WO2004011648A2

WO2004011648A2 - CYTOCHROME P450s

Info

Publication number: WO2004011648A2
Application number: PCT/GB2003/003281
Authority: WO
Inventors: 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
Original assignee: Inpharmatica Ltd
Current assignee: Inpharmatica Ltd
Priority date: 2002-07-30
Filing date: 2003-07-30
Publication date: 2004-02-05
Anticipated expiration: 2005-01-30
Also published as: AU2003255732A8; WO2004011648A3; GB0217631D0; AU2003255732A1

Abstract

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.

Description

CYTOCHROME P450s

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.

BACKGROUND

The process of drug discovery is presently undergoing a fundamental revolution as the era of functional genomics comes of age. The term "functional genomics" applies to an approach utilising bioinformatics tools to ascribe function to protein sequences of interest. Such tools are becoming increasingly necessary as the speed of generation of sequence data is rapidly outpacing the ability of research laboratories to assign functions to these protein sequences.

As 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.

Various institutions and commercial organisations are examining sequence data as they become available and significant discoveries are being made on an on-going basis. However, there remains a continuing need to identify and characterise further genes and the polypeptides that they encode, as targets for research and for drug discovery.

Recently, a remarkable tool for the evaluation of sequences of unknown function has been developed by the Applicant for the present invention. 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.

The aim behind the integration of these 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.

This data resource has made possible the accurate prediction of protein function from sequence alone. Using conventional technology, this is only possible for proteins that exhibit a high degree of sequence identity (above about 20%-30% identity) to other proteins in the same functional family. Accurate predictions are not possible for proteins that exhibit a very low degree of sequence homology to other related proteins of known function.

In the present case, 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.

Introduction to Cytochrome P450s

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 IO⁶ 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.

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. Also many 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.

Fungal and bacterial P450s are also of medical interest because of their potential as antibiotic targets. 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. For example, 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.

There is thus a great need for the identification of 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

The invention is based on the discovery that the T00364 protein and BAB 13458.1 protein function as Cytochrome P450s.

For the T00364 protein, it has been found that a region including residues 586-946 of this protein sequence adopts an equivalent fold to residues 31 to 398 of the 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 possession of an equivalent fold allows the functional annotation of this region of T00364, and therefore proteins that include this region, as possessing Cytochrome P450 activity. For the BAB13458.1 protein, it has been found that a region including residues 1112- 1350 of this protein sequence adopts an equivalent fold to residues 122 to 352 of the Pseudomonas putida Cytochrome P450CAM (PDB code 1DZ4:A). Pseudomonas putida Cytochrome P450CAM is known to function as a Cytochrome P450. Furthermore, the heme cofactor binding residue Cys347 of the Pseudomonas putida Cytochrome P450CAM is conserved as Cysl345 in BAB13458.1, respectively. Note that the 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. Thus, by reference to the Genome Threader™ alignment of BAB 13458.1 with the Pseudomonas putida Cytochrome P450CAM (1DZ4:A) Cysl345 is predicted to form the heme cofactor binding residue.

The combination of equivalent fold and conservation of the heme cofactor binding residue allows the functional annotation of this region of BAB 13458.1, and therefore proteins that include this region, as possessing Cytochrome P450 activity.

In addition, 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. In addition, 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. It is also consistent with roles for this P450 in regulating pathways associated with regulation of vascular tone of brain microcirculation implicated in cerebrovascular diseases, such as stroke and vasospastic conditions (subarachnoid haemorrhage and migraine), and in regulating pathways associated with the synthesis of neurosteroids.

The known literature has consistently reported the widespread effects of steroids on the brain (known as neurosteroids). For instance, 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. In addition, 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. The finding of a P450 domain within this previously unannotated protein (Mollet et al, Nature Genetics, 2002, 300-5 and Nature Genetics, 2002, 32, 300-5) provides an opportunity to understand the pathophysiology of juvenile nephronophthisis and thereby offer potential therapies to treat this disease with specific remedies that alter the P450 function. 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. The finding that the 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. Thus, 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.

For 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. In addition, 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). 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.

For 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. In addition, 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. Consistent with a role for BAB 13458.1 in placental function, the transcript levels for BAB13458.1 were very high in the pre-term placental sample (20 weeks). Thus, 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. The finding of high levels of the transcript in the thymus and in the cell lines derived from haematopoietic cell lines including Jurkat cells and two myeloid cell lines, HL60 cells and U937 cells indicates that modulators of activity or transcript levels may have value in treating disorders of T cell proliferation or maturation including leukaemias and lymphopenias following infections such as HIV or chemotherapy or radiotherapy. In addition, 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). Consistent with this, 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.

In a first aspect, 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).

Preferably, 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". According to this aspect of the invention, 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. As defined herein, 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". According to this aspect of the invention, 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). As defined herein, the P450G5 Cytochrome P450 region is considered to extend between residue 1112 and residue 1350 of the P450G5 polypeptide sequence.

By "Cytochrome P450 activity", as this term is used herein, 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. In particular, we mean that 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. Chem., 239; 2370). Preferably, the polypeptides and functional equivalents according to the invention function as P450 enzymes. By "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. According to this aspect of the invention, 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. As defined herein, 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. As defined herein, 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.

In a second aspect, the invention provides a purified nucleic acid molecule that encodes a polypeptide of the first aspect of the invention. Preferably, 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. In a third aspect, 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.

In a fourth aspect, 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. In a fifth aspect, 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.

In a sixth aspect, 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. For example, such ligands may bind specifically to the heme binding domain of the Cytochrome P450, thus preventing oxidation of a substrate from taking place.

In a seventh aspect, 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. Importantly, 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.

In an eighth aspect, 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.

In particular, 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 emphysema; 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, compulsive obsessive disorders, anxiety, depression; and addictive behaviours such as alcoholism, eating disorders and drug addiction including nicotine addiction. The moieties of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention may also be used in the manufacture of a medicament for the treatment of such diseases.

In a ninth aspect, 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. Such 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.

Preferably, 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 pathological conditions. In particular, 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. In a tenth aspect, 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. In particular, 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.

In an eleventh aspect, 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.

In a twelfth aspect, 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, 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.

In particular, 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 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, compulsive obsessive disorders, anxiety, depression; and addictive behaviours such as alcoholism, eating disorders and drug addiction including nicotine addiction.

In a thirteenth aspect, 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.

For diseases in which the expression of a natural gene encoding a polypeptide of the first aspect of the invention, or in which the activity of a polypeptide of the first aspect of the invention, is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist. Conversely, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist. Examples of such antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.

In a fourteenth aspect, 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.

A summary of standard techniques and procedures which may be employed in order to utilise the invention is given below. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors and reagents described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and it is not intended that this terminology should limit the scope of the present invention. The extent of the invention is limited only by the terms of the appended claims.

Standard abbreviations for nucleotides and amino acids are used in this specification. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art.

Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Transcription and Translation (B.D. Hames & SJ. Higgins eds. 1984); Animal Cell Culture (R.I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J.H. Miller and.M P. Calos eds. 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. 1987, Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer Verlag, N. Y.); and Handbook of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell eds. 1986).

As used herein, the term "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. In such polypeptides, 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. For example, it is often advantageous to include 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. Alternatively or additionally, 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. Among the known 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. Other potential modifications include acetylation, acylation, amidation, covalent attachment of flavin, covalent attachment of a haeme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, GPI anchor formation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, 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.

The modifications that occur in a polypeptide often will be a function of how the polypeptide is made. For polypeptides that are made recombinantly, 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.

The 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. Degrees of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

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. Especially preferred are 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;

Typically, greater than 30% identity between two polypeptides (preferably, over a specified region) is considered to be an indication of functional equivalence. Preferably, functionally equivalent 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.

Percentage identity, as referred to herein, 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/) [Blosum 62 matrix; gap open penalty=l 1 and gap extension penalty=l].

In the present case, 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.

In the present case, 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. 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 P450G5 polypeptide and which possess the heme cofactor binding residue Cys 1345. As discussed above, 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. For example, the Inpharmatica Genome Threader™ 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.

By "significant structural homology" is meant that the Inpharmatica Genome Threader™ 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 Threader™ is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome Threader™ 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). This resulted in a neural network score between 0 and 1. However, again as the number of proteins that are related and the number that are unrelated were known, it was possible to partition the neural network results into packets and calculate empirically the percentage of the results that were correct. In this manner, any genuine prediction in the Biopendium search database has an attached neural network score and the percentage confidence is a reflection of how successful the Inpharmatica Genome Threader™ was in the training/testing set.

