[go: up one dir, main page]

WO2004011648A2 - Cytochromes p450 - Google Patents

Cytochromes p450 Download PDF

Info

Publication number
WO2004011648A2
WO2004011648A2 PCT/GB2003/003281 GB0303281W WO2004011648A2 WO 2004011648 A2 WO2004011648 A2 WO 2004011648A2 GB 0303281 W GB0303281 W GB 0303281W WO 2004011648 A2 WO2004011648 A2 WO 2004011648A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
nucleic acid
cytochrome
disease
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB2003/003281
Other languages
English (en)
Other versions
WO2004011648A3 (fr
Inventor
Christopher Benjamin Phelps
Richard Joseph Fagan
Ellie Louise James
Valerie Nathalie Pierron
Janet Marjorie Allen
Sarah Helen Kamp
Nelly Collinet
Lindsey Reynolds
Peter William Munday
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inpharmatica Ltd
Original Assignee
Inpharmatica Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inpharmatica Ltd filed Critical Inpharmatica Ltd
Priority to AU2003255732A priority Critical patent/AU2003255732A1/en
Publication of WO2004011648A2 publication Critical patent/WO2004011648A2/fr
Publication of WO2004011648A3 publication Critical patent/WO2004011648A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/80—Cytochromes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • This invention relates to novel proteins, termed T00364 and BAB13458.1 herein identified as Cytochrome P450s and to the use of these proteins and nucleic acid sequences from the encoding genes in the diagnosis, prevention and treatment of disease. All publications, patents and patent applications cited herein are incorporated in full by reference.
  • bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
  • This tool is a database system, termed the Biopendium search database, that is the subject of co-pending International Patent Application No. PCT/GBOl/01105.
  • This database system consists of an integrated data resource created using proprietary technology and containing information generated from an all-by-all comparison of all available protein or nucleic acid sequences.
  • sequence data from separate data resources is to combine as much data as possible, relating both to the sequences themselves and to information relevant to each sequence, into one integrated resource. All the available data relating to each sequence, including data on the three-dimensional structure of the encoded protein, if this is available, are integrated together to make best use of the information that is known about each sequence and thus to allow the most educated predictions to be made from comparisons of these sequences.
  • the annotation that is generated in the database and which accompanies each sequence entry imparts a biologically relevant context to the sequence information.
  • a protein whose sequence is recorded in a publicly available database as hypothetical protein KIAA0673 (NCBI Genebank nucleotide accession number XM_030915 and a Genebank protein accession number T00364), is implicated as a novel member of the Cytochrome P450 family.
  • a second protein whose sequence is recorded in a publicly available database as KIAA1632 protein (NCBI Genebank nucleotide accession number AB046852 and a Genebank protein accession number BAB13458.1), is implicated as a novel member of the Cytochrome P450 family.
  • P450s are a large superfamily of enzymes all of which use a heme bound iron atom to catalyse the insertion of an oxygen atom into a substrate.
  • the overall reaction of a P450 converts an organic substrate, molecular oxygen and NADPH to a hydroxylated organic substrate, water and NADP+.
  • the oxidation of NADPH is generally carried out by a separate enzyme or enzymes: P45 reductase or ferredoxin and ferredoxin reductase, which subsequently transfer the electrons to the P450. Examples have been found where P450 and P450 reductase have been fused however. Subsequent rearrangements and reactions of the hydroxylated product lead to P450s catalysing over 40 known reactions.
  • P450s catalyse the oxygenation of a large range of substrates: over 1000 are known to date and there may be IO 6 in total.
  • This broad range of biochemical functions gives P450s a similarly broad range biological functions: detoxification of harmful chemicals, activation (by modification) of beneficial drug precursors and hormones, activation of harmful chemicals (such as carcinogens), breakdown and synthesis of steroids, vitamins, fatty acids, pigments, pheromones, insecticides amongst other classes of biological molecule.
  • P450s are found in nearly all known organisms including plants, animals, fungi and bacteria. In mammals P450s are found in most tissues though their concentration is highest in the liver where the detoxification of many chemicals takes place.
  • P450 genes are also implicated in growth and differentiation of cells due to their tissue and developmental specific expression patterns.
  • P450s The P450s' role in the metabolism (both activation and deactivation) of drugs and carcinogens has made them the subject of much medical interest.
  • the susceptibility of a drug to mactivation by P450s may make it biologically inactive.
  • drugs are administered in an inactive form and only become active when they have passed through the liver and been altered by P450s once and even twice.
  • P450s have been the subject of extensive site-directed mutagenesis experiments which have aimed to determine residues essential for substrate binding specificity.
  • Most P450 structures are of soluble bacterial enzymes though there have been efforts to homology model mammalian enzymes in order to aid understanding of variations in substrate binding specificities and to aid rational drug design efforts.
  • P450s catalyse the formation of many crucial biological compounds required by pathogens and inhibitors of P450 activity are usually strong antibiotics.
  • Inhibitors of P450s could stop mactivation of drugs and activation of carcinogens.
  • the drug exemestane has been approved for use as an inhibitor of aromatase P450 in breast cancer.
  • Azole antifungals such as Nizoral and Diflucan inhibit the P450 lanosterol demethylase. which catalyses the synthesis of ergosterol, a major component of fungal plasma membranes.
  • Recent studies have also crystallised a Mycobacterium tuberculosis P450 in complex with two different azole inhibitors, 4-phenylimidazole (4-PI) and FLU, helping understanding of binding of these important antifungals.
  • novel P450s As these proteins are implicated in the diseases identified above, as well as in other disease states.
  • the identification of novel P450s in bacterial, fungal and human systems is therefore extremely relevant for the treatment and diagnosis of disease, particularly those identified above.
  • the invention is based on the discovery that the T00364 protein and BAB 13458.1 protein function as Cytochrome P450s.
  • Saccharopolyspora erythraea Cytochrome P450ERYF (PDB code UIO:A). Saccharopolyspora erythraea Cytochrome P450ERYF is known to function as a Cytochrome P450. This relationship is not just to Saccharopolyspora erythraea Cytochrome P450ERYF, but rather to the Cytochrome P450 family as a whole.
  • the heme cofactor binding residue Cys347 of the Pseudomonas putida Cytochrome P450CAM is conserved as Cysl345 in BAB13458.1, respectively.
  • residue numbering for Pseudomonas putida Cytochrome P450CAM relates to the amino acid position relative to the crystal structure 1DZ4:A. This relationship is not just to Pseudomonas putida Cytochrome P450CAM, but rather to the Cytochrome P450 family as a whole.
  • Cysl345 is predicted to form the heme cofactor binding residue.
  • results presented herein clearly indicate that the T00364 and BAB 13458.1 transcripts are present at detectable levels in a variety of human tissues and cell lines. This confirms the relevance of the T00364 and BAB13458.1 proteins as important targets for further biochemical characterisation.
  • the particular tissues and cell lines identified herein as expressing the T00364 and BAB13458.1 proteins represent ideal targets for further studies of T00364 and BAB13458.1 protein function in vivo. Such studies may, for example, make use of the ligands identified using the assays and screening methods disclosed herein to investigate the effects of inducing or inhibiting T00364 and BAB13458.1 protein function.
  • the cloning of the T00364 and BAB13458.1 proteins allows for high-level expression, purification and characterisation of the polypeptides of the invention described herein.
  • the inventors have discovered that the mRNAs for the T00364 and BAB13458.1 proteins are expressed at significant levels in the human brain.
  • P450s are involved in the synthesis and metabolism of various components of metabolic pathways including steroids, fatty acids, prostaglandins, leukotrienes, bile acids and retinoids.
  • the finding of a novel P450 that is expressed preferentially in the human brain is consistent with a role for this P450 in regulating metabolic pathways associated with inflammatory conditions in the brain such as multiple sclerosis and dementia.
  • neurosteroids have been shown to influence neurotransmission particularly in the field of receptors such as those for GAB A and NMDA and Sigma receptors. Neurosteroids have been shown to play a neuroprotective role.
  • Therapeutic intervention through the development of substrates and inhibitors of the T00364 and BAB 13458.1 proteins may therefore have a role in treatment of neurodegenerative conditions such as dementia, Parkinson's disease and neurodegeneration following cerebrovascular disease such as infarction or haemorrhage (stroke) and trauma to the central nervous system and spinal cord.
  • neurodegenerative conditions such as dementia, Parkinson's disease and neurodegeneration following cerebrovascular disease such as infarction or haemorrhage (stroke) and trauma to the central nervous system and spinal cord.
  • neurosteroids have been shown to influence cognitive processing, spatial learning and memory, anxiety and behaviours such as craving which leads to addictive behaviour patterns.
  • Development of agonists and antagonists to the T00364 and BAB 13458.1 proteins may therefore lead to therapeutic intervention to treat dementias, learning difficulties, anxiety and addictive behaviours including alcoholism, eating disorders and drug addiction.
  • T00364 has been shown to be the gene to be affected in juvenile nephronophthisis type 4. As such this protein has been shown to interact with nephrocystin.
  • Nephronophthisis is a familial condition, inherited as an autosomal recessive, that cause renal failure in children and is characterised by thickening of the basement membrane, interstitial fibrosis and medullary cysts (Mollet et al, Nature Genetics, 2002, 300-5 and Nature Genetics, 2002, 32, 300-5). Nephronophthisis can be associated with ocular motor apraxis and retinitis pigmentosa.
  • P450s are involved in the synthesis and turnover of a variety of small metabolically active small molecular weight compounds in the human body. These include retinoic acids, leukotrienes, prostaglandins and steroids. These agents are involved in controlling blood flow in vascular beds, in regulating the inflammatory responses, in regulating growth of cells and in regulating metabolic functions.
  • transcripts for T00364 and BAB 13458.1 are expressed in the brain at high levels implies a role for these proteins in the synthesis and turnover of these agents in the brain.
  • Neurosteroids are known to be synthesised in the brain and play an important role in cognition, learning and addictive and repetitive behaviours.
  • identification of modulators for T00364 or BAB 13458.1 activity or transcript levels are potentially of benefit in the treatment of disorders of cognition and memory including dementias including Alzheimer's disease, extrapyramidal disorders including Parkinson's disease, neurotic behaviours including compulsive-obsessive disorders, anxiety and depression, addictive behaviours including alcoholism, smoking behaviours and nicotine dependency, drug dependency and obesity.
  • T00364 high levels of transcript were also identified in testis, thymus, ovary, lung and bladder. Modulating T00364 activity or transcript levels may therefore have value in the treatment of diseases of testis and ovary including testicular cancer, ovarian cancer and infertility. Finding high transcript levels in the thymus and in the cell lines derived from haematopoietic cell lines including Jurkat cells and HL60 cells indicates that inhibitors may have value in treating disorders of T cell proliferation or maturation including leukemias and lymphopenias following infections such as HIV or chemotherapy or radiotherapy.
  • regulating T cell maturation may have value in the treatment of autoimmune diseases including type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, psoriasis, atopic dermatitis, asthma, eczema, inflammatory bowel disease (Crohn's disease and ulcerative colitis).
  • autoimmune diseases including type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, psoriasis, atopic dermatitis, asthma, eczema, inflammatory bowel disease (Crohn's disease and ulcerative colitis).
  • Finding of the transcript in the lung suggests that modulators of T00364 activity or transcript levels may be of value in various lung diseases such as, but not exclusively, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pulmonary hypertension, chronic bronchitis and emphysema.
  • COPD chronic obstructive pulmonary disease
  • pulmonary fibrosis
  • BAB 13458.1 high levels of transcript were also found in the placenta and thymus. This is consistent with a role for BAB 13458.1 as a P450 metabolising substrates including retinoic acids, leukotrienes, steroids and prostaglandins.
  • the placenta is a very active steroidogenic organ and is required to maintain pregnancy especially at the early stages and to provide nutrients to the developing foetus.
