WO2002000840A2 - Lyases humaines - Google Patents

Lyases humaines Download PDF

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Publication number
WO2002000840A2
WO2002000840A2 PCT/US2001/019166 US0119166W WO0200840A2 WO 2002000840 A2 WO2002000840 A2 WO 2002000840A2 US 0119166 W US0119166 W US 0119166W WO 0200840 A2 WO0200840 A2 WO 0200840A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
hlya
antibody
seq
Prior art date
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PCT/US2001/019166
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English (en)
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WO2002000840A3 (fr
Inventor
Michael Thornton
Jayalaxmi Ramkumar
Catherine M. Tribouley
Henry Yue
Danniel B. Nguyen
Monique G. Yao
Chandra Patterson
Ameena R. Gandhi
Neil Burford
Kavitha Thangavelu
Mariah R. Baughn
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Incyte Genomics, Inc.
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Application filed by Incyte Genomics, Inc. filed Critical Incyte Genomics, Inc.
Priority to CA002381338A priority Critical patent/CA2381338A1/fr
Priority to EP01944529A priority patent/EP1292685A2/fr
Priority to AU2001266932A priority patent/AU2001266932A1/en
Priority to JP2002505956A priority patent/JP2004501632A/ja
Publication of WO2002000840A2 publication Critical patent/WO2002000840A2/fr
Publication of WO2002000840A3 publication Critical patent/WO2002000840A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to nucleic acid and amino acid sequences of human lyases and to the use of these sequences in the diagnosis, treatment, and prevention of cancers and autoimmune/inflammatory, neurological, and eye disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of human lyases.
  • Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-S, C-(halide),
  • the group of C-C lyases includes carboxyl-lyases (decarboxylases), aldehyde-lyases (aldolases), oxo-acid-lyases, and other lyases.
  • the C-0 lyase group includes hydro-lyases, lyases acting on polysaccharides, and other lyases.
  • the C-N lyase group includes ammonia-lyases, amidine- lyases, amine-lyases (deaminases), and other lyases.
  • Lyases are critical components of cellular biochemistry, with roles in metabolic energy production, including fatty acid metabolism and the tricarboxylic acid cycle, as well as other diverse enzymatic processes.
  • One important family of lyases are the carbonic anhydrases (CA), also called carbonate dehydratases, which catalyze the hydration of carbon dioxide in the reaction H 2 0 + C0 2 ⁇ HC0 3 " + H + .
  • CA carbonic anhydrases
  • CA accelerates this reaction by a factor of over 10 s by virtue of a zinc ion located in a deep cleft about 15A below the protein's surface and co-ordinated to the imidazole groups of three His residues. Water bound to the zinc ion is rapidly converted to HC0 3 " .
  • CAI, CAII, and CAIJJ three cytosolic isozymes
  • CAIV and CAVE two membrane-bound forms
  • CAV mitochondrial form
  • CAVT secreted salivary form
  • CAVT yet uncharacterized isozyme
  • CAI, CAII, and bovine CADI have similar secondary structure and polypeptide-chain fold
  • CAI has 6 tryptophans
  • CAII has 7
  • CADI has 8 (Boren, K. et al. (1996) Protein Sci. 5:2479-2484).
  • CAU is the predominant CA isoenzyme in the brain of mammals.
  • CAs participate in a variety of physiological processes that involve pH regulation, C0 2 and HC0 3 " transport, ion transport, and water and electrolyte balance.
  • CAH contributes to H + secretion by gastric parietal cells, by renal tubular cells, and by osteoclasts that secrete H + to acidify the bone-resorbing compartment.
  • CAH promotes HCCy secretion by pancreatic duct cells, cilary body epithelium, choroid plexus, salivary gland acinar cells, and distal colonal epithelium, thus playing a role in the production of pancreatic juice, aqueous humor, cerebrospinal fluid, and saliva, and contributing to electrolyte and water balance.
  • CAH also promotes C0 2 exchange in proximal tubules in the kidney, in erythrocytes, and in lung.
  • CAIV has roles in several tissues: it facilitates HC0 3 " reabsorption in the kidney; promotes C0 2 flux in tissues including brain, skeletal muscle, and heart muscle; and promotes C0 2 exchange from the blood to the alveoli in the lung.
  • CANT probably plays a role in pH regulation in saliva, along with CAH, and may have a protective effect in the esophagus and stomach.
  • Mitochondrial CAV appears to play important roles in gluconeogenesis and ureagenesis, based on the effects of CA inhibitors on these pathways.
  • CA inhibitors such as acetazolamide are used in the treatment of glaucoma (Stewart, W.C. (1999) Curr. Opin. Opthamol. 10:99-108), essential tremor and Parkinson's disease (Uitti, R.J. (1998) Geriatrics 53:46-48, 53-57), intermittent ataxia (Singhvi, J.P. et al. (2000) Neurology India 48:78-80), and altitude related illnesses (Klocke, D.L. et al. (1998) Mayo Clin. Proc. 73:988-992).
  • CA activity can be particularly useful as an indicator of long-term disease condition, since the enzyme reacts relatively slowly to physiological changes.
  • CAI and zinc concentrations have been observed to decrease in hyperthyroid Graves' disease (Yoshida, K. (1996) Tohoku J. Exp. Med. 178:345-356) and glycosylated CAI is observed in diabetes mellitus (Kondo, T. et al. (1987) Clin. Chim. Acta 166:227-236).
  • a positive correlation has been observed between CAI and CAH reactivity and endometriosis (Brinton, DA. et al. (1996) Ann. Clin. Lab. Sci. 26:409-420; D'Craz , O.J. et al. (1996) Fertil. Steril. 66:547-556).
  • ODC ornithine decarboxylase
  • conserveed residues include those at the PLP binding site and a stretch of glycine residues thought to be part of a substrate binding region (Prosite PDOC00685 Orn/DAP/Arg decarboxylase family 2 signatures). Mammalian ODCs also contain PEST regions, sequence fragments enriched in proline, glutamic acid, serine, and threonine residues that act as signals for intracellular degradation (Medina, supra).
  • ODC levels and activity Many chemical carcinogens and tumor promoters increase ODC levels and activity.
  • oncogenes may increase ODC levels by enhancing transcription of the ODC gene, and ODC itself may act as an oncogene when expressed at very high levels.