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. The 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.

As used herein, the term "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.

For the P450G4 polypeptide, this region is considered to extend between residue 586 and residue 946. For the P450G5 polypeptide, 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.

In one respect, the term "variant" is meant to include extended or truncated versions of this polypeptide fragment.

For extended variants, it is considered highly likely that the 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. For example, 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.

For truncated variants 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 P450G4 polypeptide.

For truncated variants of the P450G5 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. In a second respect, 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. The term "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.

Homologues include those polypeptide molecules that possess greater than 30% identity with the P450G4 and P450G5 Cytochrome P450 regions, of the P450G4 or P450G5 polypeptides, respectively. 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/) [Blosum 62 matrix; gap open penalty=Tl and gap extension penalty=l]. Preferably, 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.

The 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 Threader™. 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. When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region. For instance, 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. However, several fragments may be comprised within a single larger polypeptide.

The polypeptides of the present invention or their immunogenic fragments (comprising at least one antigenic determinant) can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides. 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. The term "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. As used herein, the term "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.

By "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.

Preferably, the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold, 10³-fold, IO⁴- fold, 10⁵-fold, 10 -fold or greater for a polypeptide of the invention than for known P450 polypeptides.

If polyclonal antibodies are desired, a selected mammal, such as a mouse, rabbit, goat or horse, 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. If desired, 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,

239: 1534 (1988); Rabat et al, J. Immunol., 147: 1709 (1991); Queen et al, Proc. Nati Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Nati Acad. Sci. USA, 88: 34181 (1991); and Hodgson et al, Bio/Technology 9: 421 (1991)). The term "humanised antibody", as used herein, 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.

In a further alternative, 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, whether polyclonal or monoclonal, have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In these applications, 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).

The 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). The term "PNA", as used herein, 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. The terminal lysine confers solubility to the composition. 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. In SEQ ID NO:l the P450G4 Cytochrome P450 region is thus encoded by a nucleic acid molecule including nucleotide 1756 to nucleotide 2838. Nucleic acid molecules encompassing this stretch of sequence, and homologues of this sequence, form a preferred embodiment of this aspect of the invention.

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. In SEQ ID NO:3 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. Such 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.

As discussed above, 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.

Functionally equivalent 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.

Among 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. For example, to screen peptide libraries for inhibitors of the activity of the polypeptide, it may be useful to express, using such a combined nucleic acid molecule, 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). Such 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).

The term "hybridization" as used here 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. [supra]). The inhibition of hybridization of a completely complementary molecule to a target molecule may be examined using a hybridization assay, as known in the art (see, for example, Sambrook et al. [supra]). A substantially homologous molecule will then compete for and inhibit the binding of a completely homologous molecule to the target molecule under various conditions of stringency, as taught in Wahl, G.M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A.R. (1987; Methods Enzymol. 152:507-511).

"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]). Preferably, 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. Accordingly, 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, as referred to herein, 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/).

Preferably, 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. In this regard, 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.

Preferably, 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. In this regard, 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.

As discussed additionally below in connection with assays that may be utilised according to the invention, 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.

In this regard, the following techniques, among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein. Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD). Preferably, 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).

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. Using these probes, 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.

In many cases, 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). Recent modifications of this technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.), for example, have significantly simplified the search for longer cDNAs. A slightly different technique, termed "restriction-site" PCR, uses universal primers to retrieve unknown nucleic acid sequence adjacent a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Inverse PCR may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T., et al. (1988) Nucleic Acids Res. 16:8186). 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.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, 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.

In one embodiment of the invention, the nucleic acid molecules of the present invention may be used for chromosome localisation. In this technique, 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. In addition, 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.

Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene encoding a polypeptide of the invention. RNA interference (RNAi) (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.

Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies. 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.

The 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). Generally, 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. The appropriate 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). Generally, 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.

Examples of 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 (HACs) 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. Introduction of 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). In eukaryotic cells, 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. These 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.

In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of 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. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportl™ 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. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.

The control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.

For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, 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.

In the baculovirus system, the materials for 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. There are many 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. Additional examples of genetic expression in plant cell culture have been described by Zenk, (1991) Phytochemistry 30, 3861-3863. In particular, all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene. Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.

Examples of particularly suitable host cells for fungal expression 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.

Also, 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.

Although the presence or absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed. For example, if the relevant sequence is inserted within a marker gene sequence, transformed cells containing the appropriate sequences can be identified by the absence of marker gene function. Alternatively, 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.

Alternatively, 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).

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing 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. Alternatively, the sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate 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, which may be used for ease of detection, 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. Examples of such 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). The inclusion of 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. 3: 263-281) while the thioredoxin or enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. A discussion of vectors which contain fusion proteins is provided in Kroll, DJ. et al. (DNA Cell Biol. 199312:441-453).

If the polypeptide is to be expressed for use in screening assays, it 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.

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. In general, such 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.

Preferably, 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. Alternatively, 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:

incubating the purified polypeptide, or microsomal preparations, reconstituted systems or cells expressing the polypeptide with putative substrates, for example, but not exlusively, steroids, prostaglandins, fatty acids, retinoic acid, vitamin D derivates, oxysterols, bile. Metabolites may then be separated by methods such as liquid chromatography (LC) followed by their analysis, such as by mass spectrometry.

In an alternative method, 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.

The polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays. Thus, 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. Although 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.

Alternatively, 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. In another embodiment, 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. A preferred method for identifying an agonist or antagonist compound of a polypeptide of the present invention comprises:

(a) contacting a cell expressing on the surface thereof the polypeptide according to the first aspect of the invention, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(b) determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the polypeptide. A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:

(a) contacting a cell expressing on the surface thereof the polypeptide, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(b) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound. 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) contacting a polypeptide immobilized on a solid support, the polypeptide being associated with a second component capable of providing a detectable signal in response to the binding of a compound to the polypeptide, with a compound to be screened under conditions to permit binding to the polypeptide; and

(b) determining whether the compound binds to and activates or inhibits the polypeptide by measuring the level of a signal generated from the interaction of the compound with the polypeptide.

A further preferred method for identifying an agonist or antagonist of a polypeptide of the invention comprises:

(b) determining whether the compound binds to and activates or inhibits the polypeptide by comparing the level of a signal generated from the interaction of the compound with the polypeptide with the level of a signal in the absence of the compound.

In further preferred embodiments, 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.

In another embodiment of 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. Preferably the ligand is labelled.

More particularly, 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,

(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;

(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.

Preferably, 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. Alternatively, 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. For example, human NADPH CYP- reductase may be used to maximise the activity of a P450G3 polypeptide of the invention in assays screening for antagonists.

The polypeptides may be found to modulate a variety of physiological and pathological processes in a dose-dependent manner in the above-described assays. Thus, 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. Although 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. In certain of the embodiments described above, 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. In another embodiment, 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. For example, 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). In 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.

Examples of suitable assays for the identification of agonists or antagonists of the polypeptides of the invention are described in Rosen et al, Curr. Opin. Drug Discov. Devel. 2003 6(2):224-30.

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). 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.

The invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.

According to the terminology used herein, 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. Preferably, 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.

The pharmaceutical compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention. The term "therapeutically effective amount" as used herein 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. For any compound, 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.

The precise 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. 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. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ. 1991). 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 the patient.

Once formulated, the 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.

The pharmaceutical 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. Typically, 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.

If the activity of the polypeptide of the invention is in excess in a particular disease state, several approaches are available. 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. Preferably, such antagonists are antibodies. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously. In another approach, soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered. Typically, the polypeptide may be administered in the form of fragments that retain the relevant portions.

In an alternative approach, 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. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). 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.

In addition, expression of the polypeptide of the invention may be prevented by using ribozymes specific to its encoding mRNA sequence. 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.

For treating abnormal conditions related to an under-expression of the polypeptide of the invention and its activity, several approaches are also available. 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. Alternatively, 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 contrast, 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. For example, 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.

In situations in which the polypeptides or nucleic acid molecules of the invention are disease-causing agents, 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"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.

Since polypeptides may be broken down in the stomach, vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for 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. For example, 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.

Genetic delivery of antibodies that bind to polypeptides according to the invention may also be effected, for example, as described in International patent application WO98/55607.

The technology referred to as jet injection (see, for example, www.powderject.com) may also be useful in the formulation of vaccine compositions.

A number of suitable methods for vaccination and vaccine delivery systems are described in International patent application WO00/29428. This invention also relates to the use of 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.

