Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to nucleic acid and amino acid sequences of human chaperone proteins and to the use of these sequences in the diagnosis, treatment, and prevention of reproductive, eye, neuromuscular, metabolic, and autoimmune/inflammatory disorders, infectious diseases, and cell proliferative disorders including cancer.
• chaperones are proteins that interact with many other cellular proteins. Chaperones are involved in normal cellular functions, such as the folding of newly synthesized polypeptides and the assembly of multisubunit protein structures, in the transport of proteins across membranes, and in the stabilization of proteins in inactive configurations. This may give chaperones a role in cell signaling, as they can limit the access of proteins to their signaling partners, and in any regulatory process dependent on oligomerization or complex protein rearrangements. Chaperones are also involved in cellular responses to stresses such as toxicity and heat shock, and are therefore called heat-shock proteins (Hsp).
• Hsp heat-shock proteins
• Chaperones are found in many cellular compartments. In the mitochondria, for example, chaperones on both sides of the membrane are involved in importing most of the nuclear encoded proteins necessary for oxidative phosphorylation. Chaperones may be divided into several classes named for their approximate molecular weights, including Hsp90, Hsp70,Hsp 60, Hsp40 (also called DnaJ), and the small Hsps (having molecular masses between 20 kD and 30 kD). Mitochondrial chaperones show a high degree of similarity to molecular chaperones in bacteria, and in general chaperones are ubiquitous and highly conserved, from bacteria to humans (Martinus, R.D. et al. (1995) FASEB J. 9(5):371-378).
• Molecular chaperone genes are activated by a variety of stresses, including glucose deprivation, ethanol, and heavy metals as well as heat shock, all of which affect protein folding and aggregation. Activation may be expected in any disorder that results in temperature elevation.
• Molecular chaperones have been suggested to play a role in the development of autoimmune conditions and have been implicated in a variety of metabolic and developmental disorders as well as in response to trauma.
• any disorder affecting metabolism may involve mitochondrial import translocases.
• Hsps Under normal or nonstressed conditions, coastitutively expressed Hsps facilitate proper protein folding and maturation, promote protein translocation across membranes, and regulate hormone receptor and protein kinase activity (Hightower, L.E. et al. (1991) Cell 66:191-197), antigen presentation, protein degradation in the lysosome, and uncoating of clathrin-coated vesicles.
• Hsps are located in all major cellular compartments and function as monomers, multimers, or in complexes with other cellular proteins, which may determine the rate and specificity of Hsp action. Different roles have been ascribed to different classes of Hsps.
• Hsp20 proteins seem to form heterooligomers that can protect other proteins against heat-induced denaturation and aggregation.
• Hsp70 is a dimeric and ubiquitous protein which binds its substrates in an extended conformation through hydrophobic interactions. Hsp70 binds to newly synthesized proteins and is required for protein transport. The strength and specificity of Hsp70's interaction with its substrates is modified by binding and hydrolysis of ATP. Hsp70 has low protein affinity in its ATP-bound state, and increased protein affinity after ATP is hydrolyzed to ADP (Burston, S.G. and Clarke, A.R. (1995) Essays Biochem. 29:125-136). DnaJ chaperones work in concert with Hsp70. In particular, DnaJ interacts with the ATPase domain of Hsp70.
• the defining characteristic of the DnaJ chaperone family is an N-terminal, approximately 70 amino acid signature called the J domain, which is required for interactions with Hsp70.
• the tripeptide HPD seems to be particularly important for this interaction (Kelley, W. (1999) Curr. Biol. (1999) 9:R305-308).
• DnaJ stimulates ATP hydrolysis, increasing the affinity of Hsp70 for its protein substrate.
• GrpE another co-chaperone, promotes dissociation of ADP from Hsp70, again modifying the Hsp70/substrate interaction and completing the cycle (Burston and Clarke, supra). Many eukaryotic DnaJ homologs have recently been described.
• Hsps heat shock proteins
• Hsps Both heat shock-induced and exogenous Hsps protect smooth muscle cells from serum deprivation-induced cell death. Overexpression of Hsps also protects murine fibroblasts from both UV light injury and proinflammatory cytokines released during UV exposure (Marber, M.S. et al. (1995) J. Clin. Invest. 95:1446-1456; Simon, M.M. et al. (1995) J. Clin. Invest. 95:926-933). Specific Hsps bind immunosuppressive drugs and may play a role in modulation of immune responses. Hsps expressed in cancer cells can protect the cancer cells from the cytotoxic effects of drugs used in anticancer therapies.
• Hsps isolated from tumor cells and used as antigens have been shown to provide immunity to the tumors from which they are isolated (Udono, H. et al. (1994) J. Immunol. 152:5398-5403; Young R.A. (1990) Annu. Rev. Immunol. 8:401-420; Marber, M.S. et al. (1995) J. Clin. Invest. 95:1446-1456; Simon, M.M. et al. (1995) J. Clin. Invest. 95:926-933). Chaperones are useful as markers of environmental stress and disease, and are associated with a variety of diseases and immune and drug responses.
• Hsp90 binds to steroid hormone receptors, represses transcription in the absence of the ligand, and provides the proper folding of the ligand-binding domain in the presence of the hormone (Burston and Clarke, supra).
• Heat shock treatment of B-cells enhances processing of antigen and the assembly and function of MHC class II molecules (Sargent, CA. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1968-1972; Fang, Y. et al. (1996) J.
• Hsp70 Abnormal transcription of Hsp70 has been associated with major depression (Shimizu, S. et al. (1996) Biochem. Biophys. Res. Commun. 219:745-752). Hsp70 expression increases in response to tobacco smoke (Vayssier, M. et al. (1998) Biochem. Biophys. Res. Commun. 252:249-256). Hsp70 is involved in drug resistance in breast cancer patients treated with combination chemotherapies (Vargas-Roig, L.M. et al.
• Hsp70 variants are associated with clozapine-induced agranulocytosis, an adverse drug reaction (Turbay, D. et al. (1997) Blood 89:4167-4174).
• Knockout mice have provided additional information on the roles of Hsp70 in reproduction. For example, female homozygous knockout mice for Hsp70 are found to undergo normal meiosis and are fertile. In contrast, the homozygous male knockout mice lack postmeiotic spermatids and mature sperm, and are infertile (Dix, D.J. et al. (1996) Proc. Natl. Acad. Sci. U.S.A.