  • a high level of ODC is found in a number of precancerous conditions, and elevation of ODC levels has been used as part of a screen for tumor-promoting compounds (Pegg, A.E. et al. (1995) J. Cell. Biochem. Suppl. 22:132-138).
  • Inhibitors of ODC have been used to treat tumors in animal models and human clinical trials, and have been shown to reduce development of tumors of the bladder, brain, esophagus, gastrointestinal tract, lung, oral cavity, mammary gland, stomach, skin and trachea (Pegg, supra; McCann, P.P. and A.E. Pegg (1992) Pharmac. Ther. 54:195-215). ODC also shows promise as a target for chemoprevention (Pegg, supra).
  • ODC inhibitors have also been used to treat infections by African trypanosom.es , malaria, and Pneumocystis carinii, and are potentially useful for treatment of autoimmune diseases such as lupus and rheumatoid arthritis (McCann, supra).
  • GAD glutamate decarboxylase
  • HDC histidine decarboxylase
  • DDC aromatic-L-amino-acid decarboxylase
  • SCD cysteine sulfinic acid decarboxylase
  • Taurine is an abundant sulfonic amino acid in brain and is thought to act as an osmoregulator in brain cells (Bitoun, M. and Tappaz, M. (2000) J. Neurochem. 75:919-924).
  • the discovery of new human lyases and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in tlie diagnosis, prevention, and treatment of cancers and autoinimune/inflammatory, neurological, and eye disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of human lyases.
  • the invention features purified polypeptides, human lyases, referred to collectively as "HLYA” and individually as “HLYA-1,” “HLYA-2,” and "HLYA-3.”
  • the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-3.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1-3.
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l- 3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3.
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ED NO: 1-3.
  • the polynucleotide is selected from the group consisting of SEQ ED NO:4-6.
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO:l-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3.
  • 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 selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherem 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 selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO.l -3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3.
  • the invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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 selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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 selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, b) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of 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 selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO:l-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and a pharmaceutically acceptable excipient.
  • the composition comprises an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3.
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of • functional HLYA, 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 selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l-3.
  • the method 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 HLYA, 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 selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO:l -3.
  • 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 HLYA, 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 selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NO: 1-3.
  • 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 that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NO : 1 -3 , b) a naturally occurring polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3, and d) an irnmunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-3.
  • 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, wherem 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 ED NO:4-6, 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, ii) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of 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 selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, ii) a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ED NO:4-6, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • a target polynucleotide selected from the group consisting of i) a polynucleotide comprising
  • the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is also shown.
  • Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.
  • Table 5 shows the representative cDNA library for polynucleotides of the invention.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
  • HLYA refers to the amino acid sequences of substantially purified HLYA 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 HLYA.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of HLYA either by directly interacting with HLYA or by acting on components of the biological pathway in which HLYA participates.
  • allelic variant is an alternative form of the gene encoding HLYA. 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. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding HLYA include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as HLYA or a polypeptide with at least one functional characteristic of HLYA. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HLYA, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HLYA.
  • 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 HLYA.
  • 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 HLYA 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 haying 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 HLYA.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of HLYA either by directly interacting with HLYA or by acting on components of the biological pathway in which HLYA 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 HLYA 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
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition capable of base-pairing with the "sense”
  • 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.
  • 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 HLYA, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5 -AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding HLYA or fragments of HLYA 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 (Applied 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 GELVTEW 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 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.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • a "fragment” is a unique portion of HLYA or the polynucleotide encoding HLYA 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 purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • 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 ED NO:4-6 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ED NO:4-6, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ED NO:4-6 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ED NO:4-6 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ED NO:4-6 and the region of SEQ ED NO:4-6 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ED NO:l-3 is encoded by a fragment of SEQ ED NO:4-6.
  • a fragment of SEQ ED NO: 1-3 comprises a region of unique amino acid sequence that specifically identifies SEQ ED NO : 1 -3.
  • a fragment of SEQ ED NO : 1 -3 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ED NO: 1-3.
  • the precise length of a fragment of SEQ ED NO : 1 -3 and the region of SEQ ED NO : 1 -3 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a “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.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blast ,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • 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 weh-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 (April-21 -2000) with blastp set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62
  • 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.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for 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/ l 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).
  • 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.
  • “hnmune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • An ''immunogenic fragment is a polypeptide or oligopeptide fragment of HLYA 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 HLYA 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 HLYA.
  • modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HLYA.
  • 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 ohgonucleotide 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 HLYA 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 HLYA.
  • Probe refers to nucleic acid sequences encoding HLYA, 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 purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the Pri OU 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/MET 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 vims, 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 HLYA, nucleic acids encoding HLYA, or fragments thereof 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.
  • 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 the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of 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.
  • 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 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymo ⁇ hic variants also may encompass "single nucleotide polymo ⁇ hisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymo ⁇ hisms
  • 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- 1 99) 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 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human lyases (HLYA), the polynucleotides encoding HLYA, and the use of these compositions for the diagnosis, treatment, or prevention of cancers and autoimmune/inflammatory, neurological, and eye disorders.
  • HLYA new human lyases
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ED NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ED) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
  • Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (Genbank ED NO:) of the nearest GenBank homolog.
  • Column 4 shows the probability score for the match between each polypeptide and its GenBank homolog.
  • Column 5 shows the annotation of the GenBank homolog along with relevant citations where applicable, all of which are expressly inco ⁇ orated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2
  • FIG. 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison WE).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein stracture/function analysis and in some cases, searchable databases to which the analytical methods were applied.
  • SEQ ED NO:l is 59% identical, from residue Ml to residue A239, to human carbonic anhydrase 1 (GenBank ED gl79793) as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 6.9e-80, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO:l also contains a eukaryotic-type carbonic anhydrase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • SEQ ED NO:l is a carbonic anhydrase.
  • SEQ ED NO:2 is 59% identical, from residue Ml to residue 1459, to Xenopus laevis ornithine decarboxylase-2 (GenBank ED g6746571) as determined by the Basic Local Alignment Search Tool (BLAST).
  • BLAST Basic Local Alignment Search Tool
  • the BLAST probability score is 1.9e-148, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance.
  • SEQ ED NO:2 also contains an ornithine decarboxylase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses provide further corroborative evidence that SEQ ID NO:2 is a carbonic anhydrase.