In one embodiment, 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.

To aid the detection of nucleic acid molecules in the above-described methods, 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.

Such diagnostics are particularly useful for prenatal and even neonatal testing. 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)). For example, 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. Further, 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).

In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al., DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane. Fluorescence in situ hybridization (FISH) is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250: 559-562 (1990), and Trask et al, Trends, Genet. 7:149-154 (1991)). In another embodiment of the invention, 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).

In one embodiment, 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. In another aspect, 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). In another aspect, 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.

In addition to the methods discussed above, 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. 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. 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) a nucleic acid molecule of the present invention;

(b) a polypeptide of the present invention; or (c) a ligand of the present invention.

In one aspect of the invention, 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.

In an alternative aspect of the invention, 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. To detect polypeptide 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.

Such 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. In particular, 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. Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the P450G4 and P450G5 polypeptides.

It will be appreciated that modification of detail may be made without departing from the scope of the invention. Brief description of the Figures

Figure 1 : Front page of the Biopendium™. Search initiated using 1 JIO:A.

Figure 2A: Inpharmatica Genome Threader™ 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.

Figure 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 Threader™ 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 Threader™ alignment of T00364 (P450G4) and 1 JIO.A.

Figure 7: Front page of the Biopendium™. Search initiated using 1DZ4:A.

Figure 8 A: Inpharmatica Genome Threader™ 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 Threader™ 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.

Figure 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 Threader™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 Threader™ 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 14: P450G4 tissue distribution

Figure 15: P450G4 cell line distribution

Figure 16: P450G4 foetal tissue distribution Figure 17: P450G5 tissue distribution

Figure 18: P450G5 cell line distribution

Figure 19: P450G5 foetal tissue distribution

Figure 20: Reduced CO-difference spectral studies on G4-P450 domain sample

Figure 21: Reduced CO-difference spectral studies on G5-P450 domain sample. Total protein concentration: 0.337mg/ml

Examples

Example 1: T00364 (P450G4)

In order to initiate a search for novel, distantly related Cytochrome P450s, an archetypal Cytochrome P450 family member, Saccharopolyspora erythraea Cytochrome P450ERYF 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.

The structure chosen is Saccharopolyspora erythraea Cytochrome P450ERYF, PDB code 1JIO.A (Figure 1). A search of the Biopendium™ for homologues of 1 JIO:A takes place and returns 3618 Inpharmatica Genome Threader™ results (selection given in Figure 2 A and 2B) and 2270 Inpharmatica PSI-Blast results (selection in Figure 2C and 2D). The 3618 Genome Threader™¹ results include examples of other Cytochrome P450 family members, such as Streptomyces spheroides Cytochrome P450 Novl (Figure 2 A, arrow). Among the known Cytochrome P450 members appears a protein of apparently unknown function, T00364 (P450G4, Figure 2B, arrow).

The Inpharmatica Genome Threader™ 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 Threader™ 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).

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. The identification of a relationsliip between lJIO:A and T00364 (P450G4) in Inpharmatica PSI-Blast iteration -4 at a highly significant E-value of 2.0E-62 strongly supports the Genome Threader annotation of T00364 (P450G4) being a Cytochrome P450.

In order to view what is known in the public domain secondary databases about T00364

(P450G4), 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.

In order to view what is known in the public domain secondary databases, the NCBI Conserved 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. Querying NCBI PSI-Blast with T00364 (P450G4) through 10 positive iterations fails to annotate any region of T00364 (P450G4) as having a statistically significant 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 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. One of these sequences, AAG29781.1 Cytochrome P450 Novl, which has been annotated in the public domain as containing a Cytochrome P450 domain, is returned in the NCBI PSI-blast results. However, 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. Thus NCBI PSI-Blast does not annotate any region of T00364 (P450G4) as having a statistically significant relationship to any known Cytochrome P450.

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.

Only the Inpharmatica Genome Threader™ and Inpharmatica PSI-Blast are able to annotate this protein as a Cytochrome P450 family member. The reverse search is now carried out. T00364 (P450G4) is now used as the query sequence in the Biopendium™. The Inpharmatica Genome Threader™¹ identifies 134 hits (Figure 6A) while Inpharmatica PSI-Blast returns 9544 hits (Figure 6B). The Inpharmatica Genome Threader™ (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.

Thus a region from residue 586 to residue 946 of T00364 (P450G4) has been identified as adopting an equivalent fold to Saccharopolyspora erythraea Cytochrome P450ERYF.

Inpharmatica PSI-Blast also identifies the same region of T00364 (P450G4) as having a relationship with known Cytochrome P450s by the third positive iteration. For example, 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. The results indicate that residues 587-946 of T00364 (P450G4) 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 Threader™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). Thus Inpharmatica PSI-Blast is in strong agreement with Inpharmatica Genome Threader™ 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 Threader™ 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. Among the Cytochrome P450 family members that the Inpharmatica Genome Threader™ returns is the original input query Saccharopolyspora erythraea Cytochrome P450ERYF (UIO:A). lJIO:A is chosen (arrow, figure 6A) against which to view the sequence alignment of T00364 (the P450G4 polypeptide). Viewing the alignment (Figure 6D) of the query protein against the protein identified as being of a similar structure helps to visualize the areas of homology.

To summarise Cytochrome P450 annotation, only Inpharmatica Genome Threader™ can identify that residues 586-946 of T00364 (P450G4) folds in a similar manner to 1 JIO:A (Saccharopolyspora erythraea Cytochrome P450ERYF) and as such is identified as being a novel Cytochrome P450. This annotation is also strongly supported by Inpharmatica PSI- Blast sequence-sequence relationships which relate this region of T00364 (P450G4) to known Cytochrome P450s.

Example 2: BAB13458.1 (P450G5) In order to initiate a search for novel, distantly related Cytochrome P450s, 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.

The structure chosen is Pseudomonas putida Cytochrome P450CAM, PDB code 1DZ4:A (Figure 7). A search of the Biopendium™ for homologues of 1DZ4:A takes place and returns 5560 Inpharmatica Genome Threader™ results (selection given in Figure 8A and 8B) and 2198 Inpharmatica PSI-Blast results (selection in Figure 8C and 8D). The 5560 Genome Threader™ results include examples of other Cytochrome P450 family members, such as Streptomyces spheroides Cytochrome P450 Novl (Figure 9A, arrow). Among the known Cytochrome P450 members appears a protein of apparently unknown function, BAB 13458.1 (P450G5, Figure 8B, arrow).

The Inpharmatica Genome Threader™ 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 Threader™ 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).

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 Threader™ that is able to identify a structural relationship between 1DZ4:A and BAB13458.1 (P450G5). It is only Inpharmatica Genome Threader™that is able to identify BAB13458.1 (P450G5) as being a Cytochrome P450.

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. In order to view what is known in the public domain secondary databases, the NCBI Conserved Domain Database (CDD) is queried with 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. None of these sequences have been annotated in the public domain as containing a Cytochrome P450 domain. None of the entries returned in NCBI PSI-Blast have been annotated in the public domain as containing a Cytochrome P450 domain. Thus NCBI PSI-Blast does not' annotate any region of BAB 13458.1 (P450G5) as having a relationship to any known Cytochrome P450.

There is no further annotation for BAB13458.1 (P450G5). The public domain information for this protein does not annotate it as containing a Cytochrome P450 domain (Figure 11). Only the Inpharmatica Genome Threader™¹ is able to annotate this protein as a Cytochrome P450 family member.

The reverse search is now carried out. BAB 13458.1 (P450G5) is now used as the query sequence in the Biopendium™. The Inpharmatica Genome Threader™¹ identifies 180 hits (Figure 12 A) while mpharmatica PSI-Blast returns 12 hits (Figure 12B). The Inpharmatica Genome Threader™ (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.

Thus a region from residue 1112 to residue 1350 of BAB13458.1 (P450G5) has been identified as adopting an equivalent fold to Pseudomonas putida Cytochrome P450CAM. Inpharmatica PSI-Blast does not identifiy any region of BAB 13458.1 (P450G5) as having a relationship with known Cytochrome P450s in any iteration (Figure 12B).

Among the Cytochrome P450 family members that the hipharmatica Genome Threader™¹ returns is the original input query Saccharopolyspora erythraea Cytochrome P450ERYF (1JIO.A). 1JI0:A is chosen (arrow, figure 12A) against which to view the sequence alignment of T00364 (the P450G4 polypeptide). Viewing the alignment (Figure 6D) of the query protein against the protein identified as being of a similar structure helps to visualize the areas of homology. Several of the highly conserved Cytochrome P450 residues are present in the BAB13458.1 (P450G5) protein. Threonine 1239 in BAB13458.1 (P450G5) 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.