• a DnaJ (Hsp40) homolog is essential for normal placental development in mice (Hunter, P. et al. (1999) Development 126:1247-1258).
• Other DnaJ homologs have been implicated in viral DNA replication, secretion, tumor suppression, microtubule formation in M phase, and influenza virus infection (Kelley, 1998, supra).
• the small Hsps are able to supress aggregation and heat inactivation of various proteins, including actin (Hickey, E. et al. (1986) Nucleic Acids Res. 14:4127-4145; Miron, T. et al. (1991) J. Cell Biol. 114:255-261).
• actin Hickey, E. et al. (1986) Nucleic Acids Res. 14:4127-4145; Miron, T. et al. (1991) J. Cell Biol. 114:255-261).
• ⁇ -Crystallin a protein abundant in the lens of the eye, is an oligomer of two subunits, ⁇ A and oB, which are 55% identical and belong to the small Hsp family.
• ⁇ -Crystallin is thought to be important for maintaining the transparency of the lens by preventing denaturation and aggregation of proteins.
• a missense mutation in the ⁇ A-crystallin gene is associated with autosomal dominant congenital cataracts (Litt, M. et al. (1998) Hum. Molec. Genet. 7:471-474). However, the functional role of ⁇ -crystallin is not confined to the eye. A missense mutation of c ⁇ 3-crystallin has been shown to cause a desmin-related myopathy (Vicart, P. et al. (1998) Nat. Genet. 20:92-95). Desmin- related myopathies are inherited neuromuscular disorders.
• the invention features purified polypeptides, human chaperone proteins, referred to collectively as “HCPN” and individually as “HCPN-1,” “HCPN-2,” “HCPN-3,” “HCPN-4,” “HCPN-5,” “HCPN- 6,” “HCPN-7,” “HCPN-8,” “HCPN-9,” “HCPN-10,” and “HCPN-11.”
• the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11 , c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1.
• the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-11.
• the invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l- 11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
• the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-11.
• the polynucleotide is selected from the group consisting of SEQ ID NO: 12-22.
• the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1.
• the invention provides a cell transformed with the recombinant polynucleotide.
• the invention provides a transgenic organism comprising the recombinant polynucleotide.
• the invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 11 , b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1.
• the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
• the invention provides an isolated antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 11 , b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11 , and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO:l-l 1.
• the invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
• the polynucleotide comprises at least 60 contiguous nucleotides.
• the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
• the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof.
• the probe comprises at least 60 contiguous nucleotides.
• the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d).
• the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
• the invention further provides a composition comprising an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and a pharmaceutically acceptable excipient
• the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
• the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional HCPN, comprising administering to a patient in need of such treatment the composition.
• the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
• the metiiod comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
• the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
• the invention provides a method of treating a disease or condition associated with decreased expression of functional HCPN, comprising administering to a patient in need of such treatment the composition.
• the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
• the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
• the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient.
• the invention provides a method of treating a disease or condition associated with overexpression of functional HCPN, comprising administering to a patient in need of such treatment the composition.
• the invention further provides a method of screening for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
• the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
• the invention further provides a method of screening for a compound mat modulates the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 11 , c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-11.
• the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
• the invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 12-22, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.
• the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of i) a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, ii) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv).
• Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, ii) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 12-22, iii) a polynucleotide sequence complementary to i), iv) a polynucleotide sequence complementary to ii), and v) an RNA equivalent of i)-iv).
• the target polynucleotide comprises a fragment of the above polynucleotide sequence; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in me amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
• Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs), cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding HCPN.
• Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of HCPN.
• Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis; diseases, disorders, or conditions associated with these tissues; and the vector into which each cDNA was cloned.
• Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding HCPN were isolated.
• Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
• HCPN refers to the amino acid sequences of substantially purified HCPN obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
• agonist refers to a molecule which intensifies or mimics the biological activity of HCPN.
• Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of HCPN either by directly interacting with HCPN or by acting on components of the biological pathway in which HCPN participates.
• An "allelic variant” is an alternative form of the gene encoding HCPN. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form.
• allelic variants are generally ascribed to natural deletions, additions, or substitutioas of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
• altered nucleic acid sequences encoding HCPN include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as HCPN or a polypeptide with at least one functional characteristic of HCPN. Included within this definition are polymo ⁇ hisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HCPN, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HCPN.
• the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HCPN.
• Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of HCPN is retained.
• negatively charged amino acids may include aspartic acid and glutamic acid
• positively charged amino acids may include lysine and arginine.
• Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
• Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine: and phenylalanine and tyrosine.
• amino acid and amino acid sequence refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
• Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
• PCR polymerase chain reaction
• Antagonist refers to a molecule which inhibits or attenuates the biological activity of HCPN.
• Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of HCPN either by directly interacting with HCPN or by acting on components of the biological pathway in which HCPN participates.
• antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 . and Fv fragments, which are capable of binding an epitopic determinant.
• Antibodies that bind HCPN polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
• the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
• an animal e.g., a mouse, a rat, or a rabbit
• RNA e.g., a mouse, a rat, or a rabbit
• antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
• an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
• antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
• Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2 -deoxyuracil, or 7-deaza-2 -deoxyguanosine.
• Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation.
• the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
• biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
• immunologically active or “immunogenic” refers to the capability of the natural, recombinant, or synthetic HCPN, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
• compositions comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
• the composition may comprise a dry formulation or an aqueous solution.
• Compositions comprising polynucleotide sequences encoding HCPN or fragments of HCPN may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
• the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
• salts e.g., NaCl
• detergents e.g., sodium dodecyl sulfate; SDS
• other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
• Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG, Madison WI) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
• Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
• the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
• Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
• a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
• derivative refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
• a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
• a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
• a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
• a “fragment” is a unique portion of HCPN or the polynucleotide encoding HCPN which is identical in sequence to but shorter in length than the parent sequence.
• a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
• a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
• a fragment used as a probe, primer, antigen, therapeutic molecule, or for other pu ⁇ oses may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
• a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence.
• these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
• a fragment of SEQ ID NO: 12-22 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 12-22, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
• a fragment of SEQ ID NO: 12-22 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO: 12-22 from related polynucleotide sequences.
• the precise length of a fragment of SEQ ID NO: 12-22 and the region of SEQ ID NO: 12-22 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
• a fragment of SEQ ID NO: 1 - 11 is encoded by a fragment of SEQ ID NO: 12-22.