  • the vascular flow (a critical role for P450s) in the placenta is critical in maintaining the health of the foetus. Impaired placental function is associated with growth retardation and prematurity in the foetus.
  • modulators for BAB13458.1 activity or transcript levels may have a role in regulating placental function and, thus, a role in ensuring the health and normal development of the foetus, in maintenance of pregnancy (treatment of premature abortion of the foetus or premature delivery of a pre-term infant) and in hypertension associated with pregnancy such as pre-eclampsia or eclampsia. Modulators of BAB 13458.1 activity and transcript levels may be valuable for therapeutic abortions.
  • regulating T cell maturation may have value in the treatment of autoimmune diseases, including type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, psoriasis, atopic dermatitis, asthma, eczema, inflammatory bowel disease (Crohn's disease and ulcerative colitis).
  • autoimmune diseases including type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, psoriasis, atopic dermatitis, asthma, eczema, inflammatory bowel disease (Crohn's disease and ulcerative colitis).
  • the transcript for BAB13458.1 was found to be significantly reduced in the sample from psoriasis skin compared to the control skin samples.
  • the finding in myeloid cells indicates a role for inflammatory diseases including, but not exclusively, chronic obstructive pulmonary disease (COPD), osteoarthritis, wound healing and resolution of infections.
  • COPD chronic obstructive pulmonary disease
  • the invention provides a polypeptide, which polypeptide:
  • (i) comprises the amino acid sequence as recited in SEQ ID NO:2 or SEQ ID NO:4;
  • (ii) is a fragment thereof having Cytochrome P450 activity or having an antigenic determinant in common with the polypeptides of (i); or (iii) is a functional equivalent of (i) or (ii).
  • a polypeptide according to the first aspect of the invention consists of the amino acid sequence as recited in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "the P450G4 polypeptide".
  • a preferred polypeptide fragment according to part ii) above includes the region of the P450G4 polypeptide that is predicted as that responsible for Cytochrome P450 activity (hereafter, the "P450G4 Cytochrome P450 region"), or is a variant thereof.
  • the P450G4 Cytochrome P450 region is considered to extend between residue 586 and residue 946 of the P450G4 polypeptide sequence.
  • a further preferred fragment is that cloned herein, which extends between residue 575 and residue 946 of the P450G4 polypeptide sequence.
  • the polypeptide having the sequence recited in SEQ ID NO:4 is referred to hereafter as "the P450G5 polypeptide".
  • a preferred polypeptide fragment according to part ii) above includes the region of the P450G5 polypeptide that is predicted as that responsible for Cytochrome P450 activity (hereafter, the "P450G5 Cytochrome P450 region"), or is a variant thereof that possesses the heme cofactor binding (Cysl345, or equivalent residues).
  • the P450G5 Cytochrome P450 region is considered to extend between residue 1112 and residue 1350 of the P450G5 polypeptide sequence.
  • Cytochrome P450 activity is meant that the protein functions as a Cytochrome P450 enzyme, for example, in metabolizing a wide variety of xenobiotic compounds such as drugs and carcinogens, or endobiotic compounds such as prostaglandins and steroids.
  • the polypeptide exhibits a characteristic P450 spectrum such that when the heme ion is complexed with carbon monoxide, the spectrum shows a Soret absorption maximum at around 450nm (Garfinkel, 1958, Arch. Biochem. Biophys. 77: 493-509; Klingberg, 1958, Arch. Biochem. Biophys; Omuar & Sato, 1964, J. Biol.
  • the polypeptides and functional equivalents according to the invention function as P450 enzymes.
  • functions as a P450 enzyme we mean that the polypeptide retains its ability to convert an organic substrate, NADPH and molecular oxygen to NADP+, a hydroxylated organic substrate and water.
  • the ability of a polypeptide to hydroxylate a substrate may be determined by using a suitable assay known in the art.
  • a preferred polypeptide fragment according to part ii) above includes the region of the P450G4 and P450G5 polypeptide that is predicted as that responsible for Cytochrome P450 activity (hereafter, the "P450G4 Cytochrome P450 region” and "P450G5 Cytochrome P450 region”), or is a variant thereof.
  • the P450G4 Cytochrome P450 region is considered to extend between residue 586 and residue 946 of the P450G4 polypeptide sequence.
  • a fragment extending between residue 575 and residue 946 of the P450G4 polypeptide sequence is also functional in this respect and such a fragment, and its coding sequence, forms an aspect of the present invention.
  • the P450G5 Cytochrome P450 region is considered to extend between residue 1112 and residue 1350 of the P450G5 polypeptide sequence.
  • a fragment extending between residue 1112 and residue 1457 is also functional in this respect and such a fragment, and its coding sequence, forms an aspect of the present invention.
  • This aspect of the invention also includes fusion proteins that incorporate polypeptide fragments and variants of these polypeptide fragments as defined above, provided that said fusion proteins possess activity as a Cytochrome P450.
  • the invention provides a purified nucleic acid molecule that encodes a polypeptide of the first aspect of the invention.
  • the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NO:l (encoding the P450G4 polypeptide), or SEQ ID NO:3 (encoding the P450G5 polypeptide), or is a redundant equivalent or fragment of any one of these sequences.
  • a preferred nucleic acid fragment is one that encodes a polypeptide fragment according to part ii) above, preferably a polypeptide fragment that includes the P450G4 Cytochrome P450 region, the P450G5 Cytochrome P450 region, or that encodes a variant of these fragments as this term is defined above.
  • the invention provides a purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
  • the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
  • the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
  • the host cells of the invention may co-express a reductase protein which forms an active complex with the polypeptides of the first aspect of the invention and thus maximises the activity of the polypeptides of the invention in the cell.
  • the invention provides a ligand which binds specifically to, and which preferably inhibits the Cytochrome P450 activity of, a polypeptide of the first aspect of the invention.
  • Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.
  • such ligands may bind specifically to the heme binding domain of the Cytochrome P450, thus preventing oxidation of a substrate from taking place.
  • the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such compounds may be identified using the assays and screening methods disclosed herein.
  • a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
  • the identification of the function of the region defined herein as the P450G4 and P450G5 Cytochrome P450 regions of the P450G4 and P450G5 polypeptides, respectively allows for the design of screening methods capable of identifying compounds that are effective in the treatment and/or diagnosis of diseases in which Cytochrome P450s are implicated.
  • the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in therapy or diagnosis.
  • These molecules may also be used in the manufacture of a medicament for the treatment of cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity; AIDS, renal disease, infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.
  • cell proliferative disorders including neoplasm, melanoma, lung, colorectal, breast, pancreas
  • these molecules may be used in the manufacture of a medicament for the treatment of: nephronophthisis, ocular motor apraxis, retinitis pigmentosa; diseases of the testis and ovary including testicular cancer, ovarian cancer and infertility; disorders of T cell proliferation or maturation including leukaemias and lymphopenias; diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); placental disorders; autoimmune diseases including type I diabetes mellitus, reheumatoid arthritis, multiple sclerosis, eczema and atopic dermatitis; inflammatory diseases including, but not exclusively, osteoarthritis, wound healing and resolution of infections; diseases associated with the lung including chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, chronic bronchitis and e
  • the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
  • a method will preferably be carried out in vitro.
  • Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
  • a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
  • the invention also provides kits that are useful in these methods for diagnosing disease.
  • a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
  • the disease diagnosed by a method of the ninth aspect of the invention is a disease in which Cytochrome P450s are implicated.
  • Diseases which may be diagnosed by a method according to the ninth aspect of the invention include treatment of cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity; AIDS, renal disease, infections including viral infection, bacterial infection, fungal infection and parasitic infection and other
  • diseases which may be diagnosed include: diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); placental disorders; inflammatory diseases including, but not exclusively, COPD, osteoarthritis, wound healing and resolution of infections; and diseases associated with neurosteroid synthesis including dementia, Parkinson's disease, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviours such as alcoholism, eating disorders and drug addiction.
  • diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia
  • diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); placental disorders
  • inflammatory diseases including, but not exclusively, COPD, osteoarthritis, wound healing and resolution of infections
  • the invention provides for the use of a polypeptide of the first aspect of the invention as a Cytochrome P450.
  • Suitable uses of the polypeptides of the invention as a P450 include use as a regulator of cellular growth, metabolism or differentiation, use as part of a receptor/ligand pair and use as a diagnostic marker for a physiological or pathological condition selected from the list given above.
  • the invention also provides use of the polypeptides of the invention to hydroxylate organic substrates, using assays described above.
  • the invention includes use of the polypeptides of the invention as drug metabolising enzymes.
  • the invention also provides for the use of a nucleic acid molecule according to the second or third aspects of the invention to express a protein that possesses Cytochrome P450 activity.
  • the invention also provides a method for effecting Cytochrome P450 activity, said method utilising a polypeptide of the first aspect of the invention.
  • Particular preferred activities of Cytochrome P450s include the modulation, (including both the potentiation, amelioration or conversion to metabolite) of pharmacological agents, particularly small molecule pharmacological agents.
  • the invention also provides a method for effecting Cytochrome P450 activity, said method utilising a polypeptide of the first aspect of the invention, or a fragment thereof.
  • the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically-acceptable carrier.
  • the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, such as cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease,
  • diseases which may be treated include: nephronophthisis, ocular motor apraxis, retinitis pigmentosa; diseases of the testis and ovary including testicular cancer, ovarian cancer and infertility; disorders of T cell proliferation or maturation including leukaemias and lymphopenias; diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); placental disorders; autoimmune diseases including type I diabetes mellitus, reheumatoid arthritis, multiple sclerosis, eczema and atopic dermatitis; inflammatory diseases including, but not exclusively, osteoarthritis, wound healing and resolution of infections; diseases associated with the lung including chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary hypertension, chronic bronchitis and emphysema; diseases associated with
  • the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a host cell of the fifth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist.
  • the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
  • antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
  • the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
  • Such transgenic animals are very useful models for the study of disease and may also be using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such a disease.
  • polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
  • the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
  • the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
  • the polypeptide of the first aspect of the invention may form part of a fusion protein.
  • a fusion protein may contain one or more additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
  • the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
  • modifications which may commonly be present in polypeptides of the present invention are glycosylation, lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
  • the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
  • polypeptides of the present invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
  • the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the P450G4 or P450G5 polypeptides.
  • Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity” indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
  • Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the P450G4 or P450G5 polypeptides.
  • Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
  • Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
  • Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group;
  • polypeptides of the first aspect of the invention have a degree of sequence identity with the P450G4 or P450G5 polypeptide, or with active fragments thereof, of greater than 30%. More preferred polypeptides have degrees of identity of greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the P450G4 or P450G5 polypeptide, or with active fragments thereof.
  • preferred active fragments of the P450G4 polypeptide are those that include the P450G4 Cytochrome P450 region. Accordingly, this aspect of the invention includes polypeptides that have degrees of identity of greater than 30%, preferably, greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively, with the Cytochrome P450 region of the P450G4 polypeptide. As discussed above, the P450G4 Cytochrome P450 region is considered to extend between residue 586 and residue 946 of the P450G4 polypeptide sequence.
  • preferred active fragments of the P450G5 polypeptide are those that include the P450G5 Cytochrome P450 region and which possess the heme cofactor binding residue Cys 1345.
  • this aspect of the invention includes polypeptides that have degrees of identity of greater than 30%, preferably, greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively, with the Cytochrome P450 region of the P450G5 polypeptide and which possess the heme cofactor binding residue Cys 1345.
  • the P450G5 Cytochrome P450 region is considered to extend between residue 1112 and residue 1350 of the P450G5 polypeptide sequence.
  • the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
  • the Inpharmatica Genome ThreaderTM technology that forms one aspect of the search tools used to generate the Biopendium search database may be used (see co-pending International patent application PCT/GB01/01105) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the P450G4 or P450G5 polypeptides, are predicted to have Cytochrome P450 activity, by virtue of sharing significant structural homology with the P450G4 or P450G5 polypeptide sequences.
  • the Inpharmatica Genome ThreaderTM predicts two proteins, or protein regions, to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above.
  • the certainty value of the Inpharmatica Genome ThreaderTM is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome ThreaderTM exclusively using sequences of known structure. Some of the comparisons were between proteins that were known to be related (on the basis of structure). A neural network was then trained on the basis that it needed to best distinguish between the known relationships and known not-relationships taken from the CATH structure classification (www.biochem.ucl.ac.uk/bsm/cath).
  • Structural homologues of P450G4 should share structural homology with the P450G4 Cytochrome P450 region. Such structural homologues are predicted to have Cytochrome P450 activity by virtue of sharing significant structural homology with this polypeptide. Structural homologues of P450G5 should share structural homology with the P450G5 Cytochrome P450 region and possess the heme cofactor binding residue Cysl345. Such structural homologues are predicted to have Cytochrome P450 activity by virtue of sharing significant structural homology with this polypeptide sequence and possessing the heme cofactor binding residue.
  • polypeptides of the first aspect of the invention also include fragments of the P450G4 and P450G5 polypeptides, functional equivalents of the fragments of the P450G4 and P450G5 polypeptides, and fragments of the functional equivalents of the P450G4 and P450G5 polypeptides, provided that those functional equivalents and fragments retain Cytocl rome P450 activity or have an antigenic determinant in common with the P450G4 or P450G5 polypeptides.
  • fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the P450G4 or P450G5 polypeptides or one of its functional equivalents.
  • the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
  • Preferred polypeptide fragments according to this aspect of the invention are fragments that include a region defined herein as the P450G4 or P450G5 Cytochrome P450 region of the P450G4 and P450G5 polypeptides, respectively. These regions are the regions that have been annotated as Cytochrome P450.
  • this region is considered to extend between residue 586 and residue 946.
  • this region is considered to extend between residue 1112 and residue 1350.
  • Variants of this/these fragment(s) are included as embodiments of this aspect of the invention, provided that these variants possess activity as a Cytochrome P450.
  • variable is meant to include extended or truncated versions of this polypeptide fragment.
  • Cytochrome P450 region of the P450G4 and P450G5 polypeptide will fold correctly and show Cytochrome P450 activity if additional residues C terminal and/or N terminal of these boundaries in the P450G4 and P450G5 polypeptide sequences are included in the polypeptide fragment.
  • an additional 5, 10, 20, 30, 40 or even 50 or more amino acid residues from the P450G4 and P450G5 polypeptide sequence, or from a homologous sequence may be included at either or both the C terminal and/or N terminal of the boundaries of the Cytochrome P450 regions of the P450G4 and P450G5 polypeptide, without prejudicing the ability of the polypeptide fragment to fold correctly and exhibit Cytochrome P450 activity.
  • one or more amino acid residues may be deleted at either or both the C terminus or the N terminus of the Cytochrome P450 region of the P450G4 polypeptide.
  • one or more amino acid residues may be deleted at either or both the C terminus or the N terminus of the Cytochrome P450 region of the P450G5 polypeptide, although the heme cofactor binding residue (Cysl345) should be maintained intact; deletions should not extend so far into the polypeptide sequence that this residue is deleted.
  • the term "variant" includes homologues of the polypeptide fragments described above, that possess significant sequence homology with the Cytochrome P450 region of the P450G4 polypeptide provided that said variants retain activity as an Cytochrome P450.
  • variant also includes homologues of the polypeptide fragments described above, that possess significant sequence homology with the Cytochrome P450 region of the P450G5 polypeptide and which possess the heme cofactor binding residue Cys 1345 or equivalent residues), provided that said variants retain activity as a Cytochrome P450.
  • variant homologues of polypeptide fragments of this aspect of the invention have a degree of sequence identity with the P450G4 and P450G5 Cytochrome P450 regions, of the P450G4 and P450G5 polypeptides, respectively, of greater than 40%. More preferred variant polypeptides have degrees of identity of greater than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the P450G4 and P450G5 Cytochrome P450 regions of the P450G4 and P450G5 polypeptides, provided that said variants retain activity as a Cytochrome P450.
  • Variant polypeptides also include homologues of the truncated forms of the polypeptide fragments discussed above, provided that said variants retain activity as a Cytochrome P450.
  • polypeptide fragments of the first aspect of the invention may be polypeptide fragments that exhibit significant structural homology with the structure of the polypeptide fragment defined by the P450G4 and P450G5 Cytochrome P450 regions, of the P450G4 or P450G5 polypeptide sequences, for example, as identified by the Inpharmatica Genome ThreaderTM. Accordingly, polypeptide fragments that are structural homologues of the polypeptide fragments defined by the P450G4 or P450G5 Cytochrome P450 regions of the P450G4 and P450G5 polypeptide sequences should adopt the same fold as that adopted by this polypeptide fragment, as this fold is defined above.
  • Structural homologues of the polypeptide fragment defined by the P450G5 Cytochrome P450 region should also retain the heme cofactor binding residue Cys 1345.
  • Such fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • the fragment of the invention when comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region.
  • certain preferred embodiments relate to a fragment having a pre- and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
  • several fragments may be comprised within a single larger polypeptide.
  • the polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides.
  • ligands such as polyclonal or monoclonal antibodies
  • Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
  • the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
  • immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab')2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention.
  • substantially greater affinity we mean that there is a measurable increase in the affinity for a polypeptide of the invention as compared with the affinity for known P450 polypeptides.
  • the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10 3 -fold, IO 4 - fold, 10 5 -fold, 10 -fold or greater for a polypeptide of the invention than for known P450 polypeptides.
  • a selected mammal such as a mouse, rabbit, goat or horse
  • a polypeptide of the first aspect of the invention may be immunised with a polypeptide of the first aspect of the invention.
  • the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically.
  • the polypeptide can be conjugated to a carrier protein.
  • Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
  • the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography.
  • Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies using hybridoma technology is well known (see, for example, Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
  • Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
  • Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Nati. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
  • the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al., Science,
  • humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
  • the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
  • the antibody may be a "bispecific" antibody, that is, an antibody having two different antigen binding domains, each domain being directed against a different epitope.
  • Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, or from naive libraries (McCafferty, J. et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, (1991) Nature 352, 624-628).
  • Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
  • the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
  • Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode the polypeptide sequences recited in SEQ ID NO:2, or SEQ ID NO:4, and functionally equivalent polypeptides, including active fragments of the P450G4 and P450G5 polypeptides, such as a fragment including the P450G4 and P450G5 Cytochrome P450 regions of the P450G4 and P450G5 polypeptide sequences, or a homologue thereof.
  • nucleic acid molecules encompassing these stretches of sequence form a preferred embodiment of this aspect of the invention. These nucleic acid molecules may be used in the methods and applications described herein.
  • the nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
  • nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof. The nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
  • the nucleic acid molecules may be double-stranded or single-stranded.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
  • the term "nucleic acid molecule” also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • PNA refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine.
  • PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2, or an active fragment thereof may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:l. These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes the polypeptide SEQ ID NO:2, or an active fragment of the P450G4 polypeptide, such as a fragment including the P450G4 Cytochrome P450 region, or a homologue thereof.
  • the P450G4 Cytochrome P450 region is considered to extend between residue 586 and 946 of the P450G4 polypeptide sequence.
  • the P450G4 Cytochrome P450 region is thus encoded by a nucleic acid molecule including nucleotide 1756 to nucleotide 2838.
  • a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:4, or an active fragment thereof may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:3. These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes the polypeptide SEQ ID NO:4, or an active fragment of the P450G5 polypeptide, such as a fragment including the P450G5 Cytochrome P450 region, or a homologue thereof.
  • the P450G5 Cytochrome P450 region is considered to extend between residue 1112 and 1350 of the P450G5 polypeptide sequence.
  • the P450G5 Cytochrome P450 region is encoded by a nucleic acid molecule including nucleotide 3334 to nucleotide 4050. Nucleic acid molecules encompassing this stretch of sequence, and homologues of this sequence, form a preferred embodiment of this aspect of the invention.
  • nucleic acid molecules that encode the polypeptide of SEQ ID NO:2 or SEQ ID NO:4 may include, but are not limited to, the coding sequence for the mature polypeptide by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
  • the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
  • the nucleic acid molecules of the second and third aspects of the invention may also encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
  • a preferred fragment of the P450G4 polypeptide is a fragment including the P450G4 Cytochrome P450 region, or a homologue thereof.
  • the Cytochrome P450 region is encoded by a nucleic acid molecule including nucleotide 1756 to nucleotide 2838 of SEQ ID NO:l.
  • a preferred fragment of the P450G5 polypeptide is a fragment including the P450G5 Cytochrome P450 region, or a homologue thereof.
  • the P450G5 Cytochrome P450 region is encoded by a nucleic acid molecule including nucleotide 3334 to nucleotide 4050 of SEQ ID O.3.
  • nucleic acid molecules according to the invention may be naturally-occurring variants such as a naturally-occurring allelic variant, or the molecules may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