To summarise Cytochrome P450 annotation, only Inpharmatica Genome Threader™ can identify that residues 1112-350 of BAB13458.1 (P450G5) folds in a similar manner to 1DZ4:A (Pseudomonas pituda Cytochrome P450CAM) and as such is identified as being a novel Cytochrome P450.

Example 3: Cloning of P450G4

A. 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 μl of the reaction was used in the subsequent PCR. Cloning of the P450G4 was performed using human brain and testis cDNA

B. Cloning of the P450 domain of P450G4 Primers 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 MgCl₂.

The primer sequences were: P450G4 F

GTCGTGGCAACTGAATACGAGCAGG

P450G4R TGGAAATGTGTGCCAGGGCGGCAGG

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.

The expression primers were as follows:

P450G4express F GGAATTCCATATGCATCATCATCATCATCATGTCGTGGCAACTGAATAC

P450G4express R CGGGATCCTCAATGATGATGATGATGATGTGGAAATGTGTGCCAGGG

Example 4: Cloning of P450G5

A. cDNA source for PCR of P450G5

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 μl of the reaction was used in the subsequent PCR. Cloning of the P450G5 was performed using human brain and placenta cDNA

B. Cloning of the P450 domain of P450G5

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 MgCl₂.

The primer sequences were:

P450G5 F CAACAGGTCACCCACAAGGTGGCAC

P450G5 R 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.

The expression primers were as follows:

P450G5express F GGAATTCCATATGCATCATCATCATCATCATCAACAGGTCACCCACAAG

P450G5express R CGGGATCCTCAATGATGATGATGATGATGAAACCTTCTTCCAATACA

Example 5: Transcript profiling data for P450G4

In order to determine the tissue expression of the proposed P450, 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. Using linear regression analysis of the standard curves, the 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, 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.

These data (Figure 14) demonstrate that P450G4 is expressed at highest levels in the brain. Transcript levels were between 15 and 25 fold higher in the brain than in the stomach. Relative high levels of expression were also found in the testis, thymus, ovary, lung and bladder. Additional samples were analysed for transcript levels (data not shown). Low levels of transcript were identified in normal skin and normal breast samples. Surprisingly, low levels were found in the adult kidney (Figure 15). The gene encoding P450G4 has been identified as being mutated in juvenile nephronophthisis type 4 (Mollet et al, Nature Genetics, 2002, 300-5).

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. Using linear regression analysis of the standard curves, the 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.

These data (Figure 15) demonstrate that P450G4 is expressed in all cell types tested except SK-N-SH. Levels were less variable amongst the cell types although highest relative levels of expression were observed HL60 (myeloid cell line) and Jurkat cells (T cell line). The absence of transcript in SK-N-SH is curious. These cells are derived from a neuroblastoma. SK-N-MC cells by contrast had relatively abundant levels of P450G4. This cell line is derived from a neuroepithelioma. Levels of transcript were not significantly altered in certain cell lines by pharmacological manipulations which included dibutyryl cAMP, interferon-γ, phorbol esters or retinoic acid.

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.

These data (Figure 16) demonstrate that P450G4 is expressed in the foetal and embryonic tissues tested. Highest relative levels of expression were observed in the foetal brain sample. P450s are involved in the synthesis of metabolically active agents such as steroids, prostaglandins, leukotrienes, retinoic acids. The presence of a novel P450 in these embryonic and foetal tissues is consistent with the proposed role for this enzyme in the synthesis and turnover of metabolically active compounds involved in embryogenesis, tissue differentiation and growth and synthesis of steroids for maintaining pregnancy. The placenta sample here is derived from a pre-term placenta at 20 weeks.

Materials and Methods A. RNA samples

Human RNA prepared from non-diseased organs was purchased from either Ambion Europe (Huntingdon, UK) or Clontech (BD, Franklin Lakes, NJ).

Cells RNA samples

4 samples of total RNA (G-401, PC-3, SaOs2 and T-24) were from Ambion Europe (Huntingdon, UK). All the other treated or untreated total RNA were extracted from cells using GeneElute™ Mammalian Total RNA kit from Sigma. Cells had been cultured according to the recommendation from EACC (European Collection of Cell Cultures).

Foetal tissues RNA

Foetal total RNA all came from Biochain - AMS technology (Abingdon, UK) B. Oligo Design

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.

Each primer and probe was analysed using BLAST^® (Basic Local Alignment Search Tool, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol. 1990 Oct 5;215(3):403-10). Results confirmed that each oligonucleotide recognised the target sequence with a specificity >3 bp when compared to other known cDNAs or genomic sequence represented in the Unigene and GoldenPath publicly available databases.

The sequence of the primers and probes were: P450G4 Fwd CCCGTGTGAACAGAAAGTGAGA

P450G4 Probe TTGTAGCACATTGCCACCGTCC

P450G4 Rev CGTTTGAGATGACCCGAGATC 18s pre-optimised primers and probe were purchased from Applied Biosystems, Foster City, CA.

Probes were covalently conjugated with a fluorescent reporter dye (e.g. όcarboxy- fluorescein [FAM]; Xem = 518nm) and a fluorescent quencher dye (e.g. 6carboxytetram- ethyl-rhodamine [TAMRA]; Mem = 582nm) at the most 5' and most 3' base, respectively. Primers were obtained from Sigma Genosys, UK and probes were obtained from Eurogentec, Belgium.

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.

C. cDNA reaction cDNA was prepared using components from Applied Biosystems, Foster City CA. 50μl reactions are prepared in 0.5ml RNase free tubes. Reactions contain 500ng total RNA; lx reverse transcriptase buffer; 5.5mM MgC12; ImM dNTP's; 2.5μl random hexamers; 20U RNase inhibitor; and 62.5U reverse transcriptase.

D. PCR reactions:

25μl 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.

E. Performance of Assay Standard procedures for the operation of the ABI Prism 7700 or similar detection system were used. This included, for example with the ABI Prism 7700, use of all default program settings with the exception of reaction volume which is changed from 50 to 25 ul. Thermal cycling conditions consisted of two min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. 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 automatically performed by the instrument for each reaction using default parameters. Assays for target sequences and ribosomal 18s (reference) sequences in the same cDNA samples were performed in separate reaction tubes.

Within each experiment, a standard curve was carried out of a typical tissue sample, from 25ng to 0.39ng of cDNA template. From this standard curve, the amount of actual starting target or 18s cD A in each test sample was determined. The levels of target cDNA in each sample were normalised to the level of expression of target in a comparative sample. The levels of internal control cDNA in each sample were normalised to the level of expression of internal control in a comparative sample. The data was then represented as fold expression of normalised target sequence relative to the level of expression in the comparative sample, which is set arbitrarily to 1. Example 6: Transcript profiling data for P450G5

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. Using linear regression analysis of the standard curves, the 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, 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.

These data (Figure 17) demonstrate that transcripts could be found in all tissues. Highest levels of expression were found in the brain, placenta and thymus. Levels were also relatively high in ovary and testis. Additional samples were analysed for transcript levels (data not shown). Low levels of transcript were identified in normal skin and normal breast samples. In one sample of psoriatic skin, the level of expression was found to be significantly reduced compared to matched normal skin.

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. Using linear regression analysis of the standard curves, the 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.

These data demonstrate that the 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-γ, 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.

This data (Figure 19) demonstrates that the transcript for P450G5 could be identified in all the tissue samples. Highest relative expression levels were observed in pre-term placenta (20 weeks) and foetal spleen. P450s are involved in the synthesis of metabolically active agents such as steroids, prostaglandins, leukotrienes, retinoic acids. The presence of a novel P450 in these embryonic and foetal tissues is consistent with the proposed role for this enzyme in the synthesis and turnover of metabolically active compounds involved in embryogenesis, tissue differentiation and growth and synthesis of steroids for maintaining pregnancy. The materials and methods used were as given above for P450G4, except for the primers and probe.