• a fragment of SEQ ID NO: 1-11 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1 -11.
• a fragment of SEQ ID NO: 1 - 11 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO: 1-11.
• the precise length of a fragment of SEQ ID NO: 1-11 and the region of SEQ ID NO: 1-11 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended pu ⁇ ose for the fragment.
• a “full-length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
• a “full- length” polynucleotide sequence encodes a "full-length” polypeptide sequence.
• Homology refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
• percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
• the "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences.
• NCBI National Center for Biotechnology Information
• BLAST Basic Local Alignment Search Tool
• the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences” can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. The "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings.
• BLAST 2 Sequences Version 2.0.12 (April-21-2000) set at default parameters.
• Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2
• Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understcxxl that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
• percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
• Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
• NCBI BLAST software suite may be used.
• BLAST 2 Sequences Version 2.0.12 (Apr-21 -2000) with blastp set at default parameters.
• Such default parameters may be, for example:
• Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• Human artificial chromosomes are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
• humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
• Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
• Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
• wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T for the specific sequence at a defined ionic strength and pH.
• T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
• blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
• Organic solvent such as formamide at a concentration of about 35-50% v/v
• RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
• Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
• hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
• a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
• a solid support e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed.
• insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
• Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
• factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
• an “immunogenic fragment” is a polypeptide or oligopeptide fragment of HCPN which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
• the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of HCPN which is useful in any of the antibody production methods disclosed herein or known in the art.
• microarray refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.
• array element refers to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
• modulate refers to a change in the activity of HCPN.
• modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HCPN.
• nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
• PNA peptide nucleic acid
• operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
• a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
• Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
• PNA protein nucleic acid
• PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
• Post-translational modification of an HCPN may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of HCPN.
• Probe refers to nucleic acid sequences encoding HCPN, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
• Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
• Primmers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
• Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
• PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that pu ⁇ ose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
• Oligonucleotides for use as primers are selected using software known in the art for such pu ⁇ ose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have inco ⁇ orated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
• the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.)
• the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
• this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
• the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
• a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
• the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
• a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
• such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
• a “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.
• Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
• RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
• sample is used in its broadest sense.
• a sample suspected of containing nucleic acids encoding HCPN, or fragments thereof, or HCPN itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
• specific binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition.
• the interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by die binding molecule.
• a particular structure of the protein e.g., the antigenic determinant or epitope
• die binding molecule e.g., die binding molecule.
• an antibody is specific for epitope "A”
• the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
• substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
• substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
• Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
• the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
• a “transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
• Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
• transformed cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
• a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
• the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
• the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
• the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants, and animals.
• the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook, J. et al. (1989), supra.
• a “variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
• Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length.
• a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymo ⁇ hic” variant.
• a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during RNA processing.
• the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
• Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
• a polymo ⁇ hic variant is a variation in d e polynucleotide sequence of a particular gene between individuals of a given species.
• Polymo ⁇ hic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
• SNPs single nucleotide polymorphisms
• the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
• a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
• Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
• the invention is based on the discovery of new human chaperone proteins (HCPN), the polynucleotides encoding HCPN, and the use of these compositions for the diagnosis, treatment, or prevention of reproductive, eye, neuromuscular, metabolic, and autoimmune/inflammatory disorders, infectious diseases, and cell proliferative disorders including cancer.
• Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding
• HCPN HCPN.
• Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively.
• Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each HCPN were identified, and column 4 shows the cDNA libraries from which these clones were isolated.
• Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA hbraries are not indicated were derived from pooled cDNA libraries. In some cases, GenBank sequence identifiers are also shown in column 5. The Incyte clones and GenBank cDNA sequences, where indicated, in column 5 were used to assemble the consensus nucleotide sequence of each HCPN and are useful as fragments in hybridization technologies.
• column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs; column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
• the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding HCPN.
• the first column of Table 3 lists the nucleotide SEQ ID NOs.
• Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO: 12-22 and to distinguish between SEQ ID NO: 12-22 and related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
• Column 3 lists tissue categories which express HCPN as a fraction of total tissues expressing HCPN.
• Column 4 lists diseases, disorders, or conditions associated with those tissues expressing HCPN as a fraction of total tissues expressing HCPN.
• Column 5 lists the vectors used to subclone each cDNA library.
• Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding HCPN were isolated.
• Column 1 references the nucleotide SEQ ID NOs
• column 2 shows the cDNA libraries from which these clones were isolated
• column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
• SEQ ID NO: 14 maps to chromosome 10 within the interval from 46.2 to 46.8 centiMorgans. This interval also contains a gene with homology to the murine leukemia viral (bmi-1) oncogene. SEQ ID NO:15 maps to chromosome 16 within the interval from 50.8 to 56.2 centiMorgans. This interval also contains a gene associated with neuronal ceroid lipofuscinosis, also known as Batten disease.
• SEQ ID NO:22 maps to chromosome 1 within the interval from 78.3 to 84.2 centiMorgans, to chromosome 6 within the interval from 91.8 to 96.1 centiMorgans, to chromosome 10 within the interval from 93.8 to 96.9 centiMorgans, and to chromosome 12 within the interval from 13.8 to 24.6 centiMorgans.
• the interval on chromosome 1 from 78.3 to 84.2 centiMorgans also contains genes and/or ESTs associated with myopathy, hypoketotic hypoglycemia and hyperthyroxinemia.
• the interval on chromosome 6 from 91.8 to 96.1 centiMorgans also contains a gene and/or EST associated with maple syrup urine disease.
• the interval on chromosome 12 from 13.8 to 24.6 centiMorgans also contains genes and or ESTs associated with T cell antigen T4 deficiency, neonatal adrenoleukodystxophy, and Zellweger syndrome.
• the invention also encompasses HCPN variants.
• a preferred HCPN variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the HCPN amino acid sequence, and which contains at least one functional or structural characteristic of HCPN.
• the invention also encompasses polynucleotides which encode HCPN.
• the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 12-22, which encodes HCPN.
• the polynucleotide sequences of SEQ ID NO: 12-22 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
• the invention also encompasses a variant of a polynucleotide sequence encoding HCPN.
• a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HCPN.
• a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 12-22 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 12-22.
• any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HCPN. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding HCPN, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HCPN, and all such variations are to be considered as being specifically disclosed.
