxy was synthesized. KLH (Sigma-Aldrich, St. Louis M by reaction with succinimide ester (MBS)
O) to enhance immunogenicity (see, eg, Ausubel, 1995, supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity and anti-HYDRL activity of the obtained antiserum, the peptide or HYDRL was bound to a substrate, blocked with 1% BSA, reacted with rabbit antiserum and washed, and then radioactive iodine was added. Labeled goat anti-rabbit Ig
React with G.

14 Purification of Spontaneous HYDRL Using Specific Antibodies Spontaneous HYDRL or recombinant HYDRL is substantially purified by immunoaffinity chromatography using an antibody specific for HYDRL. The immunoaffinity column is constructed by covalently linking an anti-HYDRL antibody with a resin for activation chromatography such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After binding, the resin is blocked and washed according to the manufacturer's instructions.

A culture solution containing HYDRL is passed through an immunoaffinity column, and the column is washed under conditions capable of preferentially adsorbing HYDRL (for example, with a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and HYDRL (for example, a buffer of pH 2-3 or a high concentration of chaotropic ion such as urea or thiocyanate ion), and HYDRL is recovered.

15 Identification of Molecules that Interact with HYDRL HYDRL, or a biologically active fragment thereof, was labeled with ¹²⁵ I Bolton-Hunter reagent (see, eg, Bolton AE and WM Hunter (1973) Biochem. J. 133: 529). Label with. Candidate molecules pre-arranged in multiwell plates were labeled with HY
Incubate with DRL, wash and assay all wells with labeled HYDRL complex. The data obtained at various HYDRL concentrations are used to calculate numerical values for the association, affinity, and number of candidate molecules with HYDRL.

Those skilled in the art will be able to make various modifications of the described methods and systems of the present invention without departing from the scope and spirit of the invention. Although the present invention has been described with reference to particular preferred embodiments, it should be understood that the scope of the invention should not be unduly limited to such particular 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 within the scope of the following claims.

BRIEF DESCRIPTION OF THE TABLES Table 1 lists the polypeptide and nucleotide sequences used to generate the full length sequence encoding HYDRL (SEQ ID NO), clone identification number, cDN
A library and a cDNA fragment are shown.

Table 2 lists the characteristics of each polypeptide sequence, including latent motifs and homologous sequences, and
The method, algorithm and searchable database used for HYDRL analysis are shown.

Table 3 shows selected fragments of each nucleic acid sequence, tissue-specific expression patterns of each nucleic acid sequence determined by Northern analysis, diseases, disorders and conditions associated with these tissues, and each DNA. Shows the cloned vector.

Table 4 shows the tissues used to create the cDNA library in which the cDNA clone encoding HYDRL was isolated.

Table 5 shows the tools, programs, and algorithms used in the analysis of HYDRL, along with a description, references, and threshold parameters.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Brief description of drawings]

Figure 1: LASERGENE software multi-sequence alignment program (DNASTE
R, Madison WI), HYDRL-1 (Incyte clone number 229376)
4; SEQ ID NO: 1) and Colobus guereza lysozyme c precursor (GI 1790927; SEQ I
D NO: 33) and Colobus angolensis lysozyme c precursor (GI 1790967; SEQ ID NO:
34) and an amino acid sequence alignment between Nasalis larvatis lysozyme c precursor (GI 1790984; SEQ ID NO: 35).

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 25/28 A61P 35/00 4C084 29/00 37/02 4H045 35/00 43/00 111 37/02 C07K 16/40 43/00 111 C12N 1/15 C07K 16/40 1/19 C12N 1/15 1/21 1/19 9/14 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z 9/14 33/50 Z C12Q 1/68 33/53 MG01N 33/15 33/566 33/50 33/573 A 33/53 C12N 15/00 ZNAA 33/566 5/00 A 33/573 A61K 37/02 (81 ) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ) , TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, C Z, DE, DK, EE, ES, FI, GB, GD, GE , GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU , ZW (72) Inventor Hillman, Jennifer El, USA 94040. Mountain View # 12 Monrode Live 230 (72) Inventor Yue, Henry 94087, California, USA 94087, Sunnyvale Lewis Avenue 826 (72) Inventor Lal, Pretty, 95054, California, Santa Clara Russ Drive 2382 (72) Inventor Bandman, Olga 94043, California, USA Mountainview Anna Ave 366 (72) Inventor Coley, Neil Sea 94040, California USA Mountain View # 30 Dale Ave 1240 (72) Inventor Gegler, Karl Jay United States California 94025 Menlo Park Oakland Avenue 1048 (72) Inventor Bogun, Mariah Earl United States California 94577 San Leandro Santiago Road 14244 (72) Akira Ryu, Dung Aina Em United States California 95136 San Jose Park Belmont Place 55 (72) Inventor Azimzai, Yalda United States California 94545 Hayward Rock Springs Drive 2045 (72) Inventor Young, Junming United States California State 95129 San Jose Clarendon Street 7136 F-term (reference) 2G045 AA34 AA35 AA40 CB01 DA12 DA13 DA14 DA20 DA36 DA77 FB02 FB03 4B024 AA01 AA11 BA11 CA04 DA02 DA05 EA03 EA04 GA11 HA03 HA03 QAQBQ CC16 CC03 DD1111 QQ44 QR32.

Claims (20)

[Claims] 1. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ
A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of ID NO: 16, and fragments thereof. 2. A substantially purified variant having at least 90% amino acid sequence identity with the amino acid sequence of claim 1. 3. An isolated and purified polynucleotide encoding the polypeptide of claim 1. 4. An isolated and purified polynucleotide variant sequence having at least 90% polynucleotide sequence identity with the polynucleotide of claim 3. 5. An isolated and purified polynucleotide which hybridizes with the polynucleotide of claim 3 under stringent conditions. 6. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 3. 7. A method of detecting a polynucleotide comprising: (a) hybridizing the polynucleotide of claim 6 with at least one nucleic acid in a sample, thereby forming a hybridization complex; b) a step of detecting the hybridization complex, the step of detecting the presence of the hybridization complex correlating with the presence of the polynucleotide in the sample. Law. 8. The detection method according to claim 7, further comprising a step of amplifying the polynucleotide before the hybridization. 9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 17-32 and fragments thereof. 10. An isolated and purified polynucleotide variant sequence having at least 90% polynucleotide sequence identity with the polynucleotide of claim 9. 11. An isolated and purified polynucleotide having a sequence complementary to the polynucleotide of claim 9. 12. An expression vector comprising at least one fragment of the polynucleotide of claim 3. 13. A host cell containing the expression vector of claim 12. 14. A method for producing a polypeptide, comprising: (a) culturing the host cell according to claim 13 under conditions suitable for expression of the polypeptide; and (b) a medium for the host cell. And a step of recovering the polypeptide from the same. 15. A pharmaceutical composition comprising the polypeptide of claim 1 together with a suitable medical carrier. 16. A purified antibody that specifically binds to the polypeptide of claim 1. 17. A purified agonist of the polypeptide of claim 1. 18. A purified antagonist of the polypeptide of claim 1. 19. A method of administering HYDRL, which comprises the step of administering an effective amount of the pharmaceutical composition of claim 15 to a patient in need of treatment or prevention of a disease associated with reduced expression or activity of human hydrolase protein. A method of treating or preventing a disease associated with decreased activity. 20. In order to increase the expression or activity of HYDRL, which comprises the step of administering an effective amount of the antagonist of claim 18 to a patient in need of treatment or prevention of a disease associated with the increase in expression or activity of HYDRL. A method of treating or preventing a related disorder.