The sequence of the primers and probes were:

P450G5 Fwd CAGTGCTCAACATGGAATCCA

P450G5 Probe CTTTGAAGAGGACTCCCAGCTCCGGA P450G5 Rev GTTTATCACCAATTCCCCTTCAAT

Example 7: Assay principles and constructs for P450G4

A. P450 Bacterial expression

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 120μg/ml ampicillin. 1 ml of this culture was added to 100ml Luria broth medium containing 120μg/ml ampicillin and 34μg/ml of chloramphenicol in a 1 litre flask. The culture was shaken at 270 rpm, 37°C for 3 hours. Heme precursor: δ-aminolevulinic acid was added to a final concentration of 80μg/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. Growth was allowed to continue for 24hr at 30°C, at a shaking rate of 120rpm. Cells were harvested by centrifugation at 2500g for 10 min at 4°C, washed with buffer A (lOniM potassium phosphate (pH 7.4), O.lmM EDTA), re-suspended in buffer A, and stored frozen at -80°C. B. Protein Extraction

After thawing, PMSF was added to a concentration of ImM, the cells were disrupted by sonication on ice, 4 x 30 seconds, and an equal volume of buffer A containing 40% glycerol was added. The suspension was centrifuged at 6000rpm for 15 min at 4°C. After 5 centrifugation the pellet was resuspended in buffer B (67mM potassium phosphate pH 8.0, 20% glycerol, ImM PMSF, ImM dithiothreitol and 0.2M NaCI) and sonicated again. Then the suspension was centrifuged again at 6000rpm for 15 min at 4°C. 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:

Tween 20, Nonidet P40, Triton 100, Chaps and Renex overnight at 4°C with stirring. 15 Then a centrifugation at 30,000g for 60min at 4°C, gave a supernatant containing solubilised protein.

C. P450 spectrum measurement

For the analysis the light absorption was recorded between 400nm and 600nm in a spectrophometer DU640.

20. 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

25 without was recorded. The result is shown in Figure 20.

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".

Example 8: Assay principles and constructs for P450G5

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 120μg/ml ampicillin. 1 ml of this culture was added to 100ml Luria broth medium containing 120μg/ml ampicillin and 34μg/ml of chloramphenicol in a 1 litre flask. The culture was shaken at 270 rpm, 37°C for 3 hours. Heme precursor: δ-aminolevulinic acid was added to a final concentration of 80μg/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.Growth was allowed to continue for 24hr at 30°C, at a shaking rate of 120rpm. Cells were harvested by centrifugation at 2500g for 10 min at 4°C, washed with buffer A (lOmM potassium phosphate (pH 7.4), O.lmM EDTA), re-suspended in buffer A, and stored frozen at -80°C. B. Protein Extraction

After thawing, PMSF was added to a concentration of ImM, the cells were disrupted by sonication on ice, 4 x 30 second, and an equal volume of buffer A containing 40% glycerol was added. The suspension was centrifuged at 6000rpm for 15 min at 4°C. After centrifugation the pellet was resuspended in buffer B (67mM potassium phosphate pH 8.0, 20% glycerol, ImM PMSF, ImM dithiothreitol and 0.2M NaCI) and sonicated again. Then the suspension was centrifuged again at 6000rpm for 15 min at 4°C. 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:

Tween 20, Nonidet P40, Triton 100, Chaps and Renex for overnight at 4°C with stirring. Then a centrifugation at 30,000g for 60min at 4°C, gave a supernatant containing solubilised protein.

C. P450 spectrum measurement

The total protein concentration was determined by a Bradford assay. For the analysis 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.

The UV spectrum of the G5-P450 reduced Carbon monoxide adduct gives the characteristic Soret absorption max of the P450 around 450nm (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.

The purpose of these experiments is to determine the specific substrates for the P450G4 and P450G5. To this end, 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.

An alternative method of expression uses the baculovirus/insect system. The 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.

Substrate determination using LC/MS

A series of putative endogenous substrates (e.g. steroids, prostaglandins, fatty acids, retinoic acid, vitamin D derivates, oxysterols, bile acids) as well as a number of small molecules are added to the reconstituted P450 system. The protein mixtures can then be separated by LC and analysed by mass spectrometry. For example 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

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.

By addition of unlabelled compound libraries a competition assay is carried out to identify putative inhibitors of the P450 protein. Examples of these experiments can be found in Roman, R.J., (2002) 82 Physiol. Rev.

List of T00364 and BAB 13458.1 specific sequences

(Sequence Listing)

SEQ ID NO:l (Nucleotide coding sequence for T00364 (P450G4) protein)

1 cagcagatac ctggcctgct tccagctcat ggagaatccg gcgacgctct ccgaaagcct 61 cgcctccaga agcccatcac ggggcacttg gatgacttat tcttcaccct gtacccctcc

121 ctggagaagt ttgaggaaga gctgctggag ctccacgtcc aggaccactt ccaggaggga

181 tgtggcccac tggacggtgg tgccctggag atcctggagc ggcgcctgcg tgtgggcgtg

241 cacaatggtc tgggcttcgt gcagaggccg caggtcgttg tactggtgcc tgagatggat

301 gtggccttga cgcgctcagc tagcttcagc aggaaagtgg tctcctcttc caagaccagc 361 tccgggagcc aagctctggt tttgagaagc cgcctccgcc tcccagagat ggtcggccac

421 cctgcatttg cggtcatctt ccagctggag tacgtgttca gcagccctgc aggagtggac

481 ggcaatgcag cttcggtcac ctctctgtcc aacctggcat gcatgcacat ggtccgctgg

541 gctgtttgga accccttgct ggaagctgat tctggaaggg tgaccctgcc tctgcagggt

601 gggatccagc ccaacccctc gcactgtctg gtctacaagg taccctcagc cagcatgagc 661 tctgaagagg tgaagcaggt ggagtcgggt acactccggt tccagttctc gctgggctca

721 gaagaacacc tggatgcacc cacggagcct gtcagtggcc ccaaagtgga gcggoggcct

781 tccaggaaac cacccacgtc cccttcgagc ccgccagcgc cagtacctcg agttctcgct

841 gccccgcaga actcacctgt gggaccaggg ttgtcaattt cccagctggc ggcctccccg

901 cggtccccga ctcagcactg cttggccagg cctacttcac agctacccca tggctctcag 961 gcctccccgg cccaggcaca ggagttcccg ttggaggccg gtatctccca cctggaagcc

1021 gacctgagcc agacctccct ggtcctggaa acatccattg ccgaacagtt acaggagctg

1081 ccgttcacgc ctttgcatgc ccctattgtt gtgggaaccc agaccaggag ctctgcaggg

1141 cagccctcga gagcctccat ggtgctcctg cagtcctccg gctttcccga gattctggat

1201 gccaataaac agccagccga ggctgtcagc gctacagaac ctgtgacgtt taaccctcag 1261 aaggaagaat cagattgtct acaaagcaac gagatggtgc tacagtttct tgcctttagc

1321 agagtggccc aggactgccg aggaacatca tggccaaaga ctgtgtattt caccttccag

1381 ttctaccgct tcccacccgc aacgacgcca cgactgcagc tggtccagct ggatgaggcc

1441 ggccagccca gctctggcgc cctgacccac atcctcgtgc ctgtgagcag agatggcacc

1501 tttgatgctg ggtctcctgg cttccagctg aggtacatgg tgggccctgg gttcctgaag 1561 ccaggtgagc ggcgctgctt tgcccgctac ctggccgtgc agaccctgca gattgacgtc

1621 tgggacggag actccctgct gctcatcgga tctgctgccg tccagatgaa gcatctcctc

1681 cgccaaggcc ggccggctgt gcaggcctcc cacgagcttg aggtcgtggc aactgaatac

1741 gagcaggaca acatggtggt gagtggagac atgctggggt ttggccgcgt caagcccatc

1801 ggcgtccact cggtggtgaa gggccggctg cacctgactt tggccaacgt gggtcacccg 1861 tgtgaacaga aagtgagagg ttgtagcaca ttgccaccgt ccagatctcg ggtcatctca

1921 aacgatggag ccagccgctt ctctggaggc agcctcctca cgactggaag ctcaaggcga

1981 aaacacgtgg tgcaagcaca gaagctggcg gacgtggaca gtgagctggc tgccatgcta

2041 ctgacccatg cccggcaggg caaggggccc caggacgtca gccgcgagtc ggatgccacc

2101 cgcaggcgta agctggagcg gatgaggtct gtgcgcctgc aggaggccgg gggagacttg 2161 ggccggcgtg ggacgagcgt gttggcgcag cagagcgtcc gcacacagca cttgcgggac