  • the substitutions, deletions or insertions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
  • the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
  • Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
  • Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
  • Such combined nucleic acid molecules are included within the second or third aspects of the invention.
  • a fusion protein that can be recognised by a commercially-available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
  • the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
  • antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
  • hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al.
  • Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
  • High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCI, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
  • Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]).
  • the conditions used for hybridization are those of high stringency.
  • Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the P450G4 polypeptide (SEQ ID NO:2), or P450G5 polypeptide (SEQ ID NO:4), and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
  • a preferred active fragment is a fragment that includes an P450G4 or P450G5 Cytochrome P450 region of the P450G4 and P450G5 polypeptide sequences, resepctively.
  • preferred nucleic acid molecules include those that are at least 70% identical over their entire length to a nucleic acid molecule encoding the Cytochrome P450 region of the P450G4 and P450G5 polypeptide sequence.
  • Percentage identity is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/).
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecule having the sequence given in SEQ ID NO:l, to a region including nucleotides 1756-2838 of this sequence, or a nucleic acid molecule that is complementary to any one of these regions of nucleic acid.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the P450G4 polypeptide.
  • a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecule having the sequence given in SEQ ID NO:3, to a region including nucleotides 3334-4050 of this sequence, or a nucleic acid molecule that is complementary to any one of these regions of nucleic acid.
  • nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
  • Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the P450G5 polypeptide.
  • the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
  • a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the P450G4 or P450G5 polypeptides and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
  • the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
  • One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the P450G4 or P450G5 polypeptides, particularly with an equivalent function to the P450G4 or P450G5 Cytochrome P450 region of the P450G4 or P450G5 polypeptides is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
  • Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:l), particularly a region from nucleotides 1756- 2838 of SEQ ID NO:l, are particularly useful probes. Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO: 3), particularly a region from nucleotides 3334-4050 of SEQ ID NO:3, are particularly useful probes.
  • Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
  • Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
  • the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
  • isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
  • Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al., Proc. Nati. Acad. Sci. USA (1988) 85: 8998-9002).
  • RACE Rapid Amplification of cDNA Ends
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1: 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055- 3060). Additionally, one may use PCR, nested primers, and PromoterFmderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron exon junctions.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • the nucleic acid molecules of the present invention may be used for chromosome localisation.
  • a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
  • the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
  • the nucleic acid molecules of the present invention are also valuable for tissue localisation.
  • Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
  • These techniques include in situ hybridization techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism.
  • comparative studies of the normal expression pattem of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.
  • RNA interference (Elbashir, SM et al, Nature 2001, 411, 494-498) is one method of sequence specific post- transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.
  • the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
  • the host cells of the invention which may be transformed, transfested or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
  • polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell.
  • Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al. (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
  • any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
  • nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those described in Sambrook et al, (supra).
  • the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
  • suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
  • Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
  • Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cos id DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
  • Cell-free translation systems can also be employed to produce the polypeptides of the invention.
  • nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998).
  • expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
  • the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
  • signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Leader sequences can be removed by the bacterial host in post-translational processing.
  • regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
  • Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
  • cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines.
  • ATCC American Type Culture Collection
  • baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
  • Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
  • plant cell culture and whole plant genetic expression systems known in the art. Examples of suitable plant cellular genetic expression systems include those described in US 5,693,506; US 5,659,122; and US 5,608,143.
  • yeast cells for example, S. cerevisiae
  • Aspergillus cells examples include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
  • any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk " or aprt ⁇ cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Nati. Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
  • marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
  • a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983) J. Exp. Med, 158, 1211-1216).
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
  • sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison Wl); and U.S. Biochemical Corp., Cleveland, OH)).
  • Suitable reporter molecules or labels include radionuclides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
  • the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
  • Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
  • purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to, facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al (1992) Prot. Exp. Purif.
  • the polypeptide may be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
  • FACS fluorescence activated cell sorting
  • the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
  • Such active compounds (which may be agonist or antagonist compounds) may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These active compounds may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al, Current Protocols in Immunology 1 (2): Chapter 5 (1991).
  • Compounds that are most likely to be good inhibitors are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
  • Potential inhibitors include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
  • the polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
  • screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
  • the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
  • Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
  • Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
  • the P450G4 and P450G5 polypeptides of the present invention may promote the metabolism of endogenous and exogenous substrates and/or drugs.
  • the ability of the P450G4 and P450G5 polypeptides to promote metabolism of these agents can be examined and the methods described above can be used to identify agonists and antagonists of the metabolic effects of the P450G4 and P450G5 polypeptides.
  • assays for identifying substrates and antagonists of the P450G4 and P450G5 polypeptides are conducted using a system in which activity of the P450G4 and P450G5 polypeptides is maximised.
  • This may be achieved by co-expressing the P450G4 or P450G5 polypeptide with a reductase protein that produces an active complex to screen for antagonists.
  • the P450G4 or P450G5 protein and the reductase protein may be produced separately and introduced into the assay system.
  • Microsomal cytochromes occur on the membrane of the ER and require NADPH cytochrome reductase and a flavoprotein for activity, whereas mitochondrial cytochromes occur on the inner membrane and ferredoxin and NADPH ferredoxin reductase for activity (Beckman, M., and DeLuca, H. (1997) Methods in Enzymol. 282, 200-223; Armbrecht, H.J., Okuda, K., Wongsurawat, N., Nemani, R., Chen, M., and Boltz, M. (1992) J. Steroid Biochem. Molec. Biol. 43, 1073-1081).
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • LC liquid chromatography
  • the polypeptide of interest in purified form, or in reconstituted systems, microsomal preparations or expressed in a cellular system, may be contacted with labelled substrates.
  • Metabolites formed in the reaction can be extracted, for example, by chemical methods, and their level analysed by measuring the amount of label.
  • This method can be used to screen for competitors of the particular polypeptides.
  • Unlabelled compound libraries can be added with the polypeptide and the labelled substrate.
  • the competition level is determined by the reduction in the metabolites detected.
  • polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose- dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • a number of different methodologies are available for presenting a polypeptide on the surface of a cell.
  • the polypeptide may, for example, be artificially anchored to the cell membrane, or form part of a chimeric receptor.
  • An alternative method may involve contacting a labelled or unlabeled compound with a polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself.
  • a labelled or unlabeled compound with a polypeptide immobilized on any solid support (for example beads, plates, matrix support, chip) and detection of the compound by measuring the label or the presence of the compound itself.
  • a preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:
  • a further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:
  • the general methods that are described above may further comprise conducting the identification of agonist or antagonist in the presence of labelled or unlabelled ligand for the polypeptide.
  • the method for identifying agonist or antagonist of a polypeptide of the present invention comprises: determining the inhibition of binding of a ligand to cells which have a polypeptide of the invention on the surface thereof, or to cell membranes containing such a polypeptide, or to any other solid support such as those described above, in the presence of a candidate compound under conditions to permit binding to the polypeptide, and determining the amount of ligand bound to the polypeptide.
  • a compound capable of causing reduction of binding of a ligand is considered to be a competitor which may act as an agonist or antagonist.
  • the ligand is labelled.
  • a method of screening for a polypeptide antagonist or agonist compound comprises the steps of: (a) incubating a labelled ligand with a whole cell expressing a polypeptide according to the invention on the cell surface, or a cell membrane containing a polypeptide of the invention, or a solid support to which the polypeptide is bound,
  • step (b) measuring the amount of labelled ligand bound to the polypeptide on the solid support, whole cell or the cell membrane; (c) adding a candidate compound to a mixture of labelled ligand and immobilized polypeptide on the solid support, the whole cell or the cell membrane of step (a) and allowing the mixture to attain equilibrium;
  • step (d) measuring the amount of labelled ligand bound to the immobilized polypeptide or the whole cell or the cell membrane after step (c); and (e) comparing the difference in the labelled ligand bound in step (b) and (d), such that the, compound which causes the reduction in binding in step (d) is considered to be an agonist or antagonist.
  • the P450G4 and P450G5 polypeptides of the present invention may promote the metabolism of drags.
  • the ability of the P450G4 and P450G5 polypeptides to promote metabolism of particular drugs can be examined and the methods described above can be used to identify agonists and antagonists of the drug metabolising effect of the P450G4 and P450G5 polypeptides.
  • assays for identifying substrates and antagonists of the P450G4 and P450G5 polypeptides are conducted using a system in which activity of the P450G4 and P450G5 polypeptides is maximised. This may be achieved by co-expressing the P450G4 or
  • P450G5 polypeptide with a reductase protein that produces an active complex to screen for antagonists may be produced separately and introduced into the assay system.