  • SEQ ED NO:3 is 58% identical, from residue E30 to residue R314, to human adenylate kinase (GenBank ID g3283386) as determined by the Basic Local Angnment Search Tool (BLAST).
  • SEQ ED NO:3 also contains a eukaryotic-type carbonic anhydrase domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains.
  • HMM hidden Markov model
  • PROFTLESCAN analyses provide further corroborative evidence that SEQ ED NO:3 is a eukaryotic-type carbonic anhydrase.
  • the algorithms and parameters for the analysis of SEQ ED NO:l-3 are described in Table 7.
  • the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and the corresponding Incyte polynucleotide consensus sequence number (incyte Polynucleotide ED) for each polynucleotide of the invention.
  • Column 3 shows the length of each polynucleotide sequence in basepairs.
  • Column 4 lists fragments of the polynucleotide sequences which are useful, for example, in hybridization or amplification technologies that identify SEQ ED NO:4-6 or that distinguish between SEQ ED NO:4-6 and related polynucleotide sequences.
  • Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention.
  • Columns 6 and 7 of Table 4 show the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.
  • the identification numbers in Column 5 of Table 4 may refer specifically, for example, to
  • Incyte cDNAs along with their corresponding cDNA libraries.
  • 2698586F6 is the identification number of an Incyte cDNA sequence
  • UTRSNOT12 is the cDNA library from which it is derived.
  • 644715H1 is the identification number of an Incyte cDNA sequence
  • BRAENOC01 is the cDNA library from which it is derived
  • incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 71672723 VI or 70996811 VI).
  • the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., gl 191247) which contributed to the assembly of the full length polynucleotide sequences.
  • the identification numbers in column 5 may refer to coding regions predicted by Genscan analysis of genomic DNA. The Genscan-predicted coding sequences may have been edited prior to assembly. (See Example EV.)
  • the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon- stretching" algorithm. (See Example V.) In some cases, incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences.
  • the tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.
  • the invention also encompasses HLYA variants.
  • a preferred HLYA 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 HLYA amino acid sequence, and which contains at least one functional or structural characteristic of HLYA.
  • the invention also encompasses polynucleotides which encode HLYA.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:4-6, which encodes HLYA.
  • the polynucleotide sequences of SEQ ED NO:4-6 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 HLYA.
  • 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 HLYA.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:4-6 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 ED NO:4-6. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HLYA.
  • nucleotide sequences which encode HLYA and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring HLYA under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HLYA 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 HLYA and HLYA 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 HLYA or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ED NO:4-6 and fragments thereof under various conditions of stringency.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ED NO:4-6 and fragments thereof under various conditions of stringency.
  • 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 (Applied Biosystems), 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 AB1 CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular 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 HLYA 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.
  • one method which may be employed restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1 93) 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 it is preferable to use libraries that have been size-selected to include larger cDNAs.
  • 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 electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied 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.
  • appropriate software e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems
  • polynucleotide sequences or fragments thereof which encode HLYA may be cloned in recombinant DNA molecules that direct expression of HLYA, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express HLYA.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HLYA-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 MOLECULARBREEDENG (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 HLYA, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds.
  • MOLECULARBREEDENG 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
  • 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 HLYA may be synthesized, in whole or in part, using chemical methods weU known in the art.
  • chemical methods See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.
  • HLYA 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 maybe achieved using the ABI 431 A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of HLYA, or any part thereof, maybe 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.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)
  • 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.
  • 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 HLYA. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HLYA.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • no additional transcriptional or translational control signals may be needed.
  • 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. CeU Differ. 20:125-162.)
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding HLYA. These include, but are not Hmited 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., baculovims); 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., baculovims); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV
  • Expression vectors derived from retrovirases, 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 ceU employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HLYA.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding HLYA can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding HLYA into the vector's multiple cloning site disrupts the lacZ 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 HLYA may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of HLYA.
  • 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 HLYA. Transcription of sequences encoding HLYA may be driven by 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). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results ProbL Cell Differ.
  • 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. 3:1311).
  • plant promoters
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated tiansfection. (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 HLYA 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 HLYA 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.
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of
  • 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.
  • HLYA in cell lines is preferred.
  • sequences encoding HLYA 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. These include, but are not limited to, the he ⁇ es simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tt and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) CeU 11 :223-232; Lowy, I. et al. (1980) CeU 22:817-823.) Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the a inoglycosides neomycin and G-418
  • als and pat confer resistance to cUorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboUtes.
  • 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. 55:121-131.)
  • 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 HLYA is inserted within a marker gene sequence
  • transformed ceUs containing sequences encoding HLYA can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HLYA 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 weU.
  • host ceUs that contain the nucleic acid sequence encoding HLYA and that express HLYA may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not Umited to, DNA-DNA or DNA-RNA hybridizations, PCR ampUfication, 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 HLYA using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-Unked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HLYA include oUgolabeUng, nick translation, end-labeUng, or PCR ampUfication using a labeled nucleotide.
  • the sequences encoding HLYA, 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 radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the Uke.
  • Host cells transformed with nucleotide sequences encoding HLYA may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed cell may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode HLYA may be designed to contain signal sequences which direct secretion of HLYA through a prokaryotic or eukaryotic cell membrane.
  • a host ceU strain may be chosen for its abiUty to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not Umited to, acetylation, carboxylation, glycosylation, phosphorylation, Upidation, 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 ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture CoUection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture CoUection
  • Manassas VA American Type Culture CoUection
  • natural, modified, or recombinant nucleic acid sequences encoding HLYA may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric HLYA protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of HLYA activity.
  • Heterologous protein and peptide moieties may also faciUtate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not Umited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmoduUn 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, calmoduUn, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that speciflcaUy recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the HLYA encoding sequence and the heterologous protein sequence, so that HLYA may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10).
  • a variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins.
  • synthesis of radiolabeled HLYA 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.
  • HLYA of the present invention or fragments thereof may be used to screen for compounds that speciflcaUy bind to HLYA. At least one and up to a pluraUty of test compounds may be screened for specific binding to HLYA.
  • test compounds include antibodies, oUgonucleotides, proteins (e.g., receptors), or smaU molecules.