2221 ctacaggtca tcgccgccta ccgggaacgc acgaaggccg agagcatcgc cagcctgctg

2281 agcctggcca tcaccacgga gcacacgctc cacgccacgc tgggggtcgc cgagttcttt

2341 gagtttgtgc ttaagaaccc ccacaacaca cagcacacgg tgactgtgga gatcgacaac 2401 cccgagctca gcgtcatcgt ggacagtcag gagtggaggg acttcaaggg tgctgctggc

2461 ctgcacacac cggtggagga ggacatgttc cacctgcgtg gcagcctggc cccccagctc

2521 tacctgcgcc cccacgagac cgcccacgtc cccttcaagt tccagagctt ctctgcaggg

2581 cagctggcca tggtgcaggc ctctcctggg ttgagcaacg agaagggcat ggacgccgtg

2641 tcaccttgga agtccagcgc agtgcccact aaacacgcca aggtcttgtt ccgagcgagt 2701 ggtggcaagc ccatcgccgt gctctgcctg actgtggagc tgcagcccca cgtggtggac

2761 caggtcttcc gcttctatca cccggagctc tccttcctga agaaggccat ccgcctgccg

2821 ccctggcaca catttccagg tgctccggtg ggaatgcttg gtgaggaccc cccagtccat

2881 gttcgctgca gcgacccgaa cgtcatctgt gagacccaga atgtgggccc cggggaacca

2941 cgggacatat ttctgaaggt ggccagtggt ccaagcccgg agatcaaaga cttctttgtc 3001 atcatttact cggatcgctg gctggcgaca cccacacaga cgtggcaggt ctacctccac

3061 tccctgcagc gcgtggatgt ctcctgcgtc gcaggccagc tgacccgcct gtcccttgtc

3121 cttcggggga cacagacagt gaggaaagtg agagctttca cctctcatcc ccaggagctg

3181 aagacagacc ccaaaggtgt cttcgtgctg ccgcctcgtg gggtgcagga cctgcatgtt

3241 ggcgtgaggc cccttagggc cggcagccgc tttgtccatc tcaacctggt ggacgtggat 3301 tgccaccagc tggtggcctc ctggctcgtg tgcctctgct gccgccagcc gctcatctcc

3361 aaggcctttg agatcatgtt ggctgcgggc gaagggaagg gtgtcaacaa gaggatcacc

3421 tacaccaacc cctacccctc ccggaggaca ttccacctgc acagcgacca cccggagctg

3481 ctgcggttca gagaggactc cttccaggtc gggggtggag agacctacac catcggcttg

3541 cagtttgcgc ctagtcagag agtgggtgag gaggagatcc tgatctacat caatgaccat 3601 gaggacaaaa acgaagaggc attttgcgtg aaggtcatct accagtga

SEQ ID NO:2 (Protein T00364; P450G4)

1 qqipgllpah gesgdalrkp rlqkpitg l ddlfftlyps lekfeeelle lhvgdhfgeg 61 cgpldggale ilerrlrvgv nglgfvqrp qwvlvpemd valtrsasfs rkwssskts 121 sgsqalvlrs rlrlpemvgh pafavifqle yvfsspagvd gnaasvtsls nlacrnh vrw 181 avwnpllead sgrvtlplqg giqpnpshcl vykvpsasms seevkqvesg tlrfqfslgs 241 eehldaptep vsgpkverrp srkpptspss ppapvprvla apqnspvgpg Isisqlaasp 301 rsptqhclar ptsqlphgsq aspaqaqefp leagishlea dlsqtslvle tsiaeqlqel 361 pftplhapiv vgtqtrssag qpsrasmvll qssgfpeild ankqpaeavs atepvtfnpq 421 keesdclqsn emvlqflafs rvaqdcrgts wpktvyftfq fyrfppattp rlqlvqldea 481 gqpssgalth ilvpvsrdgt fdagspgfql rymvgpgflk pgerrcfary lavqtlqidv 541 dgdslllig saavgmkhll rqgrpavqas helewatey eqdnmwsgd lgfgrvkpi 601 gvhswkgrl ltlanvghp ceqkvrgcst lppsrsrvis ndgasrfsgg sllttgssrr 661 khwqaqkla dvdselaaml ltharqgkgp qdvsresdat rrrklermrs vrlqeaggdl 721 grrgtsvlaq qsvrtqhlrd lqviaayrer tkaesiasll slaittehtl atlgvaeff 781 efvlknp nt qhtvtveidn pelsvivdsq e rdfkgaag lhtpveedmf hlrgslapql

841 ylrphetahv pfkfqsfsag qlamvqaspg Isnekgmdav sp kssavpt khakvlfras

901 ggkpiavlcl tvelqphwd q frfy pel sflkkairlp p htfpgapv gmlgedppvh

961 vrcsdpnvic etqnvgpgep rdiflkvasg pspeikdffv iiysdr lat ptqtwqvyl 1021 slqrvdvscv agqltrlslv lrgtqtvrkv raftshpqel ktdpkgvfvl pprgvqdlhv

1081 gvrplragsr fv lnlvdvd chqlvaswlv clccrqplis kafeimlaag egkgvnkrit

1141 ytnpypsrrt fhl sdhpel lrfredsfqv gggetytigl qfapsqrvge eeiliyindh

1201 edkneeafcv kviyq

SEQ ID NO:3 (the nucleotide coding sequence for BAB13458.1 (P450G5) protein)

1 aaaacaaacg gtgacggcgc cgcggaaggg tctatggccg aggcggtgaa gccccagcgc

61 cgggccaagg ccaaggccag ccggactaaa acaaaggaaa agaagaagta tgaaactcct

121 cagagggaag agtccagtga agtctccctt ccaaaaacct ccagagagca ggaaatccct

181 tctctagcct gtgaattcaa aggagaccat ctgaaggtgg taactgattc ccagctccag 241 gatgatgcca gtggacaaaa tgagagtgaa atgtttgatg taccactcac ctccttaact

301 ataagcaatg aagagtccct gacgtgtaac acagagcccc caaaggaagg gggagaggcc

361 agaccctgtg tgggggacag tgcagtcact ccaaaggtcc accctggaga caatgttgga

421 actaaagtag aaacccccaa gaacttcaca gaggtagagg aaaatatgtc ggtacaaggt

481 ggactttcag aaagtgcacc ccaatctaat ttttcttata ctcagccagc aatggaaaat 541 atacaagtca gagaaactca gaatagtaaa gaagacgaac aaggcctggt ttgttcttca

601 gaggtgccac agaatgttgg cttgcagagt tcttgcccag ccaaacatgg ttttcagaca

661 cctagagtga agaaactgta tccccagttg ccagctgaaa ttgctggaga agcaccagct

721 ttggtggcag tgaaaccctt gcttcgcagt gagcgactct acccagaact cccgtctcaa

781 ctggaactag taccatttac taaagaacag ctaaaaatct tggagcctgg ttcatggctg 841 gaaaatgttg agtcatattt agaagaattt gacagcatgg ctcatcaaga caggcatgaa

901 ttttatgagt tgcttttgaa ctactcacga tgtaggaagc aactgctgct ggctgaagct

961 gagctgctta ctctgacatc tgattgccaa aatgctaaaa gtcggctgtg gcagtttaag

1021 gaggaacaaa tgtctgtaca gggtatctgt gcagatcaag tgaaagtttt cagctatcat

1081 cgctaccaaa gagtagaaat gaatgaaaat gcactggtgg agctaaagaa gctattcgat 1141 gccaaatctg agcacctcca ccagaccctg gcccttcatt cttatacttc tgtgctctca

1201 agattgcaag tggagtctta catctatgca ttgctcagta gttcagctgt tctgagatct

1261 tcagcaattc accagcaagg ccgagcgtct aagcagacag aaagcattcc ctctgatctg

1321 tgtcaactaa aggaatgcat tagtgtctta ttcatgttca ccaggagagt taatgaagat

1381 actcagtttc atgatgatat tcttctctgg ctgcagaaat tggtatccgt gctacaaaga 1441 gttggctgtc ctggagatca cctcttcctt ctaaaccata ttcttcgatg ccccgctggt

1501 gttagtaaat gggctgttcc ttttatccag atcaaagtgt tgcataaccc atcaggggtc

1561 tttcatttta tgcaatccct tgccctgctg atgtctcctg tcaaaaatcg agctgagttt

1621 atgtgccaca tgaagcccag cgagcggaag ccatcctcct cagggcctgg gtctgggact

1681 tggacgctag tagacgaagg aggagaagag gatgaagacc ctgagaccag ttggattctc 1741 cttaatgaag atgatttggt taccatttta gcacagttcc cctttcatga actctttcag 1801 catcttcttg ggtttaaagc aaaaggtgat tatttacctg aaacaacaag acctcaagag

1861 atgatgaaaa tttttgcctt tgccaactca ctagtggaac ttctggctgt ggggttagaa

1921 acctttaata gagcacgcta taggcagttt gtgaagcgaa ttggttatat gatcaggatg

1981 actcttggtt atgtaagtga ccattgggca cagtatgtga gccataacca aggctcagga 2041 ttggcccaac agccctactc tatggagaaa ctacaggttg aatttgatga actgtttttg

2101 agggctgtcc tacatgtgct gaaggccaaa agacttggca tttggctgtt tatgtccgag

2161 atgccctttg ggactctgtc tgtgcaaatg ctgtggaagc tcttctacct catgcaccag

2221 gtggagagcg agaacctgca gcagctctcc tcctccctgc agcccgccca gtgcaagcag

2281 cagctccaag acccagaaca ttttaccaac tttgagaagt gtctttcttc tatgaatagc 2341 tcagaagaga tttgccttct gactaccttt gctcagatgg ctcaggccag aagaaccaat

2401 gtggacgaag acttcataaa aattattgtt ctggagatat atgaggtatc ttatgtcacc

2461 ttgtctacca gagagacttt ttcaaaggtt ggtcgagagc ttttagggac tatcacagct

2521 gttcaccctg agataatttc cgtccttctg gatagagttc aagagaccat tgaccaggtt

2581 ggaatggttt ctctatactt gtttaaagaa ctgcctttgt atctttggca accttctgca 2641 tctgagatag cggtgattcg ggattggtta ttgaattaca acctgacagt ggtgaagaat

2701 aaactggcct gtgttattct tgaaggactg aattggggat ttgctaaaca ggctaccctt

2761 catctggatc aagcagtcca tgctgaagtg gctttaatgg ttcttgaagc ttatcagaag

2821 taccttgcac agaagccata tgctgggatt ctctctgaaa gtatgaagca ggtttcatat

2881 ttggccagta ttgttcgata tggagagaca cccgagacct catttaacca atgggcctgg 2941 aatttaatct tgaggctaaa actgcacaaa aacgattatg gaatacagcc gaattgtcca

3001 gctgttccct tctccgtcac tgtgcctgac atgacagagt cacccacgtt tcatcctctc

3061 ttgaaggctg tgaaagcggg catgcccatt ggctgttatc tagccttatc tatgacagct

3121 gtgggccaca gcattgagaa gttctgtgca gaaggcatcc cactattggg aattctggtc

3181 cagtcaagac atttgagaac agtggttcat gtcctggata agattctgcc tttattttac 3241 ccttgccagt attaccttct gaagaatgag cagtttttat cgcatctcct cttgttccta

3301 cacttggaca gcggtgtccc tcagggtgtc acacaacagg tcacccacaa ggtggcacag

3361 cacctgacag gagccagcca tggggacaac gtgaagcttc tcaacagcat gatccaggca

3421 cacatatctg taagcactca gcccaatgaa gtgggccccg ttgctgtgtt ggagttctgg

3481 gttcaggctc tcataagcca gcatctctgg taccgagaac aacctatcct cttcctcatg 3541 gaccacttgt gtaaagcagc ttttcagctg atgcaggaag actgcataca gaaattactc

3601 taccaacaac ataagaatgc cttgggttac cactgtgacc ggagtctgct ctcatctttg

3661 gtaagctgga ttgtggcagg caacatcact ccttcctttg tggagggctt ggccacgccc

3721 actcaggtct ggtttgcctg gacagtgctc aacatggaat ccatctttga agaggactcc

3781 cagctccgga gagttattga aggggaattg gtgataaact ctgctttcac ccctgaccaa 3841 gctctgaaga aagcccagac ccagctgaag ctccccatcg tgccatccct ccagaggctg

3901 ctgatttatc gctgggccca ccaggctctg gtcacacctt ctgatcaccc cctcctgcca

3961 ctcatttggc agaagttctt ccttctgtat cttcaccgtc cgggaccaca gtatgggtta

4021 cccatagatg gttgtattgg aagaaggttt tttcaaagtc ctgctcatat caatttgttg

4081 aaagaaatga agagacgttt gaccgaggtg gctgacttcc accatgctgc aagcaaggcc 4141 ctccgtgttc cagcagaggg cagtgaaggg ctgccagaga gccactctgg cacccctggt 4201 tacctgactt caccagaact gcacaaggag ctggtgaggc tcttcaatgt atatatatta

4261 tggctagaag atgagaattt tcaaaaagga gatacctata tcccttctct accaaagcat

4321 tatgatattc acaggctagc aaaagtgatg cagaatcagc aggtgaaatt atag

SEQ ID NO: 4 (Protein BAB13458.1; P450G5)

1 ktngdgaaeg smaeavkpqr rakakasrtk tkekkkyetp qreessevsl pktsreqeip

61 slacefkgdh lkwtdsqlq ddasgqnese mfdvpltslt isneesltcn teppkeggea

121 rpcvgdsavt pkvhpgdnvg tkvetpknft eveeπmsvqg glsesapqsn fsytqpamen

181 iqvretqnsk edegglvcss evpqnvglqs scpak gfqt prvkklypql paeiageapa 241 lvavkpllrs erlypelpsq lelvpftkeq lkilepgs l envesyleef ds ahqdrhe

301 fyelllnysr crkqlllaea elltltsdcq naksrlwqfk eeqmsvqgic adq kvfsyh

361 ryqrvemnen alvelkklfd akse lhqtl alhsytsvls rlqvesyiya llsssavlrs

421 sai qqgras kqtesipsdl cqlkecisvl fmftrrvned tqfhddill lqklvsvlqr

481 vgcpgd lfl lnhilrcpag vsk avpfiq ikvlhnpsgv fhfmqslall mspvknraef 541 mch kpserk psssgpgsgt tlvdeggee dedpets il lneddlvtil aqfpfhelfq

601 hllgfkakgd ylpettrpqe inmkifafans lvellavgle tfnraryrqf vkrigymiπα

661 tlgyvsdh a qyvshnqgsg laqqpysmek lqvefdelfl ravlhvlkak rlgiwlfmse

721 mpfgtlsvqm lwklfylmhq vesenlqqls sslqpaqckq qlqdpe ftn fekclssmns

781 seeicllttf aq aqarrtn vdedfikiiv leiyevsyvt lstretfskv grellgtita 841 vhpeiisvll drvqetidq gmvslylfke lplyl qpsa seiavirdwl lnynltwkn

901 klacvilegl n gfakqatl hldqavhaev almvleayqk ylaqkpyagi lsesmkqvsy

961 lasivryget petsfnqwaw nlilrlklhk ndygiqpncp avpfsvtvpd mtesptfhpl

1021 lkavkagmpi gcylalsmta vghs±ekfca egipllgilv qsrhlrtwh vldkilplfy

1081 pcqyyllkne qflshlllfl hldsgvpqgv tqqvthkvaq ltgashgdn vkllnsmiqa 1141 hisvstqpne vgpvavlefw vqalisqhlw yreqpilflm dhlckaafql mqedciqkll

1201 yqqhknalgy hcdrsllssl vs ivagnit psfveglatp t vwfawtvl πtαesifeeds

1261 qlrrviegel vinsaftpdq alkkaqtqlk lpivpslqrl liyrwahqal vtpsdpllp

1321 li qkfflly lhrpgpqygl pidgcigrrf fqspahinll kemkrrltev adfhhaaska

1381 Irvpaegseg Ipeshsgtpg yltspelhke lvrlfnvyil wledenfqkg dtyipslpkh 1441 ydihrlakv qnqqvkl

Claims

1. A polypeptide, which polypeptide:

(i) comprises or consists of 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 polypeptide of (i); or

(iii) is a functional equivalent of (i) or (ii).

2. A polypeptide which is a fragment according to claim l(ii), which includes the Cytochrome P450 region of the P450G4 polypeptide, said Cytochrome P450 region being defined as including between residues 586 and 946 inclusive, of the amino acid sequence recited in SEQ ID NO:2, wherein said fragment possesses Cytochrome P450 activity.

3. A polypeptide which is a functional equivalent according to claim l(iii), is homologous to the amino acid sequence as recited in SEQ ID NO:2, has Cytochrome

P450 activity.

4. A polypeptide according to claim 3, wherein said functional equivalent is homologous to the Cytochrome P450 region of the P450G4 polypeptide.