• nucleotide sequences which encode HCPN and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring HCPN under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HCPN or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
• RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
• the invention also encompasses production of DNA sequences which encode HCPN and HCPN derivatives, or fragments thereof, entirely by synthetic chemistry.
• the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
• synthetic chemistry may be used to introduce mutations into a sequence encoding HCPN or any fragment thereof.
• polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO: 12-22 and fragments thereof under various conditions of stringency.
• Hybridization conditions including annealing and wash conditions, are described in "Definitions.”
• Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
• the methods may employ such enzymes as the Klenow fragment of
• DNA polymerase I SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
• sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosystems).
• Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEG AB ACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art.
• the resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.)
• the nucleic acid sequences encoding HCPN may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
• restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.)
• Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
• the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
• a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
• capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA.
• multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
• Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res.
• primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
• OLIGO 4.06 Primer Analysis software National Biosciences, Plymouth MN
• anneal to the template at temperatures of about 68°C to 72°C.
• Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
• Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
• capillary sequencing may employ flowable polymers for electiophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
• Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
• polynucleotide sequences or fragments thereof which encode HCPN may be cloned in recombinant DNA molecules that direct expression of HCPN, or fragments or functional equivalents thereof, in appropriate host cells.
• nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HCPN-encoding sequences for a variety of pu ⁇ oses including, but not limited to, modification of the cloning, processing, and or expression of the gene product.
• DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
• oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
• the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of HCPN, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
• MOLECULARBREEDING Maxygen Inc., Santa Clara CA; described in U.S. Patent Number 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et
• DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
• genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
• sequences encoding HCPN may be synthesized, in whole or in part, using chemical methods well known in the art.
• chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
• HCPN itself or a fragment thereof may be synthesized using chemical methods.
• peptide synthesis can be performed using various solution-phase or solid-phase techniques.
• Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (PE Biosystems). Additionally, the amino acid sequence of HCPN, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
• the peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421.)
• the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
• the nucleotide sequences encoding HCPN or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
• these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding HCPN. Such elements may vary in their strength and specificity.
• Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HCPN. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
• exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
• Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
• a variety of expression vector/host systems may be utilized to contain and express sequences encoding HCPN. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
• microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
• yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., baculovirus)
• plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV, or tobacco
• Expression vectors derived from retroviruses, adenoviruses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
• the invention is not limited by the host cell employed.
• a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HCPN.
• routine cloning, subcloning, and propagation of polynucleotide sequences encoding HCPN can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORTl plasmid (Life Technologies). Ligation of sequences encoding HCPN into the vector's multiple cloning site disrupts the lac gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
• these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
• vectors which direct high level expression of HCPN may be used.
• vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
• Yeast expression systems may be used for production of HCPN.
• a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
• such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
• Plant systems may also be used for expression of HCPN. Transcription of sequences encoding
• HCPN may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311).
• plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
• These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.)
• a number of viral-based expression systems may be utilized.
• sequences encoding HCPN may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses HCPN in host cells.
• transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
• SV40 or EBV- based vectors may also be used for high-level protein expression.
• HACs Human artificial chromosomes
• HACs may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid.
• HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic pu ⁇ oses.
• liposomes, polycationic amino polymers, or vesicles for therapeutic pu ⁇ oses.
• HCPN in cell lines is preferred.
• sequences encoding HCPN can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
• expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
• cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
• the pu ⁇ ose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
• Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines.
• he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes for use in tk ⁇ and apr cells, respectively.
• he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes for use in tk ⁇ and apr cells, respectively.
• antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
• dhfr confers resistance to methotrexate
• neo confers resistance to the aminoglycosides neomycin and G-418
• als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
• Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
• Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol.
• marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
• sequence encoding HCPN is inserted within a marker gene sequence, transformed cells containing sequences encoding HCPN can be identified by the absence of marker gene function.
• a marker gene can be placed in tandem with a sequence encoding HCPN under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
• host cells that contain the nucleic acid sequence encoding HCPN and that express HCPN may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and or quantification of nucleic acid or protein sequences.
• Immunological methods for detecting and measuring the expression of HCPN using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
• ELISAs enzyme-linked immunosorbent assays
• RIAs radioimmunoassays
• FACS fluorescence activated cell sorting
• Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HCPN include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
• the sequences encoding HCPN, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
• RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
• T7, T3, or SP6 RNA polymerase
• reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
• Host cells transformed with nucleotide sequences encoding HCPN may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
• the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
• expression vectors containing polynucleotides which encode HCPN may be designed to contain signal sequences which direct secretion of HCPN through a prokaryotic or eukaryotic cell membrane.
• a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
• Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
• Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
• Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
• ATCC American Type Culture Collection
• natural, modified, or recombinant nucleic acid sequences encoding HCPN may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
• a chimeric HCPN protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HCPN activity.
• Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
• Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
• GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
• FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
• a fusion protein may also be engineered to contain a proteolytic cleavage site located between the HCPN encoding sequence and the heterologous protein sequence, so that HCPN may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
• synthesis of radiolabeled HCPN may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
• HCPN of the present invention or fragments thereof may be used to screen for compounds that specifically bind to HCPN.
• At least one and up to a plurality of test compounds may be screened for specific binding to HCPN.
• Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
• the compound thus identified is closely related to the natural ligand of HCPN, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
• the compound can be closely related to the natural receptor to which HCPN binds, or to at least a fragment of the receptor, e.g., the ligand binding site.
• the compound can be rationally designed using known techniques.
• screening for these compounds involves producing appropriate cells which express HCPN, either as a secreted protein or on the cell membrane.
• Preferred cells include cells from mammals, yeast, Drosophila. or E. coli. Cells expressing HCPN or cell membrane fractions which contain HCPN are then contacted with a test compound and binding, stimulation, or inhibition of activity of either HCPN or the compound is analyzed.
• An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
• the assay may comprise the steps of combining at least one test compound with HCPN, either in solution or affixed to a solid support, and detecting the binding of HCPN to the compound.
• the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
• the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.
• HCPN of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of HCPN.
• Such compounds may include agonists, antagonists, or partial or inverse agonists.
• an assay is performed under conditions permissive for HCPN activity, wherein HCPN is combined with at least one test compound, and the activity of HCPN in the presence of a test compound is compared with the activity of HCPN in the absence of the test compound. A change in the activity of HCPN in the presence of the test compound is indicative of a compound that modulates the activity of HCPN.