  • Microsomal cytochromes occur on the membrane of the ER and require NADPH cytochrome reductase and a flavoprotein for activity, whereas mitochondrial cytochromes occur on the inner membrane and ferredoxin and NADPH ferredoxin reductase for activity
  • human NADPH CYP- reductase may be used to maximise the activity of a P450G3 polypeptide of the invention in assays screening for antagonists.
  • polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays.
  • the "functional equivalents" of the polypeptides of the invention include polypeptides that exhibit any of the same modulatory activities in the above-described assays in a dose- dependent manner.
  • the degree of dose-dependent activity need not be identical to that of the polypeptides of the invention, preferably the "functional equivalents" will exhibit substantially similar dose-dependence in a given activity assay compared to the polypeptides of the invention.
  • simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
  • competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
  • Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
  • an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
  • Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signalling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
  • This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
  • One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
  • a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
  • the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy.
  • Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
  • the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
  • the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
  • compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
  • a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85% by weight of the total X+Y in the composition is X.
  • X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
  • compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
  • therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targetted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The 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.
  • an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
  • a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
  • a pharmaceutically acceptable carrier for administration of a therapeutic agent.
  • Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by
  • compositions of the invention can be administered directly to the subject.
  • the subjects to be treated can be animals; in particular, human subjects can be treated.
  • compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
  • Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
  • the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • antagonists are antibodies.
  • such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
  • soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question may be administered.
  • the polypeptide may be administered in the form of fragments that retain the relevant portions.
  • expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered.
  • Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
  • Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
  • RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
  • a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
  • Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject. Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
  • Gene therapy of the present invention can occur in vivo or ex vivo.
  • Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient.
  • in vivo gene therapy does not require isolation and purification of a patient's cells.
  • the therapeutic gene is typically "packaged" for administration to a patient.
  • Gene delivery vehicles may be non-viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479.
  • a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
  • This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
  • These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).
  • Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
  • the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
  • Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (i.e. to treat disease after infection).
  • Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
  • vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
  • parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
  • sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • jet injection see, for example, www.powderject.com
  • jet injection may also be useful in the formulation of vaccine compositions.
  • nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
  • Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al., J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291-294 (1990)) prior to analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
  • the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); c) and detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
  • a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and, c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
  • an amplification step for example using PCR, may be included. Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
  • the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
  • Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)).
  • a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags.
  • Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
  • point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
  • DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al., Proc. Nati. Acad. Sci. USA (1985) 85: 4397-4401).
  • FISH Fluorescence in situ hybridization
  • an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996) 274: 610-613).
  • the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Nati. Acad. Sci. 93: 10614-10619).
  • Oligonucleotide pairs may range from two to over one million.
  • the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
  • the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
  • an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/25116 (Baldeschweiler et al).
  • a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
  • An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
  • diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
  • This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
  • Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
  • Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
  • Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
  • reporter molecules A wide variety of reporter molecules known in the art may be used, several of which are described above. Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a diagnostic kit of the present invention may comprise:
  • a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
  • the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
  • a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
  • a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
  • kits will be of use in diagnosing a disease or susceptibility to disease, particularly cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, breast, pancreas, head and neck and other solid tumours; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, and organ transplant rejection; cardiovascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including, central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity; AIDS, renal disease, infections including viral infection, bacterial infection, fungal infection and parasitic infection and other pathological conditions.
  • cell proliferative disorders including neoplasm, melanoma, lung, colorectal, breast, pancre
  • diseases which may be diagnosed include: diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia; diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); placental disorders; inflammatory diseases including, but not exclusively, COPD, osteoarthritis, wound healing and resolution of infections; and diseases associated with neurosteroid synthesis including dementia, Parkinson's disease, neurodegeneration following cerebrovascular diseases such as infarction or haemorrhage (stroke) or trauma to the central nervous system and spinal cord, learning difficulties, anxiety and addictive behaviours such as alcoholism, eating disorders and drug addiction.
  • diseases associated with inflammatory conditions in the brain such as multiple sclerosis and dementia
  • diseases associated with regulation of vascular tone of brain microcirculation such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine); placental disorders
  • inflammatory diseases including, but not exclusively, COPD, osteoarthritis, wound healing and resolution of infections
  • Figure 1 Front page of the BiopendiumTM. Search initiated using 1 JIO:A.
  • Figure 2A Inpharmatica Genome ThreaderTM results of search using 1 JIO.A. The arrow points to AAF67502.1, Streptomyces spheroides Cytochrome P450 Novl a typical Cytochrome P450 family member.
  • Figure 2B Inpharmatica Genome Threader results of search using 1JI0:A. The arrow points to the T00364 (P450G4) protein.
  • Figure 2C Inpharmatica PSI-Blast results from search using 1 JIO:A. The arrow points to BAA96945.1, Arabidopsis thaliana Cytochrome P450, a typical Cytochrome P450 family member.
  • Figure 2D Inpharmatica PSI-Blast results from search using 1 JIO:A. The arrow points to the T00364 (P450G4) protein.
  • FIG. 3 A InterPro search results for T00364 (P450G4).
  • Figure 3B NCBI Conserved Domain Database search results for T00364 (P450G4).
  • Figure 4A Graphical view of NCBI PSI-Blast (10 iterations) results for T00364 (P450G4).
  • Figure 4B List of NCBI PSI-Blast (10 iterations) results for T00364 (P450G4).
  • Figure 5A NCBI protein report for T00364 (P450G4).
  • Figure 5B NCBI protein report for BAA31648.1, a public domain sequence which is equivalent to T00364 (P450G4).
  • Figure 6A Inpharmatica Genome ThreaderTM results of search using T00364 (P450G4) as the query sequence.
  • the arrow points to lJ!O:A, the structure of Saccharopolyspora erythraea Cytochrome P450ERYF.
  • Figure 6B Inpharmatica PSI-Blast results from search using T00364 (P450G4) as the query sequence.
  • the arrow points to AAG29781.1, Sfreptomyces rishiriensis Cytochrome P450, a known member of the Cytochrome P450 family.
  • Figure 6C Inpharmatica PSI-Blast results from search using T00364 (P450G4) as the query sequence.
  • the arrow points to lJIO:A, the structure of Saccharopolyspora erythraea Cytochrome P450ERYF.
  • Figure 6D Genome ThreaderTM alignment of T00364 (P450G4) and 1 JIO.A.
  • Figure 7 Front page of the BiopendiumTM. Search initiated using 1DZ4:A.
  • Figure 8 A Inpharmatica Genome ThreaderTM results of search using 1DZ4:A. The arrow points to AAF67502.1, Streptomyces spheroides Cytochrome P450 Novl a typical Cytochrome P450 family member.
  • Figure 8B Inpharmatica Genome ThreaderTM results of search using 1DZ4:A. The arrow points to the BAB 13458.1 (P450G5) protein.
  • Figure 8C Inpharmatica PSI-Blast results from search using 1 JIO.A.
  • FIG. 9A InterPro search results for BAB13458.1 (P450G5).
  • Figure 9B NCBI Conserved Domain Database search results for BAB13458.1 (P450G5).
  • Figure 10A Graphical view of NCBI PSI-Blast (10 iterations) results for BAB13458.1 (P450G4).
  • Figure 10B Selection of NCBI PSI-Blast (10 iterations) results for BAB 13458.1 (P450G4).
  • Figure 11 NCBI protein report for BAB13458.1 (P450G5).
  • Figure 12A Inpharmatica Genome ThreaderTM 1 results of search using BAB13458.1 (P450G5) as the query sequence.
  • the arrow points to 1DZ4:A.
  • Figure 12B Reverse-Maximised PSI-Blast results from search using BAB 13458.1 (P450G5) as the query sequence.
  • Figure 12C Genome ThreaderTM alignment of BAB13458.1 (P450G5) and 1DZ4:A. The heme cofactor binding residue has been highlighted.
  • Figure 12D LigEye for 1DZ4:A which illustrates the sites of interaction of the heme cofactor with 1DZ4:A.
  • Figure 13 iRasMol view of 1DZ4:A. The coloured ball represents the heme cofactor binding residue in 1DZ4:A that is conserved in BAB13458.1 (P450G5).
  • Figure 16 P450G4 foetal tissue distribution
  • Figure 17 P450G5 tissue distribution
  • Figure 21 Reduced CO-difference spectral studies on G5-P450 domain sample.
  • the structure chosen is Saccharopolyspora erythraea Cytochrome P450ERYF, PDB code 1JIO.A ( Figure 1).
  • a search of the BiopendiumTM for homologues of 1 JIO:A takes place and returns 3618 Inpharmatica Genome ThreaderTM results (selection given in Figure 2 A and 2B) and 2270 Inpharmatica PSI-Blast results (selection in Figure 2C and 2D).
  • the 3618 Genome ThreaderTM 1 results include examples of other Cytochrome P450 family members, such as Streptomyces spheroides Cytochrome P450 Novl ( Figure 2 A, arrow).
  • the Inpharmatica Genome ThreaderTM has identified residues 586-946 of a sequence, T00364 (P450G4), as having an equivalent structure to residues 31-398 of Saccharopolyspora erythraea Cytochrome P450ERYF (PDB code: UIO:A). Having a structure equivalent to 1 JIO.A suggests that T00364 (P450G4) is a protein that functions as a Cytochrome P450. The Inpharmatica Genome ThreaderTM identifies this with 100% confidence.
  • the 2270 Inpharmatica PSI-Blast results include examples of known Cytochrome P450s, such as Arabidopsis thaliana, Cytochrome P450 ( Figure 2C, arrow).
  • Inpharmatica PSI-Blast Forward iterations of Inpharmatica PSI-Blast are unable to identify the relationship between 1 JIO.A and T00364 (P450G4). It is only in negative iterations that Inpharmatica PSI-Blast can identify Saccharopolyspora erythraea Cytochrome P450ERYF (PDB code: lJIO:A) as having a sequence relationship to residues 586-946 of T00364 (P450G4).
  • the ability to identify relationships via negative iterations of PSI-Blast is a product of the all-by-all sequence comparison (reverse-maximisation) that underlies the Biopendium and is unique to Inpharmatica.
  • T00364 the InterPro database is queried with T00364 (P450G4; Figure 3A). InterPro returns zero hits (no matches) to T00364 (P450G4). Returning zero hits means that InterPro does not identify any region of T00364 (P450G4) as containing Cytochrome P450 identity. Thus T00364 (P450G4) is unidentifiable as a Cytochrome P450 family member using InterPro.