5. A polypeptide which is a fragment according to claim l(ii), which includes the Cytochrome P450 region of the P450G5 polypeptide, said Cytochrome P450 region being defined as including between residue 1112 and residue 1350 of the amino acid sequence recited in SEQ ID NO:4, wherein said fragment possesses the catalytic residue Cysl345, or equivalent residues, and possesses Cytochrome P450 activity.

6. A polypeptide which is a functional equivalent according to claim l(iii), is homologous to the amino acid sequence as recited in SEQ ID NO:4, possesses the catalytic residue Cysl345, or equivalent residues, and has Cytochrome P450 activity.

7. A polypeptide according to claim 6, wherein said functional equivalent is homologous to the Cytochrome P450 region of the P450G5 polypeptide.

8. A fragment or functional equivalent according to any one of claims 1-7, which has greater than 30% sequence identity with an amino acid sequence as recited in any one of SEQ ID NO:2 and SEQ ID NO:4, or with a fragment thereof that possesses Cytochrome P450 activity, preferably greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity, 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/) [Blosum 62 matrix; gap open penalty=l 1 and gap extension penalty=l].

9. A functional equivalent according to any one of claims 1 -8, which exhibits significant structural homology with a polypeptide having the amino acid sequence given in any one of SEQ ID NO:2 and SEQ ID NO:4, or with a fragment thereof that possesses Cytochrome P450 activity.

10. A fragment as recited in claim 1, 2, 5, or 8, having an antigenic determinant in common with the polypeptide of claim l(i), which consists of 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more) amino acid residues from the sequence of SEQ ID

NO:2, or SEQ ID NO:4.

11. A purified nucleic acid molecule which encodes a polypeptide according to any one of the preceding claims.

12. A purified nucleic acid molecule according to claim 11, which has the nucleic acid sequence as recited in SEQ ID NO:l or SEQ ID NO:3, or is a redundant equivalent or fragment thereof.

13. A fragment of a purified nucleic acid molecule according to claim 11 or claim 12, which comprises between nucleotides 1756 and 2838 of SEQ ID NO:l, or is a redundant equivalent thereof.

14. A fragment of a purified nucleic acid molecule according to claim 11 or claim 12, which comprises between nucleotides 3334 and 4050 of SEQ ID NO:3, or is a redundant equivalent thereof.

15. A purified nucleic acid molecule which hybridizes under high stringency conditions with a nucleic acid molecule according to any one of claims 11-14.

16. A vector comprising a nucleic acid molecule as recited in any one of claims 11-15.

17. A host cell transformed with a vector according to claim 16.

18. A ligand which binds specifically to, and which preferably inhibits the Cytochrome P450 activity of, a polypeptide according to any one of claim s 1-10.

19. A ligand according to claim 18, which is an antibody.

20. A compound that either increases or decreases the level of expression or activity of a polypeptide according to any one of claims 1-10.

21. A compound according to claim 20 that binds to a polypeptide according to any one of claims 1-10 without inducing any of the biological effects of the polypeptide.

22. A compound according to claim 20 or claim 21, which is a natural or modified substrate, ligand, enzyme, receptor or structural or functional mimetic.

23. A polypeptide according to any one of claim 1-10, a nucleic acid molecule according to any one of claims 11-15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or 19, or a compound according to any one of claims 20-22, for use in therapy or diagnosis of disease.

24. A method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to any one of claim 1- 10, or assessing the activity of a polypeptide according to any one of claim 1-10, 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.

25. A method according to claim 24 that is carried out in vitro.

26. A method according to claim 24 or claim 25, which comprises the steps of: (a) contacting a ligand according to claim 18 or claim 19 with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.

27. A method according to claim 24 or claim 25, comprising 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 according to any one of claims 11-15 and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) 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.

28. A method according to claim 24 or claim 25, comprising: a) contacting a sample of nucleic acid from tissue of the patient with a nucleic acid primer under stringent conditions that allow the fonnation of a hybrid complex between a nucleic acid molecule according to any one of claims 11-15 and the primer; b) contacting a control sample with said primer under the same conditions used in step a); and c) amplifying the sampled nucleic acid; and d) detecting the level of amplified nucleic acid from both patient and control samples; wherein detection of levels of the amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in the control sample is indicative of disease.

29. A method according to claim 24 or claim 25 comprising: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to any one of claims 11-15 from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation which is associated with disease in the nucleic acid molecule as an indication of the disease.

30. The method of claim 29, further comprising amplifying the nucleic acid molecule to form an amplified product and detecting the presence or absence of a mutation in the amplified product.

31. The method of either claim 29 or 30, wherein the presence or absence of the mutation in the patient is detected by contacting said nucleic acid molecule with a nucleic acid probe that hybridises to said nucleic acid molecule 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.

32. A method according to any one of claims 24-31, wherein said disease is a 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, organ transplant rejection, COPD, osteoarthritis, wound healing and resolution of infections; 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; placental 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.

33. A method according to any one of claims 24-31, wherein said disease is: 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 dennatitis; 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 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, compulsive obsessive disorders, anxiety, depression; and addictive behaviours such as alcoholism, eating disorders and drug addiction including nicotine addiction.

34. Use of a polypeptide according to any one of claims 1-10 as a Cytochrome P450, such as for the modulation (including both the potentiation, amelioration or conversion to metabolite) of pharmacological agents, particularly small molecule pharmacological agents.

35. Use of a nucleic acid molecule according to any one of claims 11-15 to express a protein that possesses Cytochrome P450 activity.

36. A method for effecting cell-cell adhesion, utilising a polypeptide according to any one of claims 1-10.

37. A pharmaceutical composition comprising a polypeptide according to any one of claims 1-10, a nucleic acid molecule according to any one of claims 11-15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or 19, or a compound according to any one of claims 20-22.

38. A vaccine composition comprising a polypeptide according to any one of claims 1-10 or a nucleic acid molecule according to any one of claims 11-15.

39. A polypeptide according to any one of claims 1-10, a nucleic acid molecule according to any one of claims 11-15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or 19, a compound according to any one of claims 20-22, or a pharmaceutical composition according to claim 37 for use in the manufacture of a medicament for the treatment of a 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, organ transplant rejection, COPD, osteoarthritis, wound healing and resolution of infections; 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; placental disorders; 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.

40. A polypeptide according to any one of claims 1-10, a nucleic acid molecule according to any one of claims 11-15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or 19, a compound according to any one of claims 20-22, or a pharmaceutical composition according to claim 37 for use 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 emphysema; 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, compulsive obsessive disorders, anxiety, depression; and addictive behaviours such as alcoholism, eating disorders and drug addiction including nicotine addiction.

41. A method of treating a disease in a patient, comprising administering to the patient a polypeptide according to any one of claims 1-10, nucleic acid molecule according to any one of claims 11-15, a vector according to claim 16, a host cell according to claim 17, a ligand according to claim 18 or 19, a compound according to any one of claims 20-22, or a pharmaceutical composition according to claim 37.

42. A method according to claim 41, wherein, for diseases in which the expression of the natural gene or the activity of the polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an agonist.

43. A method according to claim 41, wherein, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an antagonist.

44. A method of monitoring the therapeutic treatment of disease in a patient, comprising monitoring over a period of time the level of expression or activity of a polypeptide according to any one of claims 1-10, or the level of expression of a nucleic acid molecule according to any one of claims 11-16 in tissue from said patient, wherein altering said level of expression or activity over the period of time towards a control level is indicative of regression of said disease.

45. A method for the identification of a compound that is effective in the treatment and/or diagnosis of disease, comprising contacting a polypeptide according to any one of claims 1-10, a nucleic acid molecule according to any one of claims 11-15, or a host cell according to claim 17 with one or more compounds suspected of possessing binding affinity for said polypeptide or nucleic acid molecule, and selecting a compound that binds specifically to said nucleic acid molecule or polypeptide.

46. A kit useful for diagnosing disease comprising a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to any one of claims 11-15; a second container containing primers useful for amplifying said nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.

47. The kit of claim 46, further comprising a third container holding an agent for digesting unhybridised RNA.

48. A kit comprising an array of nucleic acid molecules, at least one of which is a nucleic acid molecule according to any one of claims 11-15.

49. A kit comprising one or more antibodies that bind to a polypeptide as recited in any one of claims 1-10 and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.

50. A transgenic or knockout non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide according to any one of claims 1-10.

51. A method for screening for a compound effective to treat disease, by contacting a non-human transgenic animal according to claim 50 with a candidate compound and determining the effect of the compound on the disease of the animal.