• a test compound is combined with an in vitro or cell-free system comprising HCPN under conditions suitable for HCPN activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of HCPN may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
• polynucleotides encoding HCPN or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
• ES embryonic stem
• Such techniques are well known in the art and are useful for the generation of animal models of human disease.
• mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
• the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
• a marker gene e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
• the vector integrates into the corresponding region of the host genome by homologous recombination.
• homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
• Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain.
• the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
• Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
• Polynucleotides encoding HCPN may also be manipulated in vitro in ES cells derived from human blastocysts.
• Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282: 1145-1147).
• Polynucleotides encoding HCPN can also be used to create "knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
• knockin technology a region of a polynucleotide encoding HCPN is injected into animal ES cells, and the injected sequence integrates into the animal cell genome.
• Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
• Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
• HCPN overexpress HCPN
• a mammal inbred to overexpress HCPN e.g., by secreting HCPN in its milk
• may also serve as a convenient source of that protein Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
• HCPN Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of HCPN and human chaperone proteins.
• the expression of HCPN is closely associated with reproductive disorders and with cancerous, proliferating, inflamed, and hematopoietic/immune tissues. Therefore, HCPN appears to play a role in reproductive, eye, neuromuscular, metabolic, and autoimmune inflammatory disorders, infectious diseases, and cell proliferative disorders including cancer.
• HCPN In the treatment of disorders associated with decreased HCPN expression or activity, it is desirable to increase the expression or activity of HCPN.
• HCPN or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCPN.
• disorders include, but are not limited to, a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hype ⁇ lasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and g
• a reproductive disorder
• composition comprising a substantially purified HCPN in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCPN including, but not limited to, those provided above.
• an agonist which modulates the activity of HCPN may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCPN including, but not limited to, those listed above.
• an antagonist of HCPN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HCPN. Examples of such disorders include, but are not limited to, those reproductive, eye, neuromuscular, metabolic, and autoimmuneinflammatory disorders, infectious diseases, and cell proliferative disorders, including cancer, described above.
• an antibody which specifically binds HCPN may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express HCPN.
• a vector expressing the complement of the polynucleotide encoding HCPN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HCPN including, but not limited to, those described above.
• any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
• the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
• An antagonist of HCPN may be produced using methods which are generally known in the art.
• purified HCPN may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HCPN.
• Antibodies to HCPN may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
• various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HCPN or with any fragment or oligopeptide thereof which has immunogenic properties.
• various adjuvants may be used to increase immunological response.
• adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
• BCG Bacilli Calmette-Guerin
• Corynebacterium parvum are especially preferable.
• the oligopeptides, peptides, or fragments used to induce antibodies to HCPN have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of HCPN amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
• Monoclonal antibodies to HCPN may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
• chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
• techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce HCPN-specific single chain antibodies.
• Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
• Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the Uterature. (See, e.g., Oriandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
• Antibody fragments which contain specific binding sites for HCPN may also be generated.
• fragments include, but are not limited to, F(ab ⁇ ) 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
• Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
• immunoassays may be used for screening to identify antibodies having the desired specificity.
• Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
• Such immunoassays typically involve the measurement of complex formation between HCPN and its specific antibody.
• a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HCPN epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
• Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for HCPN.
• K a is defined as the molar concentration of HCPN-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions.
• the K determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple HCPN epitopes, represents the average affinity, or avidity, of the antibodies for HCPN.
• the K ⁇ determined for a preparation of monoclonal antibodies, which are monospecific for a particular HCPN epitope, represents a true measure of affinity.
• High-affinity antibody preparations with K ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the HCPN-antibody complex must withstand rigorous manipulations.
• Low-affinity antibody preparations with K ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of HCPN, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach. IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
• polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
• a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generally employed in procedures requiring precipitation of HCPN-antibody complexes.
• Procedures for evaluating antibody specificity, liter, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al, supra.)
• the polynucleotides encoding HCPN may be used for therapeutic pu ⁇ oses.
• modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding HCPN.
• complementary sequences or antisense molecules DNA, RNA, PNA, or modified oligonucleotides
• antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HCPN. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics.
• Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
• Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
• viral vectors such as retrovirus and adeno-associated virus vectors.
• Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art.
• polynucleotides encoding HCPN may be used for somatic or germline gene therapy.
• Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
• SCID severe combined immunodeficiency
• ADA adenosine deaminase
• HCPN hepatitis B or C virus
• fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
• protozoan parasites such as Plasmodium falciparum and Trvpanosoma cruzi.
• diseases or disorders caused by deficiencies in HCPN are treated by constructing mammalian expression vectors encoding HCPN and introducing these vectors by mechanical means into HCPN-deficient cells.
• Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) Cell 91 :501-510; Boulay, J-L. and H. R ⁇ cipon (1998) Curr. Opin. Biotechnol. 9:445- 450).
• Expression vectors that may be effective for the expression of HCPN include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF,
• HCPN may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al.
• CMV cytomegalovirus
• RSV40 virus SV40 virus
• TK thymidine kinase
• ⁇ -actin genes e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al.
• liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
• PERFECT LIPID TRANSFECTION KIT available from Invitrogen
• transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
• the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
• diseases or disorders caused by genetic defects with respect to HCPN expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding HCPN under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cw-acting RNA sequences and coding sequences required for efficient vector propagation.
• Retrovirus vectors e.g., PFB and PFBNEO
• Retrovirus vectors are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
• the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VS Vg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471 ; Zufferey, R. et al.
• VPCL vector producing cell line
• U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell lines and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al.
• an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding HCPN to cells which have one or more genetic abnormalities with respect to the expression of HCPN.
• the construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S.
• Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby inco ⁇ orated by reference.
• adenoviral vectors see also Antinozzi, P.A. et al. (1999) Annu. Rev. Nutr. 19:511-544; and Verma, I.M. and N. Somia (1997) Nature 18:389:239-242, both inco ⁇ orated by reference herein.
• a he ⁇ es-based, gene therapy delivery system is used to deliver polynucleotides encoding HCPN to target cells which have one or more genetic abnormalities with respect to the expression of HCPN.
• HSV he ⁇ es simplex virus
• the use of he ⁇ es simplex virus (HSV)-based vectors may be especially valuable for introducing HCPN to cells of the central nervous system, for which HSV has a tropism.