  • NCBI conserveed Domain Database (CDD) is queried with T00364 (P450G4; Figure 3B). CDD returns zero hits to T00364 (P450G4). Returning zero hits means that CDD does not identify any region of T00364 (P450G4) as containing a Cytochrome P450 domain. Returning zero hits from CDD means that T00364 (P450G4) is unidentifiable as a Cytochrome P450 family member using CDD. NCBI provides a public domain PSI-Blast server.
  • Figure 4A shows the graphical display of NCBI PSI-Blast results for T00364 (P450G4).
  • the horizontal axis corresponds to N-terminal to C-terminal residue numbering along the T00364 (P450G4) protein.
  • the accession codes of the sequences hit in NCBI PSI- Blast are listed in Figure 4B.
  • NCBI PSI-Blast identifies this relationship with an E-value of 0.054 and places this in the 'below the significance threshold' category of PSI-Blast results.
  • NCBI PSI-Blast does not annotate any region of T00364 (P450G4) as having a statistically significant relationship to any known Cytochrome P450.
  • T00364 There is no further annotation for T00364.
  • the public domain information for this protein does not annotate it as containing a Cytochrome P450 domain ( Figure 5A).
  • the public domain does not annotate T00364 (P450G4) as being a Cytochrome P450.
  • An equivalent sequnce, BAA31648.1 also exists in the public domain.
  • the public domain information for BAA31648.1 does not annotate it as containing a Cytochrome P450 domain ( Figure 5B).
  • the public domain does not annotate BAA31648.1 as being a Cytochrome P450.
  • the Inpharmatica Genome ThreaderTM ( Figure 6A, arrow) identifies residues 586-946 of T00364 (P450G4) as having a structure the same as Saccharopolyspora erythraea Cytochrome P450ERYF (PDB code: 1JI0:A) with 100% confidence.
  • Inpharmatica PSI-Blast also identifies the same region of T00364 (P450G4) as having a relationship with known Cytochrome P450s by the third positive iteration.
  • Figure 6B shows a selection of Inpharmatica PSI-Blast results and it can be seen that the sequence AAG29781.1, Sfreptomyces rishiriensis Cytochrome P450 has a highly significant relationship to T00364 (P450G4), being found in the third positive iteration with an E-value of 4.0E-94.
  • residues 587-946 of T00364 are related to residues 46-402 of AAG29781.1, Streptomyces rishiriensis Cytochrome P450.
  • Residues 587-946 includes almost all of the T00364 (P450G4) Cytochrome P450 region identified by Genome ThreaderTM 1 (residues 586-946), and matches them to a region of AAG29781.1, Streptomyces rishiriensis Cytochrome P450 which contains a known Cytochrome P450 domain (residues 31-371, as determined by PFAM).
  • Inpharmatica PSI-Blast is in strong agreement with Inpharmatica Genome ThreaderTM at annotating a region between residues 586 to 946 of T00364 (P450G4) as being a Cytochrome P450. This is in contrast to public domain NCBI PSI-Blast which fails to identify any statistically significant relationship between T00364 (P450G4) and known Cytochrome P450s ( Figures 5A and 5B). Only Inpharmatica Genome ThreaderTM and Inpharmatica PSI-Blast are able to identify T00364 (P450G4) as being a Cytochrome P450.
  • Inpharmatica PSI-Blast also identifies a relationship between T00364 (P450G4) and the original query structure 1JI0:A (Saccharopolyspora erythraea Cytochrome P450ERYF), Figure 6C arrow.
  • the relationship between T00364 (P450G4) and UIO:A is found in the fourth positive iteration and has a significant E-value of 2.0E-62. This further consolidates the Genome Threader annotation of T00364 (P450G4) as being a Cytochrome P450.
  • Example 2 BAB13458.1 (P450G5)
  • P450G5 an archetypal Cytochrome P450 family member, Pseudomonas putida Cytochrome P450CAM is chosen. More specifically, the search is initiated using a structure from the Protein Data Bank (PDB) which is operated by the Research Collaboratory for Structural Bioinformatics.
  • PDB Protein Data Bank
  • the structure chosen is Pseudomonas putida Cytochrome P450CAM, PDB code 1DZ4:A ( Figure 7).
  • a search of the BiopendiumTM for homologues of 1DZ4:A takes place and returns 5560 Inpharmatica Genome ThreaderTM results (selection given in Figure 8A and 8B) and 2198 Inpharmatica PSI-Blast results (selection in Figure 8C and 8D).
  • the 5560 Genome ThreaderTM results include examples of other Cytochrome P450 family members, such as Streptomyces spheroides Cytochrome P450 Novl ( Figure 9A, arrow).
  • BAB 13458.1 P450G5, Figure 8B, arrow).
  • the Inpharmatica Genome ThreaderTM has identified residues 1112-1350 of a sequence, BAB13458.1 (P450G5), as having an equivalent structure to residues 122-352 of Pseudomonas putida Cytochrome P450CAM (PDB code: 1DZ4:A). Having a structure equivalent to 1DZ4:A suggests that BAB13458.1 (P450G5) is a protein that functions as a Cytochrome P450. The Inpharmatica Genome ThreaderTM identifies this with 85% confidence.
  • the 2198 Inpharmatica PSI-Blast results include examples of known Cytochrome P450s, such as 1C8J:A Pseudomonas putida Cytochrome P450CAM ( Figure 8C, arrow).
  • Inpharmatica PSI-Blast Forward iterations of Inpharmatica PSI-Blast are unable to identify the relationship between 1DZ4:A and BAB13458.1 (P450G5). Negative iterations of Inpharmatica PSI-Blast are also unable to identify the relationship between 1DZ4:A and BAB 13458.1 (P450G5). Therefore, it is only Inpharmatica Genome ThreaderTM that is able to identify a structural relationship between 1DZ4:A and BAB13458.1 (P450G5). It is only Inpharmatica Genome ThreaderTMthat is able to identify BAB13458.1 (P450G5) as being a Cytochrome P450.
  • BAB 13458.1 In order to view what is known in the public domain secondary databases about BAB 13458.1 (P450G5), the InterPro database is queried with BAB 13458.1 (P450G5; Figure 9A). InterPro returns one hit, ProSite match PS00237, to BAB 13458.1 (P450G5). Prosite is based on a pattern of regular expressions and because of this can often return false positive results. Irrespective of whether or not this is a false positive result, InterPro does not identify any region of BAB 13458.1 (P450G5) as containing Cytochrome P450 identity. Thus BAB13458.1 (P450G5) is unidentifiable as a Cytochrome P450 family member using InterPro.
  • CDD NCBI conserveed Domain Database
  • BAB13458.1 P450G5; Figure 9B
  • CDD returns zero hits to BAB 13458.1 (P450G5).
  • Returning zero hits means that CDD does not identify any region of BAB13458.1 (P450G5) as containing a Cytochrome P450 domain.
  • Returning zero hits from CDD means that BAB13458.1 (P450G5) is unidentifiable as a Cytochrome P450 family member using CDD.
  • NCBI provides a public domain PSI-Blast server. Querying NCBI PSI-Blast with BAB 13458.1 (P450G5) through 10 positive iterations fails to annotate any region of BAB13485.1 (P450G5) as having a relationship to any known Cytochrome P450 (note that NCBI PSI-Blast cannot provide data on negative iterations because no all-by-all calculation is performed).
  • Figure 10A shows the graphical display of NCBI PSI-Blast results for BAB13458.1 (P450G5). The horizontal axis corresponds to N-terminal to C-terminal residue numbering along the BAB 13458.1 (P450G5) protein.
  • the accession codes of the top sequences hit in NCBI PSI-Blast are listed in Figure 10B.
  • NCBI PSI-Blast does not' annotate any region of BAB 13458.1 (P450G5) as having a relationship to any known Cytochrome P450.
  • BAB 13458.1 (P450G5) is now used as the query sequence in the BiopendiumTM.
  • the Inpharmatica Genome ThreaderTM 1 identifies 180 hits ( Figure 12 A) while mpharmatica PSI-Blast returns 12 hits ( Figure 12B).
  • the Inpharmatica Genome ThreaderTM ( Figure 12A, arrow) identifies residues 1112-1350 of BAB13458.1 (P450G5) as having a structure the same as Pseudomonas putida Cytochrome P450CAM (PDB code: 1DZ4:A) with 85% confidence.
  • Threonine 1239 in BAB13458.1 aligns with threonine 342 in the 1DZ4 sequence.
  • Thr342 points into the active site of the Cytochrome P450 molecule and is required for activation of an oxygen molecule.
  • the glycine residue 3 amino acids ammo-terminal to the Threonine residue (Glyl236 in BAB13458.1 (P450G5) and Gly239 in 1DZ4) is also a highly conserved residue across the Cytochrome P450 superfamily.
  • cDNA source for PCR of P450G4 1 ng total RNA (Ambion) from different human tissues was used to generate cDNA using the superscript RT (Invitrogen) and oligo dT primer following the manufacturer's protocol. 2 ⁇ l of the reaction was used in the subsequent PCR. Cloning of the P450G4 was performed using human brain and testis cDNA
  • P450G4 F and P450G4 R were used to amplify the proposed P450 domain encompassing amino acids 575-946 based on the numbering of the KIAA0673 clone. PCR was carried out using the Roche Expand Polymerase (Roche Diagnostics Ltd, Lewes, UK) in 1.5 mM MgCl 2 .
  • the primer sequences were: P450G4 F
  • the resulting PCR products from brain and testis cDNAs were pooled and then cloned into the vector pGEMTEasy (Promega UK Ltd, Southampton, UK) and verified by sequence analysis. Sequences were identical to the proposed sequence of P450G4. Inserts were then re-amplified using primers P450G4express F and P450G4 express R and sub- cloned into the vector pET-3b (CN Biosciences, Nottingham, UK) by restriction digest with the enzymes Nde I and BaniHI. The construct can then be expressed with both N- and C- terminal His Tags. The construct was once again verified by sequencing.
  • RNA 1 ng total RNA (Ambion) from different human tissues was used to generate cDNA using the superscript RT (Invitrogen) and oligo dT primer following the manufacturer's protocol. 2 ⁇ l of the reaction was used in the subsequent PCR. Cloning of the P450G5 was performed using human brain and placenta cDNA
  • Primers P450G5 F and P450G5 R were used to amplify the proposed P450 domain encompassing amino acids 1112-1350 based on the numbering of the KIAA1632 clone. PCR was carried out using the Roche Expand Polymerase (Roche Diagnostics Ltd, Lewes, UK) in 1.5 mM MgCl 2 .
  • the primer sequences were:
  • P450G5 AAACCTTCTTCCAATACAACCATCT
  • the resulting PCR products from brain and placenta cDNAs were pooled and then cloned into the vector pGEMTEasy (Promega UK Ltd, Southampton, UK) and verified by sequence analysis. Sequences were identical to the proposed sequence of P450G4. Inserts were then re-amplified using primers P450G5express F and P450G5express R and sub- cloned into the vector pET-3b (CN Biosciences, Nottingham, UK) by restriction digest with the enzymes Nde I and BamHI. The construct can then be expressed with both N- and C- terminal His Tags. The construct was once again verified by sequencing.
  • P450G5express R CGGGATCCTCAATGATGATGATGATGATGAAACCTTCTTCCAATACA
  • Taqman RT-PCR quantitation was used.
  • the TaqMan 3'- 5' exonuclease assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a doublelabeled fluorogenic probe that hybridises to the target template at a site between the two primer recognition sequences (cf. U. S. Patent 5,876,930).
  • the ABI Prism 7700 automates the detection and quantitative measurement of these signals, which are stoichiometrically related to the quantities of amplicons produced, during each cycle of amplification. In addition to providing substantial reductions in the time and labour requirements for PCR analyses, this technology permits simplified and potentially highly accurate quantification of target sequences in the reactions.
  • Figure 14 shows normalised expression of P450G4 in 22 normal human tissues.
  • Taqman RT-PCR was carried out using 15ng of the indicated cDNA using primers/probes specific for P450G4 and 18s rRNA as described in the detailed description.
  • a standard curve for target and internal control was also carried out, using between 25ng to 0.39ng of cDNA template of a typical tissue sample.
  • Cycle threshold (Ct) determinations i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were performed by the instrument for each reaction using default parameters.
  • Ct Cycle threshold
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, stomach.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in stomach.
  • the expression levels of P450G4 were then normalised to the expression levels of 18s.
  • Figure 14 represents the fold expression of normalised target sequence relative to the level of expression in stomach cDNA, which is set arbitrarily to 1. Each sample was quantitated in 2 individual experiments. Figure 14 shows the mean ⁇ SEM for the multiple experiments.
  • Figure 15 shows normalised expression of P450G4 in 27 normal or treated cell lines.
  • Taqman RT-PCR was carried out using 25ng of the indicated cDNA using primers/probes specific for P450G4 and 18s rRNA as described in the detailed description.
  • a standard curve for target and internal control was also carried out, using between 50ng to 0.78ng of cDNA template of a typical cell sample.
  • Cycle threshold (Ct) detemiinations i.e. non- integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were performed by the instrument for each reaction using default parameters.
  • Ct values were used to calculate the amount of actual starting target or 18s cDNA in each test sample.
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, A172 cells.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in A172 cells.
  • the expression levels of P450G4 were then normalised to the expression levels of 18s.
  • Figure 15 represents the fold expression of normalised target sequence relative to the level of expression in A172 cells cDNA, which is set arbitrarily to 1. Each sample was quantitated in 3 individual experiments. Figure 15 shows the mean ⁇ SEM for the multiple experiments.
  • Figure 16 shows normalised expression of P450G4 in embryo, placenta and 8 human foetal tissues.
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, foetal skin.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in foetal skin.