  • the compound thus identified is closely related to the natural Ugand of
  • HLYA e.g., a Ugand 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 HLYA binds, or to at least a fragment of the receptor, e.g., the Ugand binding site. In either case, the compound can be rationaUy designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate ceUs which express HLYA, either as a secreted protein or on the ceU membrane.
  • ceUs include ceUs from mammals, yeast, Drosophila, or E. coU. CeUs expressing HLYA or ceU membrane fractions which contain HLYA are then contacted with a test compound and binding, stimulation, or inhibition of activity of either HLYA 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 HLYA, either in solution or affixed to a soUd support, and detecting the binding of HLYA to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • HLYA of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of HLYA.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for HLYA activity, wherein HLYA is combined with at least one test compound, and the activity of HLYA in the presence of a test compound is compared with the activity of HLYA in the absence of the test compound. A change in the activity of HLYA in the presence of the test compound is indicative of a compound that modulates the activity of HLYA.
  • a test compound is combined with an in vitro or ceU-free system comprising HLYA under conditions suitable for HLYA activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of HLYA may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraUty of test compounds may be screened.
  • polynucleotides encoding HLYA or their mammaUan 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 ceUs such as the mouse 129/SvJ ceU Une, are derived from the early mouse embryo and grown in culture.
  • the ES ceUs 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).
  • 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) CUn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy 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 HLYA may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceUs have the potential to differentiate into at least eight separate ceU Uneages including endoderm, mesoderm, and ectodermal ceU types. These ceU Uneages differentiate into, for example, neural ceUs, hematopoietic Uneages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding HLYA 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 HLYA is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred Unes are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress HLYA e.g., by secreting HLYA in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) BiotechnoL Annu. Rev. 4:55-74). THERAPEUTICS
  • HLYA Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of HLYA and human lyases.
  • the expression of HLYA is closely associated with diseased breast tissue as weU as adult and fetal brain tissue. Therefore, HLYA appears to play a role in cancers and autoimmune/inflammatory, neurological, and eye disorders.
  • HLYA In the treatment of disorders associated with increased HLYA expression or activity, it is desirable to decrease the expression or activity of HLYA.
  • HLYA 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 HLYA.
  • a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gangUa, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AEDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy
  • AEDS acquired immunodeficiency syndrome
  • composition comprising a substantiaUy purified HLYA 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 HLYA including, but not Umited to, those provided above.
  • an agonist which modulates the activity of HLYA may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HLYA including, but not Umited to, those Usted above.
  • an antagonist of HLYA may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HLYA.
  • disorders include, but are not Umited to, those cancers and autoimmune/inflammatory, neurological, and eye disorders described above.
  • an antibody which speciflcaUy binds HLYA may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express HLYA.
  • a vector expressing the complement of the polynucleotide encoding HLYA may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HLYA including, but not Umited 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 skiU in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy 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 HLYA may be produced using methods which are generally known in the art.
  • purified HLYA may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which speciflcaUy bind HLYA.
  • Antibodies to HLYA may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • NeutraUzing antibodies i.e., those which inhibit dimer formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HLYA or with any fragment or oUgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Umited 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 especiaUy preferable.
  • the oUgopeptides, peptides, or fragments used to induce antibodies to HLYA have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of HLYA 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 HLYA may be prepared using any technique which provides for the production of antibody molecules by continuous ceU Unes in culture.
  • chimeric antibodies such as the spUcing 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 "chimeric antibodies” such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used.
  • techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HLYA-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn Ubraries or panels of highly specific binding reagents as disclosed in the Uterature.
  • Antibody fragments which contain specific binding sites for HLYA may also be generated.
  • such fragments include, but are not Umited 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 Ubraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • K ⁇ association constant
  • High-affinity antibody preparations with 1L ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the HLYA- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of HLYA, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, ERL Press, Washington DC; LiddeU, 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 quaUty and suitabiUty of such preparations for certain downstream appUcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of HLYA-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guideUnes for antibody quaUty and usage in various appUcations are generally available. (See, e.g., Catty, supra, and CoUgan et al. supra.)
  • the polynucleotides encoding HLYA 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 oUgonucleotides) to the coding or regulatory regions of the gene encoding HLYA.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
  • antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HLYA. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press Inc., Totawa NJ.)
  • Antisense sequences can be deUvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein.
  • Slater J.E. et al. (1998) J. AUergy CU. Immunol. 102(3):469-475; and Scanlon, K.J. et al.
  • Antisense sequences can also be infroduced intiaceUularly 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 deUvery mechanisms include Uposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • polynucleotides encoding HLYA may be used for somatic or germUne gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCED)-Xl disease characterized by X- Unked 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.
  • SCED severe combined immunodeficiency
  • ADA adenosine deaminase
  • conditionaUy lethal gene product e.g., in the case of cancers which result from unregulated ceU proUferation
  • a protein which affords protection against intraceUular parasites e.g., against human retroviruses, such as human immunodeficiency virus (HTV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA.
  • HLYA hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiUensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi.
  • diseases or disorders caused by deficiencies in HLYA are treated by constructing mammaUan expression vectors encoding HLYA and introducing these vectors by mechanical means into HLYA-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual ceUs, (ii) balUstic gold particle deUvery, (Mi) Uposome-mediated tiansfection, (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) CeU 91:501-510; Boulay, J-L. and H. Recipon (1998) Curr. Opin. BiotechnoL 9:445-450).
  • Expression vectors that may be effective for the expression of HLYA include, but are not Umited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Envitrogen, Carlsbad CA), PCMV-SCREPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • HLYA 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 tetracycUne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V and H.M. Blau (1998) Curr. Opin.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • FK506/rapamycin inducible promoter or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and Blau, H.M. supra)), or (Ui) a tissue-specific promoter or the native promoter of the endogenous gene encoding HLYA from a normal individual.
  • Uposome transformation kits e.g., the PERFECT LEPID TRANSFECTION KIT, available from Envitrogen
  • PERFECT LEPID TRANSFECTION KIT available from Envitrogen
  • 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 ceUs requires modification of these standardized mammaUan tiansfection protocols.
  • diseases or disorders caused by genetic defects with respect to HLYA expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding HLYA under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional retrovirus cM-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy available (Stratagene) and are based onpubUshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci.
  • the vector is propagated in an appropriate vector producing ceU Une (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. ViroL 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. MiUer (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R.
  • VPCL ceU Une
  • U.S. Patent Number 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU Unes and is hereby inco ⁇ orated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T-ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J.
  • an adenoviras-based gene therapy deUvery system is used to deUver polynucleotides encoding HLYA to ceUs which have one or more genetic abnormaUties with respect to the expression of HLYA.
  • the construction and packaging of adenovirus-based vectors are well known to those with ordinary skiU in the art.
  • RepUcation 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). PotentiaUy useful adenoviral vectors are described in U.S.
  • Patent Number 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"), hereby incorporated 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 deUvery system is used to deUver polynucleotides encoding HLYA to target ceUs which have one or more genetic abnormaUties with respect to the expression of HLYA.
  • HSV simplex virus
  • the use of he ⁇ es simplex virus (HSV)-based vectors may be especiaUy valuable for introducing HLYA to ceUs of the central nervous system, for which HSV has a tropism.
  • HSV simplex virus
  • HSV he ⁇ es simplex virus
  • 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 ceU 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. For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby inco ⁇ orated by reference.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encoding HLYA to target ceUs.
  • SFV SemUki Forest Virus
  • SFV SemUki Forest Virus
  • SFV SemUki Forest Virus
  • alphavirus RNA repUcation a subgenomic RNA is generated that normaUy encodes the viral capsid proteins.
  • This subgenomic RNA repUcates to higher levels than the fuU length genomic RNA, resulting in the ove ⁇ roduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • inserting the coding sequence for HLYA into the alphavirus genome in place of the capsid-coding region results in the production of a large number of HLYA-coding RNAs and the synthesis of high levels of HLYA in vector transduced cells.
  • alphavirus infection is typicaUy associated with ceU lysis within a few days
  • the abiUty to estabUsh a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SEN) indicates that the lytic repUcation of alphaviruses can be altered to suit the needs of the gene therapy appUcation (Dryga, S. A. et al. (1997) Virology 228:74-83).
  • the wide host range of alphaviruses wiU allow the introduction of HLYA into a variety of ceU types.
  • the specific transduction of a subset of ceUs 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 weU known to those with ordinary skiU in the art.
  • OUgonucleotides derived from the transcription initiation site may also be employed to inhibit gene expression.
  • inhibition can be achieved using triple heUx base-pairing methodology.
  • Triple heUx pairing is useful because it causes inhibition of the abiUty of the double heUx to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Recent therapeutic advances using triplex DNA have been described in the Uterature. (See, e.g., Gee, J.E. et al. (1994) in Huber, B.E. and B.I. Carr, Molecular and Ifnmunologic Approaches. Futura PubUshing, Mt. Kisco NY, pp. 163-177.)
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may speciflcaUy and efficiently catalyze endonucleolytic cleavage of sequences encoding HLYA.
  • RNA target Specific ribozyme cleavage sites within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oUgonucleotide inoperable. The suitabiUty of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides 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 oUgonucleotides such as soUd phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HLYA. 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 ceU Unes, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stabiUty and half-Ufe. Possible modifications include, but are not Umited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase Unkages 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 HLYA.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not Umited to, oUgonucleotides, antisense oUgonucleotides, triple heUx-forming oUgonucleotides, 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 speciflcaUy inhibits expression of the polynucleotide encoding HLYA may be therapeuticaUy useful, and in the treatment of disorders associated with decreased HLYA expression or activity, a compound which speciflcaUy promotes expression of the polynucleotide encoding HLYA may be therapeuticaUy useful.
  • At least one, and up to a pluraUty, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary Ubrary of naturaUy-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 Ubrary of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding HLYA is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding HLYA are assayed by any method commonly known in the art.
  • typingUy 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 HLYA.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU Une such as HeLa ceU (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13).
  • a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU Une such as HeLa ceU (Clarke, M.L. et al. (2000)
  • a particular embodiment of the present invention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into ceUs or tissues are available and equaUy suitable for use in vivo, in vitro, and ex vivo.
  • oUgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient.
  • DeUvery by tiansfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art. (See, e.g., Goldman, C.K. et al. (1997) Nat. BiotechnoL 15:462-466.)
  • compositions which generaUy comprises an active ingredient formulated with a pharmaceutically acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA).
  • Such compositions may consist of HLYA, antibodies to HLYA, and mimetics, agonists, antagonists, or inhibitors of HLYA.
  • compositions utiUzed in this invention may be administered by any number of routes including, but not Umited to, oral, intravenous, intiamuscular, intra-arterial, intrameduUaiy, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subUngual, or rectal means.
  • Compositions for pulmonary administration may be prepared in Uquid or dry powder form.
  • compositions are generaUy aerosoUzed immediately prior to inhalation by the patient.
  • aerosol deUvery of fast- acting formulations is well-known in the art.
  • macromolecules e.g. larger peptides and proteins
  • Pulmonary deUvery has the advantage of administration without needle injection, and obviates the need for potentiaUy 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 piupose.
  • the determination of an effective dose is weU within the capabiUty of those skiUed in the art.
  • SpeciaUzed forms of compositions may be prepared for direct intraceUular deUvery of macromolecules comprising HLYA or fragments thereof.
  • Uposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule.
  • HLYA or a fragment thereof may be joined to a short cationic N- terminal portion from the HEV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, 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 therapeuticaUy effective dose refers to that amount of active ingredient, for example
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in ceU cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD J0 (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 ceU 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 Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • the exact dosage wiU be determined by the practitioner, in Ught of factors related to the subject requiring treatment. Dosage and administiation 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 combinations), 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-Ufe 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 administiation.
  • Guidance as to particular dosages and methods of deUvery is provided in the Uterature and generaUy available to practitioners in the art. Those skiUed in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, deUvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
  • antibodies which speciflcaUy bind HLYA may be used for the diagnosis of disorders characterized by expression of HLYA, or in assays to monitor patients being treated with HLYA or agonists, antagonists, or inhibitors of HLYA.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HLYA include methods which utilize the antibody and a label to detect HLYA in human body fluids or in extiacts of ceUs 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.
  • HLYA human immunosorbent assays
  • FACS fluorescence-activated cell sorting
  • HLYA expression estabUshed by combining body fluids or cell extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to HLYA under conditions suitable for complex formation.
  • the amount of standard complex formation may be quantitated by various methods, such as photometric means.
  • Quantities of HLYA expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • the polynucleotides encoding HLYA may be used for diagnostic pu ⁇ oses.
  • the polynucleotides which may be used include oUgonucleotide 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 HLYA may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of HLYA, and to monitor regulation of HLYA levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HLYA or closely related molecules may be used to identify nucleic acid sequences which encode HLYA.
  • 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 ampUfication wiU determine whether the probe identifies only naturally occurring sequences encoding HLYA, aUeUc 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 HLYA encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ED NO:4-6 or from genomic sequences including promoters, enhancers, and intions of the HLYA gene.
  • Means for producing specific hybridization probes for DNAs encoding HLYA include the cloning of polynucleotide sequences encoding HLYA or HLYA derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commerciaUy 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 radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaUne phosphatase coupled to the probe via avidin/biotin coupUng systems, and the Uke.
  • Polynucleotide sequences encoding HLYA may be used for the diagnosis of disorders associated with expression of HLYA.
  • a cancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangUa, gastrointestinal tract, heart, kidney, Uver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, saUvary glands, skin, spleen, testis, thymus, thyroid, and uterus; an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy
  • AIDS acquired immunodeficiency syndrome
  • the polynucleotide sequences encoding HLYA may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-Uke assays; and in microarrays utiUzing fluids or tissues from patients to detect altered HLYA expression.
  • quaUtative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding HLYA may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding HLYA may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is 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 HLYA in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabhshed. This may be accompUshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HLYA, under conditions suitable for hybridization or ampUfication.
  • 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 substantiaUy 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 estabUsh 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.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual cUnical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment eariier thereby preventing the development or further progression of the cancer.
  • Additional diagnostic uses for oUgonucleotides designed from the sequences encoding HLYA may involve the use of PCR. These oUgomers may be chemically synthesized, generated enzymatically, or produced in vitro.
  • OUgomers will preferably contain a fragment of a polynucleotide encoding HLYA, or a fragment of a polynucleotide complementary to the polynucleotide encoding HLYA, and will be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from the polynucleotide sequences encoding HLYA 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 Umited to, single-stranded conformation polymo ⁇ hism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymo ⁇ hism
  • fSSCP fluorescent SSCP
  • oUgonucleotide primers derived from the polynucleotide sequences encoding HLYA are used to ampUfy 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 Uke.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stianded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the oUgonucleotide primers are fluorescently labeled, which aUows detection of the ampUmers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in siUco 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 spectiometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • Methods which may also be used to quantify the expression of HLYA include radiolabeUng or biotinylating nucleotides, coampUfication of a control nucleic acid, and inte ⁇ olating results from standard curves.
  • radiolabeUng or biotinylating nucleotides See, e.g., Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oUgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oUgonucleotides 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 below.
  • 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.
  • HLYA HLYA, fragments of HLYA, or antibodies specific for HLYA 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 ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU 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 totaUty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in gh-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a pluraUty 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, ceU Unes, 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 ceU Une.
  • 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 precUnical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds.
  • AU 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)
  • 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 tianscript 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 the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or ceU 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 ceU' s proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU 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 visuaUzed 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 generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples 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 foUowed 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 HLYA to quantify the levels of HLYA expression.
  • 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. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). 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 paraUel with toxicant signatures at the tianscript level.
  • There is a poor correlation between tianscript 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 tianscript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiUng may be more reUable 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 compaied 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 appUcation W095/251116; Shalon, D. et al. (1995) PCT appUcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and HeUer, M.J.
  • nucleic acid sequences encoding HLYA may be used to generate hybridization probes useful in mapping the naturaUy 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 potentiaUy 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 Ubraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA Ubraries.
  • nucleic acid sequences of the invention may be used to develop genetic Unkage 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
  • Unkage analysis using estabUshed chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammaUan 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 locaUzed by genetic Unkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A.
  • 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.
  • HLYA its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries 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 soUd support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between HLYA 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.
  • En this method large numbers of different smaU test compounds are synthesized on a soUd substrate. The test compounds are reacted with HLYA, or fragments thereof, and washed. Bound HLYA is then detected by methods weU known in the art. Purified HLYA can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
  • nucleotide sequences which encode HLYA 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 Umited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs were derived from cDNA Ubraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto CA) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centiifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A)+ RNA was isolated using oUgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constructed the corresponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the
  • UNIZAP vector system (Stiatagene) 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 oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • the cDNA was size-selected (300- 1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were Ugated into compatible restriction enzyme sites of the polyUnker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stiatagene), or pINCY (Incyte Genomics, Palo Alto CA), or derivatives thereof.
  • Recombinant plasmids were transformed into competent E. coU ceUs including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies.
  • Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stiatagene) or by ceU lysis. Plasmids were purified using at least one of the foUowing: 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, QEAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids were resuspended in 0.1 ml of distiUed water and stored, with or without lyophiUzation, at 4°C
  • plasmid DNA was ampUfied from host cell lysates using direct Unk PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycUng steps were carried out in a single reaction mixture. Samples were processed and stored in 384-weU plates, and the concentration of ampUfied plasmid DNA was quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN H fluorescence scanner (Labsystems Oy, Helsinki, Finland).
  • Electiophoretic 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 (AppUed Biosystems) in conjunction with standard ABI protocols and base calUng 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).
  • Example VEIL The polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, Unker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRENTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • PubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases
  • BLOCKS, PRENTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • HMM is a probabiUstic approach which analyzes consensus primary structures of gene famiUes. See, for example, Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.)
  • the queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs GenBank ESTs, stitched sequences, stretched sequences, or
  • Genscan-predicted coding sequences were used to extend Incyte cDNA assemblages to fuU length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the fuU length polynucleotide sequences were translated to derive the corresponding fuU length polypeptide sequences.
  • a polypeptide of the invention may begin at any of the methionine residues of the fuU length translated polypeptide.
  • FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM.
  • FuU length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as inco ⁇ orated into the MEGALIGN multisequence aUgnment program (DNASTAR), which also calculates the percent identity between aUgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides appUcable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where appUcable, the scores, probabiUty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiUty value, the greater the identity between two sequences).
  • Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode human lyases, the encoded polypeptides were analyzed by querying against PFAM models for human lyases.
  • Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis was also used to find any Incyte cDNA or pubUc cDNA coverage of the Genscan- predicted sequences, thus providing evidence for transcription. When Encyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence.
  • FuU length polynucleotide sequences were obtained by assembUng Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubUc cDNA sequences using the assembly process described in Example HI. Alternatively, fuU length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.
  • a chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the tianslated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubUc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome' s p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 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.
  • 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.
  • 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 normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed 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 quaUty in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotide sequences encoding HLYA are analyzed with respect to the tissue sources from which they were derived. For example, some fuU length sequences are assembled, at least in part, with overlapping incyte cDNA sequences (see Example EEQ. Each cDNA sequence is derived from a cDNA Ubrary constructed from a human tissue.
  • Each human tissue is classified into one of the foUowing organ tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU Une, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding HLYA.
  • cDNA sequences and cDNA Ubrary/tissue information are found in the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII. Extension of HLYA Encoding Polynucleotides
  • FuU length polynucleotide sequences were also produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer was synthesized to initiate 3' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result in hai ⁇ in structures and primer-primer dimerizations was avoided.
  • Selected human cDNA Ubraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
  • the concentration of DNA in each weU was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each weU of an opaque fluorimeter plate (Corning Costar, Acton MA), aUowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan EE (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aUquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WE), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Pharmacia Biotech).
  • 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 reUgated using T4 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coU ceUs. Transformed ceUs 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 Uquid media.
  • the cells were lysed, and DNA was ampUfied by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the foUowing 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 reampUfied using the same conditions as described above.
  • Hybridization probes derived from SEQ ID NO:4-6 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeUng of oUgonucleotides, consisting of about 20 base pairs, is speciflcaUy described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oUgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech).
  • An aUquot 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 foUowing endonucleases: Ase I, Bgl El, Eco RI, 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 (Nytian Plus, Schleicher & SchueU, Durham NH). Hybridization is carried out for 16 hours at 40 °C. To remove nonspecific signals, blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saUne sodium citiate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visuaUzed using autoradiography or an alternative imaging means and compared. X. Microarrays
  • Unkage or synthesis of array elements upon a microarray can be achieved utiUzing photoUthography, piezoelectiic printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof.
  • the substiate in each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena (1999), supra).
  • Suggested substrates include siUcon, siUca, glass sUdes, glass chips, and siUcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and Unk 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 weU known to those of ordinary skill in the art and may contain any appropriate number of elements.
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers suitable for hybridization can be selected using software weU 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 oUgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oUgo-(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 contiol 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 labeUng) 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 SPEN 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., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • Sequences of the present invention are used to generate array elements.
  • Each array element is ampUfied from bacterial ceUs containing vectors with cloned cDNA inserts.
  • PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampUfied in thirty cycles of PCR from an initial quantity of 1 -2 ng to a final quantity greater than 5 ⁇ g. AmpUfied array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech).
  • Purified array elements are immobiUzed on polymer-coated glass sUdes.
  • Glass microscope sUdes (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments.
  • Glass sUdes are etched in 4% hydrofluoric acid (VWR Scientific Products Co ⁇ oration (VWR), West Chester PA), washed extensively in distiUed water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol.
  • Coated sUdes are cured in a 110°C oven.
  • Array elements are appUed to the coated glass substiate using a procedure described in US Patent No. 5,807,522, inco ⁇ orated herein by reference.
  • 1 ⁇ l of the array element DNA 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 sUde.
  • Microarrays are UV-crossUnked using a STRATALINKER UV-crossUnker (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 saUne (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes in 0.2% SDS and distiUed water as before. Hybridization
  • Hybridization reactions contain 9 ⁇ l 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 aUquoted onto the microarray surface and covered with an 1.8 cm 2 coversUp.
  • the arrays are transferred to a wate ⁇ roof chamber having a cavity just sUghtly larger than a microscope sUde.
  • the chamber is kept at 100% humidity internaUy 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 Unes at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Ught is focused on the array using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the sUde containing the array is placed on a computer-contioUed 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 multiUne laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipUer 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 typicaUy 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 typicaUy caUbrated using the signal intensity generated by a cDNA contiol species added to the sample mixture at a known concentration.
  • a specific location on the array contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1 : 100,000.
  • the caUbration is done by labeUng samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipUer tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) instaUed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a Unear 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).
  • Sequences complementary to the HLYA-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring HLYA.
  • oUgonucleotides comprising from about 15 to 30 base pairs is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of HLYA.
  • a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary oUgonucleotide is designed to prevent ribosomal binding to the HLYA-encoding transcript.
  • HLYA HLYA expression and purification of HLYA 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 Umited 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 HLYA upon induction with isopropyl beta-D- thiogalactopyranoside (EPTG).
  • HLYA in eukaryotic ceUs is achieved by infecting insect or mammaUan ceU Unes with recombinant Autographica caUfornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovims.
  • AcMNPV Autographica caUfornica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovims is replaced with cDNA encoding HLYA 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 tianscription.
  • baculovims Recombinant baculovims is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovims. (See Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
  • HLYA is synthesized as a fusion protein with, e.g., glutathione S- tiansferase (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 ceU lysates.
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobiUzed glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
  • the GST moiety can be proteolyticaUy cleaved from HLYA at speciflcaUy engineered sites.
  • 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 HLYA obtained by these methods can be used directly in the assays shown in Examples XVI, XVEJ, and XVTU, where appUcable. XIII. Functional Assays
  • HLYA function is assessed by expressing the sequences encoding HLYA at physiologicaUy elevated levels in mammaUan ceU culture systems.
  • cDNA is subcloned into a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU Une, for example, an endotheUal or hematopoietic cell Une, using either Uposome formulations or electioporation.
  • 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 tiansfected ceUs from nontransfected cells and is a reUable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;
  • FCM Flow cytometry
  • HLYA The influence of HLYA on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encoding HLYA and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobuUn G (IgG).
  • Transfected ceUs are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods weU known by those of skiU in the art. Expression of mRNA encoding HLYA and other genes of interest can be analyzed by northern analysis or microarray techniques.
  • PAGE polyacrylamide gel electrophoresis
  • the HLYA amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
  • oUgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (AppUed 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.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-HLYA activity by, for example, binding the peptide or HLYA to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • HLYA Naturally Occurring HLYA Using Specific Antibodies
  • HLYA is substantially purified by immunoaffinity chromatography using antibodies specific for HLYA.