• the construction and packaging of he ⁇ es-based vectors are well known to those with ordinary skill in the art.
• a replication-competent he ⁇ es simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp.
• HSV-1 virus vector has also been disclosed in detail in U.S. Patent Number 5,804,413 to DeLuca ("He ⁇ es simplex virus strains for gene transfer"), which is hereby inco ⁇ orated by reference.
• U.S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for pu ⁇ oses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
• HSV vectors see also Goins, W.F. et al.
• an alphavirus positive, single-stranded RNA virus
• an alphavirus vector is used to deliver polynucleotides encoding HCPN to target cells.
• Semliki Forest Virus has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotech. 9:464-469).
• alphavirus RNA replication a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
• HCPN-coding RNAs results in the production of a large number of HCPN-coding RNAs and the synthesis of high levels of HCPN in vector transduced cells.
• alphavirus infection is typically associated with cell lysis within a few days
• BHK-21 hamster normal kidney cells
• SIN Sindbis virus
• the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S.A. et al. (1997) Virology 228:74-83).
• the wide host range of alphaviruses will allow the introduction of HCPN into a variety of cell types.
• the specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction.
• the methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.
• Oligonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRN A by preventing the transcript from binding to ribosomes.
• Ribozymes enzymatic RNA molecules
• Ribozymes may also be used to catalyze the specific cleavage of RNA.
• the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
• engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HCPN.
• RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
• the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
• RNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
• RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HCPN. Such DNA sequences may be inco ⁇ orated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
• these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.
• RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
• An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding HCPN.
• Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
• a compound which specifically inhibits expression of the polynucleotide encoding HCPN may be therapeutically useful, and in the treament of disorders associated with decreased HCPN expression or activity, a compound which specifically promotes expression of the polynucleotide encoding HCPN may be therapeutically useful.
• test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
• a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly.
• a sample comprising a polynucleotide encoding HCPN is exposed to at least one test compound thus obtained.
• the sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system.
• Alterations in the expression of a polynucleotide encoding HCPN are assayed by any method commonly known in the art.
• the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding HCPN.
• the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds.
• a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaecharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, CM. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys.
• a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S.
• vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat.
• any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
• An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
• Excipients may include, for example, sugars, starches, celluloses, gums, and proteins.
• Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
• Such compositions may consist of HCPN, antibodies to HCPN, and mimetics, agonists, antagonists, or inhibitors of HCPN.
• compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
• compositions for pulmonary administration may be prepared in liquid or dry powder form.
• compositions are generally aerosolized immediately prior to inhalation by the patient.
• small molecules e.g. traditional low molecular weight organic drugs
• aerosol delivery of fast-acting formulations is well-known in the art.
• macromolecules e.g. larger peptides and proteins
• recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J.S. et al., U.S.
• Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.
• compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
• the determination of an effective dose is well within the capability of those skilled in the art.
• compositions may be prepared for direct intracellular delivery of macromolecules comprising HCPN or fragments thereof.
• liposome preparations containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule.
• HCPN or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
• the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
• An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
• a therapeutically effective dose refers to that amount of active ingredient, for example HCPN or fragments thereof, antibodies of HCPN, and agonists, antagonists or inhibitors of HCPN, which ameliorates the symptoms or condition.
• Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
• the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 ED 50 ratio.
• Compositions which exhibit large therapeutic indices are preferred.
• the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
• the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, die sensitivity of the patient, and the route of administration.
• Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
• Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
• Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
• antibodies which specifically bind HCPN may be used for the diagnosis of disorders characterized by expression of HCPN, or in assays to monitor patients being treated with HCPN or agonists, antagonists, or inhibitors of HCPN.
• Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HCPN include methods which utilize the antibody and a label to detect HCPN in human body fluids or in extracts of cells or tissues.
• the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
• a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
• HCPN HCPN-specific plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated plasminogen activator-associated containing HCPN.
• the polynucleotides encoding HCPN may be used for diagnostic pu ⁇ oses.
• the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
• the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of HCPN may be correlated with disease.
• the diagnostic assay may be used to determine absence, presence, and excess expression of HCPN, and to monitor regulation of HCPN levels during therapeutic intervention.
• hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HCPN or closely related molecules may be used to identify nucleic acid sequences which encode HCPN.
• the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding HCPN, allelic variants, or related sequences.
• Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the HCPN encoding sequences.
• the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 12-22 or from genomic sequences including promoters, enhancers, and introns of the HCPN gene.
• Means for producing specific hybridization probes for DNAs encoding HCPN include the cloning of polynucleotide sequences encoding HCPN or HCPN derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
• Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
• reporter groups for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
• Polynucleotide sequences encoding HCPN may be used for the diagnosis of disorders associated with expression of HCPN.
• disorders include, but are not limited to, a reproductive disorder such as a disorder of prolactin production, infertility, including tubal disease, ovulatory defects, and endometriosis, a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, an endometrial or ovarian tumor, a uterine fibroid, autoimmune disorders, an ectopic pregnancy, and teratogenesis, cancer of the breast, fibrocystic breast disease, and galactorrhea, a disruption of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hype ⁇ lasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a
• Sjogren's syndrome systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythemia, thrombocytopenic pu ⁇ ura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extraco ⁇ oreal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma; a viral infection, such as those caused by adenoviruses (acute respiratory disease, pneumonia), arenaviruses (lymphocytic choriomeningitis), bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses (hepatitis), he ⁇ esviruses (he ⁇ es simplex virus, varicella-zoster virus,
• the polynucleotide sequences encoding HCPN may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered HCPN expression. Such qualitative or quantitative methods are well known in the art.
• the nucleotide sequences encoding HCPN may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
• the nucleotide sequences encoding HCPN may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
• the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HCPN in the sample indicates the presence of the associated disorder.
• assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient. In order to provide a basis for the diagnosis of a disorder associated with expression of HCPN, a normal or standard profile for expression is established.
• Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
• hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
• the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
• oligonucleotides designed from the sequences encoding HCPN may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro.
• Oligomers will preferably contain a fragment of a polynucleotide encoding HCPN, or a fragment of a polynucleotide complementary to the polynucleotide encoding HCPN, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
• oligonucleotide primers derived from the polynucleotide sequences encoding HCPN may be used to detect single nucleotide polymo ⁇ hisms (SNPs).
• SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
• Methods of SNP detection include, but are not limited to, single-stranded conformation polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
• SSCP single-stranded conformation polymo ⁇ hism
• fSSCP fluorescent SSCP
• oligonucleotide primers derived from the polynucleotide sequences encoding HCPN are used to amplify DNA using the polymerase chain reaction (PCR).
• the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
• SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
• the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
• sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymo ⁇ hisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
• SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
• Methods which may also be used to quantify the expression of HCPN include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
• radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves.
• the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
• oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray.
• the microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484, inco ⁇ orated herein by reference.
• the microarray may also be used to identify genetic variants, mutations, and polymo ⁇ hisms.
• This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
• this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
• therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
• antibodies specific for HCPN, or HCPN or fragments thereof may be used as elements on a microarray.
• the microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.
• a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type.
• a transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent Number 5,840,484, expressly inco ⁇ orated by reference herein.)
• a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type.
• the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
• the resultant transcript image would provide a profile of gene activity.
• Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.
• Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular finge ⁇ rints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly inco ⁇ orated by reference herein).
• a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties.
• These finge ⁇ rints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in inte ⁇ retation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity.
• the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound.
• Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified.
• the transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in die treated sample.
• proteome refers to the global pattern of protein expression in a particular tissue or cell type.
• proteome expression patterns, or profiles are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time.
• a profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type.
• the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
• the proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
• the optical density of each protein spot is generally proportional to the level of the protein in the sample.
• the optical densities of equivalently positioned protein spots from different samples for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment.
• the proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry.
• the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.
• a proteomic profile may also be generated using antibodies specific for HCPN to quantify the levels of HCPN expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem.
• Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino- reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
• Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level.
• There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
• the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
• Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified.
• the amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
• Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
• the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
• Microarrays may be prepared, used, and analyzed using methods known in the art.
• methods known in the art See, e.g., Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150- 2155; and Heller, M.J. et al.
• nucleic acid sequences encoding HCPN may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping.
• sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
• HACs human artificial chromosomes
• YACs yeast artificial chromosomes
• BACs bacterial artificial chromosomes
• PI constructions or single chromosome cDNA libraries.
• the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymo ⁇ hism (RFLP).
• RFLP restriction fragment length polymo ⁇ hism
• FISH Fluorescent in situ hybridization
• Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding HCPN on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
• OMIM Online Mendelian Inheritance in Man
• In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
• nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
• HCPN in another embodiment, HCPN, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
• the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HCPN and the agent being tested may be measured.
• Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
• This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with HCPN, or fragments thereof, and washed. Bound HCPN is then detected by methods well known in the art. Purified HCPN can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
• nucleotide sequences which encode HCPN may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
• RNA was treated with DNase.
• poly(A+) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
• RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
• RNA was provided with RNA and constructed the corresponding cDNA libraries.
• cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
• cDNA was size-selected (300-1000 bp) using SEPHACRYL SI 000, SEPH AROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
• cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORTl plasmid (Life Technologies), pcDNA2.1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics, Palo Alto CA).
• Recombinant plasmids were transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones
• Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophi ⁇ zation, at 4°C
• plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384- well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
• Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems).
• Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (PE Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VI. The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art.
• Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters.
• the first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences).
• Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
• Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as inco ⁇ orated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
• polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS.
• the sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
• the full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM.
• HMM Hidden Markov Model
• Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.)
• the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
• the product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
• the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
• the product score represents a balance between fractional overlap and quality in a BLAST alignment.
• a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared.
• a product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other.
• a product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
• the results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding HCPN occurred.
• Analysis involved the categorization of cDNA libraries by organ tissue and disease.
• the organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic.
• the disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
• the cDNA sequences which were used to assemble SEQ ID NO: 12-22 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ID NO:12-22 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 5). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and G ⁇ nethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
• SHGC Stanford Human Genome Center
• WIGR Whitehead Institute for Genome Research
• G ⁇ nethon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in
• SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO:22 are described in The Invention as ranges, or intervals, of human chromosomes. More than one map location is reported for SEQ ID NO:22, indicating that previously mapped sequences having similarity, but not complete identity, to SEQ ID NO:22 were assembled into their respective clusters.
• the map position of an interval, in centiMorgans, is measured relative to die terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers.
• cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
• Mb megabase
• the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Diseases associated with the public and Incyte sequences located within the indicated intervals are also reported in the Invention where applicable.
• the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
• Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed. High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.).
• the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C
• the parameters for primer pair T7 and SK+ were as follows: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6:
• the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
• a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
• the extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
• CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
• sonicated or sheared prior to religation into pUC 18 vector
• the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
• Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
• the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
• polynucleotide sequences of SEQ ID NO: 12-22 are used to obtain 5' regulatory sequences using the procedure above, along with oligonucleotides designed for such extension, and an appropriate genomic library. VII. Labeling and Use of Individual Hybridization Probes
• Hybridization probes derived from SEQ ID NO: 12-22 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
• state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech
• the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco Rl, Pst I, Xba I, or Pvu II (DuPont NEN).
• the DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. VIII. Microarrays
• the linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof.
• the substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
• a typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
• Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
• the array elements are hybridized with polynucleotides in a biological sample.
• the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
• a fluorescence scanner is used to detect hybridization at each array element.
• laser desorbtion and mass spectrometry may be used for detection of hybridization.
• the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed.
• microarray preparation and usage is described in detail below.
• Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
• Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech).
• the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMB RIGHT kits (Incyte).
• Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 °C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc.
• reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.
• the sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Hoi brook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
• Microarray Preparation Sequences of the present invention are used to generate array elements.
• Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
• PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
• Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
• Purified array elements are immobilized on polymer-coated glass slides.
• Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
• Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110°C oven.
• Array elements are applied to the coated glass substrate using a procedure described in US Patent No. 5,807,522, inco ⁇ orated herein by reference.
• 1 ⁇ l of the array element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.
• Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 °C followed by washes in 0.2% SDS and distilled water as before. Hybridization
• Hybridization reactions contain 9 ul of sample mixture consisting of 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5X SSC, 0.2% SDS hybridization buffer.
• the sample mixture is heated to 65 °C for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip.
• the arrays are transferred to a wate ⁇ roof chamber having a cavity just slightly larger than a microscope slide.