  • the expression levels of P450G4 were then normalised to the expression levels of 18s.
  • Figure 16 represents the fold expression of normalised target sequence relative to the level of expression in foetal skin cDNA, which is set arbitrarily to 1. Each sample was quantitated in 3 individual experiments. Figure 16 shows the mean ⁇ SEM for the multiple experiments.
  • RNA prepared from non-diseased organs was purchased from either Ambion Europe (Huntingdon, UK) or Clontech (BD, Franklin Lakes, NJ).
  • RNA samples were from Ambion Europe (Huntingdon, UK). All the other treated or untreated total RNA were extracted from cells using GeneEluteTM Mammalian Total RNA kit from Sigma. Cells had been cultured according to the recommendation from EACC (European Collection of Cell Cultures).
  • Oligonucleotide primers and probes were designed using Primer Express software (Applied Biosystems, Foster City CA) with a GC-content of 40-60%, no G-nucleotide at the 5'-end of the probe, and no more than 4 contiguous Gs.
  • the sequence of the primers and probes were: P450G4 Fwd CCCGTGTGAACAGAAAGTGAGA
  • P450G4 Rev CGTTTGAGATGACCCGAGATC 18s pre-optimised primers and probe were purchased from Applied Biosystems, Foster City, CA.
  • Primer/probe concentrations were titrated in the range of 50nM to 900nM and optimal concentrations for efficient PCR reactions are determined. Optimal primer and probe concentrations vary in between lOOnM and 900nM depending on the target gene amplified.
  • cDNA reaction cDNA was prepared using components from Applied Biosystems, Foster City CA. 50 ⁇ l reactions are prepared in 0.5ml RNase free tubes. Reactions contain 500ng total RNA; lx reverse transcriptase buffer; 5.5mM MgC12; ImM dNTP's; 2.5 ⁇ l random hexamers; 20U RNase inhibitor; and 62.5U reverse transcriptase.
  • reaction 25 ⁇ l reactions were prepared in 0.5 ml thin-walled, optical grade PCR 96 well plates (Applied Biosystems, Foster City CA). Reactions contain: lx final concentration of TaqMan Universal Master Mix (a proprietary mixture of AmpliTaq Gold DNA polymerase, AmpEraseX UNG, dNTPs with UTP, passive reference dye and optimised buffer components, Applied Biosystems, Foster City CA); lOOnM Taqman probe; 900nM forward primer; 900nM reverse primer and 15ng of cDNA template.
  • TaqMan Universal Master Mix a proprietary mixture of AmpliTaq Gold DNA polymerase, AmpEraseX UNG, dNTPs with UTP, passive reference dye and optimised buffer components, Applied Biosystems, Foster City CA
  • lOOnM Taqman probe 900nM forward primer
  • 900nM reverse primer 15ng of cDNA template.
  • Taqman RT-PCR quantitation was used.
  • the TaqMan 3'- 5' exonuclease assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a doublelabeled fluorogenic probe that hybridises to the target template at a site between the two primer recognition sequences (cf. U. S. Patent 5,876,930).
  • the ABI Prism 7700 automates the detection and quantitative measurement of these signals, which are stoichiometrically related to the quantities of amplicons produced, during each cycle of amplification. In addition to providing substantial reductions in the time and labour requirements for PCR analyses, this technology permits simplified and potentially highly accurate quantification of target sequences in the reactions.
  • Figure 17 shows normalised expression of P450G5 in 22 normal human tissues.
  • Taqman RT-PCR was carried out using 15ng of the indicated cDNA using primers/probes specific for P450G5 and 18s rRNA as described in the detailed description.
  • a standard curve for target and internal control was also carried out, using between 25ng to 0.39ng of cDNA template of a typical tissue sample.
  • Cycle threshold (Ct) determinations i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were performed by the instrument for each reaction using default parameters.
  • Ct Cycle threshold
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, stomach.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in stomach.
  • the expression levels of P450G5 were then normalised to the expression levels of 18s.
  • Figure 17 represents the fold expression of normalised target sequence relative to the level of expression in stomach cDNA, which is set arbitrarily to 1. Each sample was quantitated in 2 individual experiments. Figure 17 shows the mean ⁇ SEM for the multiple experiments.
  • Figure 18 shows normalised expression of P450G5 in 27 normal or treated cell lines.
  • Taqman RT-PCR was carried out using 25ng of the indicated cDNA using primers/probes specific for P450G5 and 18s rRNA as described in the detailed description.
  • a standard curve for target and internal control was also carried out, using between 50ng to 0.78ng of cDNA template of a typical tissue sample.
  • Cycle threshold (Ct) determinations i.e. non-integer calculations of the number of cycles required for reporter dye fluorescence resulting from the synthesis of PCR products to become significantly higher than background fluorescence levels were performed by the instrument for each reaction using default parameters.
  • Ct values were used to calculate the amount of actual starting target or 18s cDNA in each test sample.
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, A 172 cells.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in A172 cells.
  • the expression levels of P450G5 were then normalised to the expression levels of 18s.
  • Figure 19 represents the fold expression of normalised target sequence relative to the level of expression in A 172 cells cDNA, which is set arbitrarily to 1. Each sample was quantitated in 3 individual experiments. Figure 19 shows the mean + SEM for the multiple experiments.
  • transcript could be detected in all cell types except SK- N-SH (neuroblastoma cell line). Highest levels of expression were observed in HL60 cells (undifferentiated myeloblastic cell line). Levels of transcript in the other cells were relatively invariant and no induction in expression was observed in various cells treated with pharmacological agents including dibutyryl cAMP, interferon- ⁇ , phorbol esters or retinoic acid.
  • Figure 19 shows normalised expression of P450G5 in embryo, placenta and 8 human foetal tissues.
  • the levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample, in this case, foetal skin.
  • the levels of 18s cDNA in each sample were also normalised to the level of expression of 18s in foetal skin.
  • the expression levels of P450G5 were then normalised to the expression levels of 18s.
  • Figure 3 represents the fold expression of normalised target sequence relative to the level of expression in foetal skin cDNA, which is set arbitrarily to 1. Each sample was quantitated in 3 individual experiments.
  • Figure 19 shows the mean ⁇ SEM for the multiple experiments.
  • P450G5 Probe CTTTGAAGAGGACTCCCAGCTCCGGA P450G5 Rev GTTTATCACCAATTCCCCTTCAAT
  • the predicted P450 domain of P450G4 was cloned into a bacterial expression vector see Example 3 section B.
  • the E.coli expressed protein enables a P450 reduced CO spectrum assay which demonstrates that the protein has P450 activity. If the sample contains an active P450 enzyme a characteristic Soret peak at 450nm is observed (Omura & Sato JBC 239, 2370, 1964).
  • a 5ml culture was prepared, starting from a single colony (BL21 DE3), and the bacteria were grown overnight at 37°C in Luria broth medium containing 120 ⁇ g/ml ampicillin. 1 ml of this culture was added to 100ml Luria broth medium containing 120 ⁇ g/ml ampicillin and 34 ⁇ g/ml of chloramphenicol in a 1 litre flask. The culture was shaken at 270 rpm, 37°C for 3 hours. Heme precursor: ⁇ -aminolevulinic acid was added to a final concentration of 80 ⁇ g/ml. After 1-hour incubation at 30°C, 120rpm, T7 RNA polymerase expression was induced by addition of IPTG to a final concentration of 0.5mM.
  • the resulting supernatant was pooled with the previous supernatant and centrifuged at 75,000g for 60min at 4°C.
  • the resulting pellet was bright red and gelatinous corresponding to the 10 membrane-bound fraction, this is highly indicative of a heme binding protein.
  • the P450G4 in the membrane bound fraction was solubilized with detergent. Pellets were re-suspended in buffer B and the solubilisation was achieved by addition a of variable percentage of detergents (0.1 -1%) such as:
  • the UV spectrum of the G4-P450 reduced carbon monoxide adduct gives two peaks ( Figure 20), one at 420nm and one smaller at 450nm.
  • the peak at 450nm is characteristic of a cytochrome P450 protein.
  • the peak at 420nm is also characteristic of a cytochrome P450, but an unstable form. It is well known that large number of reagents and enzyme react with cytochrome P450 to produce a biologically inert heme protein termed "cytochrome P420".
  • A. P450 Bacterial expression The predicted P450 domain of P450G5 was cloned into a bacterial expression vector see Example 4 section B.
  • the E.coli expressed protein enables a P450 reduced CO spectrum assay which demonstrates that the protein has P450 activity. If the sample contains an active P450 enzyme a characteristic Soret peak at 450nm is observed (Omura & Sato JBC 239, 2370, 1964).
  • a 5ml culture was prepared, starting from a single colony (BL21 DE3), and the bacteria were grown overnight at 37°C in Luria broth medium containing 120 ⁇ g/ml ampicillin.
  • 1 ml of this culture was added to 100ml Luria broth medium containing 120 ⁇ g/ml ampicillin and 34 ⁇ g/ml of chloramphenicol in a 1 litre flask.
  • the culture was shaken at 270 rpm, 37°C for 3 hours.
  • Heme precursor: ⁇ -aminolevulinic acid was added to a final concentration of 80 ⁇ g/ml.
  • T7 RNA polymerase expression was induced by addition of IPTG to a final concentration of 0.5mM.Growth was allowed to continue for 24hr at 30°C, at a shaking rate of 120rpm.
  • the resulting supernatant was pooled with the previous supernatant and centrifuged at 75,000g for 60min at 4°C.
  • the resulting pellet was bright red and gelatinous corresponding to the membrane-bound fraction, this is highly indicative of a heme binding protein.
  • the P450G5 in the membrane bound fraction was solubilized with detergent. Pellets were re-suspended in buffer B and the solubilisation was achieved by addition of variable percentage of detergents (0.1 -1%) such as:
  • the total protein concentration was determined by a Bradford assay.
  • the light absorption was recorded between 400nm and 600nm in a spectrophometer DU640.
  • Aliquots of protein preparation were added to two cuvettes (1ml each), and reduced by addition of few milligrams of solid sodium dithionite. Carbon monoxide was bubbled slowly into one of the sample cuvette for 30s, allowing the CO to ligate the heme iron of P450 previously reduced. Both cuvette samples were analysed. The reduced CO difference spectrum between the sample with the Carbon monoxide and the sample without was recorded. The result is shown in Figure 21.
  • Example 9 Mass spectrometry analysis of heme binding for P450G4 and P450G5
  • Purified protein can be analysed using mass spectrometry. This method utilises measurements of the mass to charge ratio of a sample to identify groups contained within the sample. The heme group of P450s can be identified by its characteristic charge to mass ratio. Trypsin digestion of the protein into peptide fragments and analysis of the fragments via MS/MS spectrometry also provides confirmation of the protein sequence and any possible post-translational modifications.
  • Example 10 Expression for substrate determination and inhibitor screening for P450G4 and P450G5
  • Cytochrome P450s comprise a large family of heme-containing proteins which present a variety of enzymatic activities towards exogenous and endogenous substrates. Most isoforms are assigned to a certain subfamily based on homology at the amino acid level. However, due to the low level of sequence similarity, the P450G4 and P450G5 cannot be assigned to a known class of P450s.
  • a pcDNA-DEST40 vector (Invitrogen) containing the P450G4 or P450G5 is transfected into mammalian COS-1 cells using Fugene reagent (Roche) according to the manufacturer's instructions. After 48-hours post-transfection, cells are washed and a microsomal preparation carried out.
  • BaculoDirect kit Invitrogen directly transfers the P450G4 or P450G5 cDNA into the baculovirus genome in vitro.
  • the resulting recombinant baculovirus DNA is used to transfect the SF9 insect cells.
  • Microsomal preparations or protein purifications are carried out. Proteins may be purified using affinity chromatography using the V5 and His tags contained in the vectors. Reconstituted systems containing relative amounts of P450 protein, P450 reductase, cytochrome b5 and NADPH were prepared for the in vitro assays.
  • a series of putative endogenous substrates e.g. steroids, prostaglandins, fatty acids, retinoic acid, vitamin D derivates, oxysterols, bile acids
  • putative endogenous substrates e.g. steroids, prostaglandins, fatty acids, retinoic acid, vitamin D derivates, oxysterols, bile acids
  • the protein mixtures can then be separated by LC and analysed by mass spectrometry.
  • Arachidonic acid incubation with the novel P450s may lead to specific metabolites of the HETE family that can directly detected via a reverse phase HPLC column.
  • Other classes of metabolites may also be detected via LC or with specific antibodies by ELISA based methodology.
  • Labelled substrate assays e.g. steroids, prostaglandins, fatty acids, retinoic acid, vitamin D derivates, oxysterols, bile acids
  • Radioactive or fluorescently labelled putative substrates are incubated with either intact cells overexpressing the protein of interest or microsomal preparations. These assays can be conducted in 96-well plates and therefore have the advantage of a higher throughput. The level of metabolites present in the reaction is measured by chemical separation and quantification of the label.
  • SEQ ID NO:l Nucleotide coding sequence for T00364 (P450G4) protein
  • SEQ ID NO:3 the nucleotide coding sequence for BAB13458.1 (P450G5) protein