  • An immunoaffinity column is constructed by covalently coupUng anti-HLYA antibody to an activated chromatographic resin, such as CNBr- activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupUng, the resin is blocked and washed according to the manufacturer's instructions. Media containing HLYA are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of HLYA (e.g., high ionic strength buffers in the presence of detergent).
  • activated chromatographic resin such as CNBr- activated SEPHAROSE (Amersham Pharmacia Biotech).
  • the column is eluted under conditions that disrupt antibody/HLYA 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 HLYA is coUected.
  • a chaotrope such as urea or thiocyanate ion
  • HLYA or biologicaUy 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 weUs of a multi-weU plate are incubated with the labeled HLYA, washed, and any weUs with labeled HLYA complex are assayed. Data obtained using different concentrations of HLYA are used to calculate values for the number, affinity, and association of HLYA with the candidate molecules.
  • molecules interacting with HLYA are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • HLYA may also be used in the PATHCALLENG process (CuraGen Co ⁇ ., New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • An assay for carbonic anhydrase activity of HLYA uses the fluorescent pH indicator 8- hydroxypyrene-l,3,6-tiisulfonate (pyranine) in combination with stopped-flow fluorometry to measure carbonic anhydrase activity (Shingles, et al. 1997, Anal. Biochem. 252: 190-197).
  • a pH 6.0 solution is mixed with a pH 8.0 solution and the initial rate of bicarbonate dehydration is measured. Addition of carbonic anhydrase to the pH 6.0 solution enables the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. Shingles et al.
  • the fluorescent technique's sensitivity allows the determination of initial rates with a protein concentration as Uttle as 65 ng/ml.
  • Oinithine decarboxylase activity of HLYA is assayed by measuring the release of C0 2 from L-[l- 14 C]-ornithine (Reddy, S.G et al. (1996) J. Biol. Chem. 271:24945-24953). Activity is measured in 200 ⁇ l assay buffer (50 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 2 mM dithiothreitol, 5 mM NaF, 0.1% Brij35, 1 mM PMSF, 60 ⁇ M pyridoxal-5-phosphate) containing 0.5 mM L-orrdthine plus 0.5 ⁇ Ci L-[l- 14 C]ornithine.
  • assay buffer 50 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 2 mM dithiothreitol, 5 mM NaF, 0.1% Brij35, 1 mM PMSF, 60
  • the reactions are stopped after 15-30 minutes by addition of 1 M citric acid, and the 14 C0 2 evolved is trapped on a paper disk filter saturated with 20 ⁇ l of 2 N NaOH.
  • the radioactivity on the disks is determined by Uquid scintillation spectography.
  • the amount of 14 C0 2 released is proportional to ornithine decarboxylase activity of HLYA.
  • Agonists or antagonists of HLYA activation or inhibition may be tested using the assay described in section XVEE. Agonists cause an increase in HLYA activity and antagonists cause a decrease in HLYA activity.
  • ABI FACTURA A program that removes vector sequences and masks Applied Biosystems, Foster City, CA. ambiguous bases in nucleic acid sequences.
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing and annotating Applied Biosystems, Foster City, CA; Mismatch ⁇ 50% amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA.
  • ABI AutoAssembler A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA.
  • HMM hidden Markov model
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8: 175-185; Ewing, B. and P. Green (1998) Genome Res. 8: 186-194.

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Abstract

La présente invention concerne des lyases humaines (HLYA) et des polynucléotides qui identifient et codent HLYA. Par ailleurs, cette invention concerne des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. De plus, cette invention concerne des méthodes diagnostiques, thérapeutiques ou prophylactiques de troubles associés à l'expression aberrante de HLYA.
PCT/US2001/019166 2000-06-23 2001-06-13 Lyases humaines WO2002000840A2 (fr)

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AU2001266932A AU2001266932A1 (en) 2000-06-23 2001-06-13 Human lyases
JP2002505956A JP2004501632A (ja) 2000-06-23 2001-06-13 ヒトリアーゼ

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046385A2 (fr) * 2000-12-07 2002-06-13 Incyte Genomics, Inc. Enzymes
WO2002053716A2 (fr) * 2001-01-03 2002-07-11 Bayer Aktiengesellschaft Regulation de l'ornithine decarboxylase humaine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988004688A1 (fr) * 1986-12-23 1988-06-30 Howard Florey Institute Of Experimental Physiology Isoenzyme d'anhydrase carbonique
US5972684A (en) * 1997-11-25 1999-10-26 Incyte Pharmaceuticals, Inc. Carbonic anhydrase VIII
WO2000061748A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 48 proteines humaines secretees
WO2001062927A2 (fr) * 2000-02-24 2001-08-30 Incyte Genomics Inc Molecules utilisees a des fins diagnostiques et therapeutiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988004688A1 (fr) * 1986-12-23 1988-06-30 Howard Florey Institute Of Experimental Physiology Isoenzyme d'anhydrase carbonique
US5972684A (en) * 1997-11-25 1999-10-26 Incyte Pharmaceuticals, Inc. Carbonic anhydrase VIII
WO2000061748A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 48 proteines humaines secretees
WO2001062927A2 (fr) * 2000-02-24 2001-08-30 Incyte Genomics Inc Molecules utilisees a des fins diagnostiques et therapeutiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HEWETT-EMMETT DAVID ET AL: "Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families." MOLECULAR PHYLOGENETICS AND EVOLUTION, vol. 5, no. 1, 1996, pages 50-77, XP002190992 ISSN: 1055-7903 cited in the application *
LOWE N ET AL: "STRUCTURE AND METHYLATION PATTERNS OF THE GENE ENCODING HUMAN CARBONIC ANHYDRASE I" GENE (AMSTERDAM), vol. 93, no. 2, 1990, pages 277-283, XP002190991 ISSN: 0378-1119 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046385A2 (fr) * 2000-12-07 2002-06-13 Incyte Genomics, Inc. Enzymes
WO2002046385A3 (fr) * 2000-12-07 2003-06-26 Incyte Genomics Inc Enzymes
WO2002053716A2 (fr) * 2001-01-03 2002-07-11 Bayer Aktiengesellschaft Regulation de l'ornithine decarboxylase humaine
WO2002053716A3 (fr) * 2001-01-03 2003-02-27 Bayer Ag Regulation de l'ornithine decarboxylase humaine

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