• the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC in a corner of the chamber.
• the chamber containing the arrays is incubated for about 6.5 hours at 60 °C.
• the arrays are washed for 10 min at 45 °C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45 °C in a second wash buffer (0.1X SSC), and dried.
• Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
• the excitation laser light is focused on the array using a 20X microscope objective (Nikon, Inc., Melville NY).
• the slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
• the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.
• a mixed gas multiline laser excites die two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
• the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
• Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
• the sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
• a specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
• the calibration is done by labeling samples of the calibrating cDNA wi ⁇ i the two fluorophores and adding identical amounts of each to the hybridization mixture.
• the output of die photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
• the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
• the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore' s emission spectrum.
• a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
• the fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal.
• the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).
• IX. Complementary Polynucleotides Sequences complementary to the HCPN-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HCPN. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments.
• oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of HCPN.
• a complementary oligonucleotide is designed from the most unique 5 ' sequence and used to prevent promoter binding to the coding sequence.
• a complementary oligonucleotide is designed to prevent ribosomal binding to the HCPN-encoding transcript.
• HCPN HCPN expression and purification of HCPN is achieved using bacterial or virus-based expression systems.
• cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
• promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
• Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
• Antibiotic resistant bacteria express HCPN upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
• HCPN HCPN in eukaryotic cells
• AcMNPV Autographica californica nuclear polyhedrosis virus
• the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HCPN by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
• Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
• HCPN is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
• GST glutathione S- transferase
• a peptide epitope tag such as FLAG or 6-His
• FLAG an 8-amino acid peptide
• 6- His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified HCPN obtained by these methods can be used directly in the assays shown in Examples XI and XV.
• HCPN induction by heat or toxins may be demonstrated using primary cultures of human fibroblasts or human cell lines such as CCL-13, HEK293, or HEP G2 (ATCC).
• To heat induce HCPN expression aliquots of cells are incubated at 42°C for 15, 30, or 60 minutes. Control aliquots are incubated at 37 °C for the same time periods.
• To induce HCPN expression by toxins aliquots of cells are treated with 100 ⁇ M arsenite or 20 mM azetidine-2-carboxylic acid for 0, 3, 6, or 12 hours. After exposure to heat, arsenite, or the amino acid analogue, samples of the treated cells are harvested and cell lysates prepared for analysis by western blot.
• Cells are lysed in lysis buffer containing 1 % Nonidet P-40, 0.15 M NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml pepstatin. Twenty micrograms of the cell lysate is separated on an 8% SDS-PAGE gel and transferred to a nitrocellulose membrane.
• the membrane After blocking with 5% nonfat dry milk/phosphate-buffered saline for 1 h, the membrane is incubated overnight at 4°C or at room temperature for 2-4 hours with a 1 :1000 dilution of anti-HCPN serum in 2% nonfat dry milk/phosphate-buffered saline. The membrane is then washed and incubated with a 1 : 1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG in 2% dry milk/phosphate-buffered saline. After washing with 0.1% Tween 20 in phosphate-buffered saline, the HCPN protein is detected and compared to controls by using chemiiuminescence. Induction of HCPN under stress conditioas is evidence of HCPN activity.
• HCPN activity can be determined by measuring the ability to promote ATP hydrolysis by Hsp70. Briefly, 1 ⁇ g Hsp70 protein is incubated with 1 nmol unlabled ATP and 0.01 ⁇ Ci of OC32P-ATP in ATPase buffer (50 mM HEPES, pH 7.4, 50 mM NaCl, 10 mM DTT, and 2 mM MgCl 2 ) in a total volume of 20 ⁇ l at 30 °C with or without HCPN. After 1 hr, 1 ⁇ l of the reaction is spotted on polyethyleneimine cellulose TLC plates and developed in IM formic acid with 0.5 M LiCl. Plates are examined for conversion of ⁇ 32P-ATP to ⁇ 32P-ADP by phosphorimager (Hunter, supra).
• HCPN function is assessed by expressing the sequences encoding HCPN at physiologically elevated levels in mammalian cell culture systems.
• cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
• Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation.
• 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
• Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
• Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
• FCM Flow cytometry
• FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down- regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. ( 1994) Flow Cytometry. Oxford, New York NY.
• HCPN The influence of HCPN on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HCPN and either CD64 or CD64-GFP.
• CD64 and CD64- GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
• Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
• mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding HCPN and other genes of interest can be analyzed by northern analysis or microarray techniques.
• HCPN Specific Antibodies HCPN substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g.,
• the HCPN amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
• LASERGENE software DNASTAR
• Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
• oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
• ABI 431 A peptide synthesizer PE Biosystems
• KLH Sigma- Aldrich, St. Louis MO
• MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
• Rabbits are immunized with the oligopeptide-KLH complex in complete Freund s adjuvant.
• Resulting antisera are tested for antipeptide and anti-HCPN activity by, for example, binding the peptide or HCPN to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
• Naturally occurring or recombinant HCPN is substantially purified by immunoaffinity chromatography using antibodies specific for HCPN.
• An immunoaffinity column is constructed by covalently coupling anti-HCPN antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
• HCPN Media containing HCPN are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HCPN (e.g., high ionic strength buffers in the presence of detergent).
• the column is eluted under conditions that disrupt antibody/HCPN binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HCPN is collected.
• HCPN or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent.
• Bolton-Hunter reagent See, e.g., Bolton A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.
• Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HCPN, washed, and any wells with labeled HCPN complex are assayed. Data obtained using different concentrations of HCPN are used to calculate values for the number, affinity, and association of HCPN with the candidate molecules.
• molecules interacting with HCPN are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989, Nature 340:245-246), or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
• HCPN may also be used in the PATHCALLING process (CuraGen Co ⁇ ., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
• ABI FACTURA A program that removes vector sequences and PE Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences.
• ABI/PARACEL FDF A Fast Data Finder useful in comparing and PE Biosystems, Foster City, CA; Mismatch ⁇ 50% annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
• ABI AutoAssembler A program that assembles nucleic acid sequences. PE Biosystems, Foster City, CA.
• fastx score 100 or greater
• Phred A base-calling algorithm that examines automated Ewing, B et al. (1998) Genome Res sequencer traces with high sensitivity and 8 : 175- 185 , Ewing, B . and P. Green probability. (1998) Genome Res. 8: 186-194.

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