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention se rapporte à de nouvelles protéines, appelées T00364 and BAB13458.1, identifiées ici sous le nom de cytochromes P450, ainsi qu'à l'utilisation de ces protéines et de séquences nucléotidiques des gènes codants, dans le diagnostic, la prévention et le traitement de maladies.
PCT/GB2003/003281 2002-07-30 2003-07-30 Cytochromes p450 Ceased WO2004011648A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003255732A AU2003255732A1 (en) 2002-07-30 2003-07-30 CYTOCHROME P450s

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0217631A GB0217631D0 (en) 2002-07-30 2002-07-30 Proteins
GB0217631.1 2002-07-30

Publications (2)

Publication Number Publication Date
WO2004011648A2 true WO2004011648A2 (fr) 2004-02-05
WO2004011648A3 WO2004011648A3 (fr) 2004-06-03

Family

ID=9941368

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2003/003281 Ceased WO2004011648A2 (fr) 2002-07-30 2003-07-30 Cytochromes p450

Country Status (3)

Country Link
AU (1) AU2003255732A1 (fr)
GB (1) GB0217631D0 (fr)
WO (1) WO2004011648A2 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053312A1 (fr) * 1999-12-23 2001-07-26 Hyseq, Inc. Nouveaux acides nucleiques et polypeptides
AU2002215776A1 (en) * 2000-12-15 2002-06-24 Cytochroma Inc. P450rai-2 (p450 cytochrome 26b), encoding nucleic acid molecules and methods and uses thereof

Also Published As

Publication number Publication date
AU2003255732A8 (en) 2004-02-16
WO2004011648A3 (fr) 2004-06-03
GB0217631D0 (en) 2002-09-11
AU2003255732A1 (en) 2004-02-16

Similar Documents

Publication Publication Date Title
AU2006221859B2 (en) Lipocalin protein
US20040249123A1 (en) Nuclear hormone receptor ligand binding domain
WO2004026904A1 (fr) Proteines precurseurs de peptides associes d'amide rf et peptides d'amide rf
WO2003099865A1 (fr) Proteines canaux cationiques
WO2004011648A2 (fr) Cytochromes p450
WO2004101618A2 (fr) Proteines associees au recepteur de progestine yol002c-cgi-45
WO2004003190A1 (fr) Polypeptide identifie comme cytochrome p450
US20060234218A1 (en) P450 proteins
US20040132038A1 (en) Nuclear hormone receptor ligand binding domain
CA2475230A1 (fr) Proteine p450 cytochrome
WO2003072769A1 (fr) Proteine appartenant aux cytochromes p450
WO2002029023A1 (fr) Proteine appelee nicastrine
WO2003089469A2 (fr) Proteines-canaux cationiques
WO2002062845A2 (fr) Molecules d'adhesion
WO2004081170A2 (fr) Proteine de type recepteur de la progestine
WO2002046380A2 (fr) Proteine kinase
WO2004009624A2 (fr) Proteine a repliement du type toxine a trois doigts
WO2002034901A2 (fr) Proteine kinase
WO2002008393A2 (fr) Serine protease
WO2002062976A2 (fr) Proteases a serine
WO2003072605A1 (fr) Proteine comprenant un domaine de liaison aux ligands d'un recepteur d'hormones nucleaire

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP