WO2002064765A2

WO2002064765A2 - Cloning and characterization of a human cytochrome p450, cyp 27c1, and a hybrid homolog from xenopus lævis

Info

Publication number: WO2002064765A2
Application number: PCT/CA2002/000163
Authority: WO
Inventors: Jan Wisniewski
Original assignee: Cytochroma Inc
Current assignee: Cytochroma Inc
Priority date: 2001-02-09
Filing date: 2002-02-11
Publication date: 2002-08-22
Anticipated expiration: 2003-08-09
Also published as: WO2002064765A3; AU2002231531A1

Abstract

The present invention relates to a novel cytochrome P450 protein, human CYP 27C1, and to the nucleic acid molecule comprising a nucleotide sequence encoding same. The invention also relates to species homologs thereto. A human/Xenopus lævis CYP 27C1 hybrid protein and nucleic acid molecule encoding same is also provided by the invention. In addition, the invention relates to vectors comprising the nucleic acid molecules of the invention, host cells and antibodies directed to CYP 27C1 protein and to recombinant methods directed to produce same. Furthermore, the invention relates to diagnostic and therapeutic methods for detecting and treating CYP 27C1-related disorders and to screening methods for identifying modulators, agonists and antagonists of CYP 27C1 expression and activity.

Description

Title: Cloning and characterization of a novel human cytochrome P450,

CYP 27C1, and a hybrid homolog from Xenopus laevis RELATED APPLICATIONS This application claims priority from US Provisional Patent Application

No. 60/267,410, filed February 9, 2002. As February 9, 2002 fell on a Saturday, this application was filed on February 11 , 2002 in accordance with Article 4 of the Stockholm Act of the Paris Convention for the Protection of Industrial Property, and Article 8 of the Patent Cooperation Treaty. FIELD OF THE INVENTION

The invention relates to the cloning and characterization of a novel human cytochrome P450, CYP 27C1 , and a novel hybrid human/Xenopus laevis CYP 27C1 homolog. In an embodiment, the invention relates to both the polypeptides and fragments and modifications of cytochrome P450 CYP 27C1 and the said hybrid homolog and to polynucleotides encoding the same. Related vectors, host cells, and antibodies and methods and uses of the polynucleotides, polypeptides, vectors, host cells and antibodies are also encompassed within the scope of the invention. BACKGROUND OF THE INVENTION

The cytochrome P450s comprise a large gene superfamily that encodes over 500 distinct heme-thiolate proteins that catalyze the oxidation of drugs and numerous other compounds in the body. It is of considerable interest in the pharmaceutical and other fields to identify cytochrome P450s and the role they play in the metabolism of individual compounds. Cytochrome P450s are heme - containing enzymes that strongly absorb at a wavelength of 450nm when the heme is bound to a molecule of carbon monoxide (1). Most often known for their ability to catalyze the metabolism of a wide variety of drugs, xenobiotics, carcinogens, mutagens and pesticides, and are also involved in catalyzing reactions that make or degrade cholesterol, steroids, and other lipids (2). The reactions performed by these enzymes are generally oxidations, hydroxylations, acetylations, and demethylations (1). Mutations in cytochrome P450s can cause a number of human diseases such as glaucoma and breast cancer (2). It has been known that cytochrome P450s are also involved in Vitamin D₃ and D₂ metabolism. Vitamin D₃ and D₂, both seco-steroids, are activated by cytochrome P450s.

The vitamin D metabolic pathway is part of a vital endocrine system that is highly regulated at certain stages and produces metabolites that control the secretion of the parathyroid gland hormones (3,4). 1α,25(OH) ₂D₃, a hormone produced in the vitamin D pathway, regulates phosphate and calcium levels in the blood which in turn control bone mass, the state of bones, and affect cellular differentiation in the skin and the immune system (5). In the vitamin D pathway, cytochrome P450s introduce functional groups by hydroxylation usually at positions 1, 25, and 24 of the steroid (3). The metabolism of vitamin D begins with 25-hydroxlyation of vitamin D₃ or D₂ in the liver (5) to 25(OH)D₃. A P450 thought to be responsible for this hydroxylation, CYP 27A1 , has been cloned in the mitochondrial fractions of human, rabbit, and rat liver tissue (5). Previous studies have also found 25-hydroxylase activity in the microsomal extracts of the liver but the enzyme responsible has never been cloned (4,6,7,8,11- 14). CYP 27A1 has been functionally determined to be a 25-hydroxylase enzyme involved in the vitamin D pathway as well as a protein responsible for initiation of the acidic pathway in bile acid biosynthesis (5,8,9). CYP 27B1 is the mitochondrial cytochrome P450 responsible for the second hydroxylation to form 1 ,25(OH)₂D₃ and has been found in the renal proximal tubular cell and extra-renally in the bone, alveolar macrophages, placenta, keratinocytes, and nonsmall cell lung carcinomas (4,10). 25(OH)D₃ and 1α,25(OH)₂D₃ are converted to 24,25(OH)₂D₃ and 1 ,24,25(OH)₃D₃ by a third mitochondrial P450 involved in the vitamin D pathway; CYP 24 (11). CYP 24 is induced by 1 ,25(OH)₂D₃ and is found in the kidney as well as other vitamin D target tissues such as the parathyroid cells, keratinocytes, osteoblasts, and enteroctyes (4). Microsomal cytochromes occur on the membrane of the ER and require NADPH cytochrome P450 reductase and a flavoprotein for activity, whereas mitochondrial cytochromes occur on the inner mitochondrial membrane and require ferredoxin and NADPH ferredoxin reductase for activity (3,5).

Findings by Guo et al. (9) indicate that CYP 27A1 hydroxylates 1α-hydroxylated compounds more readily than non-hydroxylated vitamin D compounds, and reveal the absence of CYP 27A1 mRNA in Hep3B cells which still have 25-hydroxylase activity. Results that also support a second 25-hydroxylase have shown that CYP 27A1 does not appear to hydroxylate vitamin D₂, which is activated by the same pathway as vitamin D₃ (4). Studies of the 5' flanking region of CYP 27A1 have determined that the cytochrome is not transcriptionally regulated by vitamin D (13). The vitamin D response element is absent in the promoter region of CYP 27A1 leading researchers to further postulate that the microsomal 25-hydroxylase could be responsible for the physiological metabolism of vitamin D₃ and D₂ (13). Mutations in the CYP 27A1 gene decrease cholesterol metabolism, supporting its role in bile acid biosynthesis, but do not result in vitamin D deficiency diseases such as rickets or osteomalacia which would be expected if this enzyme played a key role in the vitamin D metabolic pathway (4,9). It has been suggested that CYP 27A1 is present when vitamin D levels are high in circulation, whereas the microsomal 25- hydroxylase is present in low levels, but has high specificity for vitamin D compounds (9). A number of benefits to vitamin D as well as a number of diseases and conditions related to vitamin D deficiency exist. Diseases or conditions associated with vitamin D or its metabolites include but are not limited to: (i) in the parathyroid - hyper- and hypo-parathyroidism, Osudohypo- parathyroidism, Secondary hyperparathyroidism; (ii) in the pancreas - diabetes;

(iii) in the thyroid - medullary carcinoma; (iv) in the skin - psoriasis; (v) in the lung - sarcoidosis and tuberculosis;

(vi) in the kidney - chronic renal disease, hypophosphtatemic VDRR, vitamin D dependent rickets; (vii) in the bone - anitconvulsant treatment, fibrogenisis imperfecta ossium, osteitits fibrosa cystica, osteomalacia, osteporosis, osteopenia, osteosclerosis, renal osteodytrophy, rickets; (viii) in the intestine - glucocorticoid antagonism, idopathic hypercalcemia, malabsorption syndrome, steatorrhea, tropical sprue. More common conditions related to vitamin D or vitamin D metabolite deficiency are obesity problems, hyperhoshatemic turmoral calcinosis, sarcoidosis, tuberculosis, primary hyperparathyroidism, vitamin D dependent rickets type II, cholestatic or paremchymal liver disease.

As such, there is a need to further characterize the metabolism and mode of action of vitamin D and its metabolites. The search continues for P450 polypeptides that play a role in vitamin D metabolism.

Further cytochrome P450s are involved in many metabolic pathways as noted herein, including that of apoptosis, cholesterol, steroids, retinoic acid and other lipids. The identification and characterization of novel cytochrome P450s may shed light into these pathways and any related conditions, such as cerebrotendinous xanthomatosis (CX) [related to metabolism of cholesterol], and various forms of cancers and autoimmune diseases.

SUMMARY OF THE INVENTION The present inventors have cloned and characterized for the first time human CYP 27C1. In one embodiment the CYP 27C1 is a human hydroxylase. In another embodiment it is a mitochondrial hydroxylase. In one embodiment the CYP 27C1 is expressed in or isolated from hepatic, colonic or fetal tissue. In another embodiment, CYP 27C1 is expressed in or isolated from human brain, heart, kidney, lung, muscle, stomach, testis, adrenal gland, ovary, prostate, skin, fetal brain but more preferably the testis, adrenal gland or fetal brain. On yet another embodiment CYP 27C1 is expressed in prostate, lung or breast cancer, such as DV 145, WTE, SKMES, SK-luci-6, PC-3, MCF-7, HEP3B and NT-2 cells.

These findings have important implications in terms of increased understanding of the vitamin D pathway and their application to various disease states related to cytochrome P450 activity, such as those related to the vitamin D pathway (for example, those noted above), the vitamin A or retinoic acid pathway, apoptosis, various cancers, or cholesterol, steroid or other lipid metabolic pathways.

In another embodiment the invention provides a hybrid CYP 27C1 polynucleotide (isolated, preferably a human/Xenopus laevis CYP 27C1 hybrid, and polypeptide, preferably a CYP 27C1 polypeptide homolog, encoding the same.

Although, the CYP 27C1 and encoding nucleic acid sequence of the invention can be isolated and characterized from any tissue, in one embodiment it is isolated and characterized from fetal kidney, fetal brain, fetal liver and fetal lung, colon and uterus tissue. In another embodiment the CYP 27C1 is isolated and characterized from NT-2, colon, fetal brain and Hep 3B cells.

Accordingly, in one aspect, the present invention provides an isolated polynucleotide or isolated nucleic acid molecule comprising a nucleotide sequence encoding a CYP 27C1 , preferably a human CYP 27C1 or human/Xenopus laevis hybrid CYP 27C1 and to variants, homologs, analogs thereof and to fragments thereof. Complimentary polynucleotide sequences to the polynucleotides of the invention are also encompassed within the scope of the invention.

In a preferred embodiment, an isolated polynucleotide is provided having a nucleic acid sequence as shown in Figures 2A (SEQ. ID.. NO:39) or 11A(SEQ. ID. NO:44). Most preferably, the purified and isolated polynucleotide comprises: (a) a nucleic acid sequence as shown in Figures 2A(SEQ. ID. NO: 39) or 11A(SEQ. ID. NO:44) wherein T can also be U; (b) nucleic acid sequences complementary to (a); (c) nucleic acid sequences which are homologous to (a) or (b); or, (d) a fragment of (a) to (c) that is at least 10, preferably at least 15 bases, most preferably 20 to 30 bases, and which will hybridize to (a) to (c) under stringent hybridization conditions. In a further embodiment, the invention provides polynucleotides that consist of the isolated polynucleotides noted above, more preferably that consists of the polynucleotide sequence that encodes human CYP 27C1 or human/xenpus laevis hybrid CYP 27C1 protein.

The present invention also includes the CYP 27C1 polypeptide and human/Xenopi/s laevis CYP 27C1 hybrid polypeptide. In one embodiment, the invention provides a polypeptide having an amino acid sequence as shown in Figure 2B(SEQ. ID. NO:40) or 11B(SEQ. ID. NO.45) and to variants, homologs, and analogs, insertions, deletions, substitutions and mutations thereto. The invention also comprises polypeptides comprising fragments of the amino acid sequence of Figure 2B(SEQ. ID. NO:40) or 11 B(SEQ. ID. NO:45) or to their respective variants, homologs, analogs, insertions, deletions, substitutions and mutations. In another embodiment the fragments preferably comprise at least 14 amino acid residues and are most preferably antigenic. In another embodiment the invention provides polypeptides encoded by a polynucleotide having the sequence of Figure 2A(SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44), or to variants, homologs, analogs or fragments thereof.

Accordingly, in one embodiment the invention provides vectors, and host cells comprising the polynucleotides of the invention or that can express the polypeptides of the invention. Antibodies to the polypetides of the invention are also encompassed within the scope of this invention. The invention further provides recombinant methods for producing CYP 27C1 and the human/Xenopi/s laevis hybrid polypeptides and polynucleotides of the invention. In one embodiment, the invention provides a polynucleotide of the invention operationally linked to an expression control sequence in a suitable expression vector. In another embodiment, the expression vector comprising a polynucleotide of the invention is capable of being activated to express the peptide which is encoded by the polynucleotide and is capable of being transformed or transfected into a suitable host cell. Such transformed or transfected cells are also encompassed with the scope of this invention.

The invention also provides a method of preparing a polypeptide of the invention utilizing a polynucleotide of the invention. In one embodiment, a method for preparing the polypeptide, preferably CYP 27C1 or hybrid thereof is provided comprising: transforming a host cell with a recombinant expression vector comprising a polynucleotide of the invention; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the protein; and (d) isolating the protein.

In yet another embodiment, the invention also includes diagnostic methods for detecting and screening for disorders related to CYP 27C1 gene expression and polypeptides and to therapeutic methods for treating such disorders. As such, the invention also includes a method for detecting a disease associated with CYP 27C1 expression in an animal. "A disease associated with CYP 27C1 expression" as used herein means any disease or condition which can be affected or characterized by the level of CYP 27C1 expression. This includes, without limitation, diseases affected by, high, normal, reduced or non-existent expression of CYP 27C1 or expression of mutated CYP 27C1. A disease associated with CYP 27C1 expression includes but is not limited to diseases associated with vitamin D metabolism or cholesterol metabolism. The method comprises assaying for CYP 27C1 from a sample, such as a biopsy, or other cellular or tissue sample, from an animal susceptible of having such a disease. In one embodiment, the method comprises contacting the sample with an antibody of the invention which binds CYP 27C1 , and measuring the amount of antibody bound to CYP 27C1 in the sample, or unreacted antibody. In another embodiment, the method involves detecting the presence of a nucleic acid molecule having a sequence encoding a CYP 27C1 , comprising contacting the sample with a nucleotide probe which hybridizes with the nucleic acid molecule, preferably mRNA or cDNA to form a hybridization product under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.

The invention further includes a kit for detecting a disease associated with CYP 27C1 expression in a sample comprising an antibody of the invention, preferably a monoclonal antibody. Preferably directions for its use is also provided. The kit may also contain reagents that are required for binding of the antibody to a CYP 27C1 protein in the sample.

The invention also provides a kit for detecting the presence of a polypeptide having a sequence encoding a polypeptide of, related to or analogous to a polypeptide of the invention, comprising a nucleotide probe which hybridizes with the nucleic acid molecule, reagents required for hybridization of the nucleotide probe with the nucleic acid molecule, and directions for its use.

The invention also includes screening methods for identifying binding partners of CYP 27C1. In addition, the invention relates to screening methods for identifying modulators, such as agonists and antagonists, of CYP 27C1 activity. In one embodiment such modulators of CYP 27C1 activity or expression can include antibodies to CYP 27C1 and antisense polynucleotides to the CYP 27C1 gene or fragment thereof. The invention further provides a method of treating or preventing a disease associated with CYP 27C1 expression comprising administering an effective amount of an agent that activates, simulates or inhibits CYP 27C1 expression, as the situation requires, to an animal in need thereof. In a preferred embodiment, CYP 27C1 , a therapeutically active fragment thereof, or an agent which activates or simulates CYP 27C1 expression is administered to the animal in need thereof to treat a disease or condition associated with vitamin D deficiency, such as those mentioned herein above or to cholesterol metabolism, such as cerebrotendinous xanthomatosis (CX) or to cancer. In another embodiment the disease is associated with over expression of CYP 27C1 and the method of treatment comprises administration of an effective amount of an agent that inhibits CYP 27C1 expression such as an antibody to CYP 27C1 , a mutation thereof, or an antisense nucleic acid molecule to all or part of the CYP 27C1 gene.

In another embodiment the invention provides pharmaceutical compositions comprising a modulator of CYP 27C1 activity and a pharmaceutical acceptable carrier. In another embodiment, the pharmaceutical composition of the invention comprises CYP 27C1

(preferably a soluble form thereof) or a therapeutically effective fragment thereof and a pharmaceutically acceptable carrier. In another embodiment the pharmaceutical compositions of the invention comprise both a modulator of CYP 27C1 activity and CYP 27C1 (preferably a soluble form thereof) or a therapeutically effective fragment thereof. In a further embodiment the pharmaceutical compositions of the invention can further comprise any one or more of: (a) vitamin D or its metabolites, (b) NADPH ferredoxin reductase and/or ferredoxin; or (c) NADPH cytochrome P450 reductase and/or a flavoprotein.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

Figure 1A is the nucleotide sequence of genomic clone RP11- 30F3(SEQ. ID. NO:21) and Figure 1 B is an alignment map of nucleic acid sequence of predicted exons of CYP 27C1 on human genomic 188 888bp clone RP11-30F3 (Figure 1 B). Positions of exons 3,4 and 6-9 (SEQ. ID. NOS:24,25, and 27-30)are shown. Exon 5(SEQ. ID. NO:26) is not contained in the genomic sequence that was on the database. Solid, bolded lines represent the 40 unordered segments of the RP11-30F3 clone. The numbers in brackets beside the exon designations show the position of the exon with respect to the cDNA sequence of Figure 2A(SEQ. ID. NO:39). Figure 1C shows the predicted exon domains of CYP27C1 from Dr. Nelson's web site http://www.drnelson.utmem.edu/whatsnew.html (Feb1 , 2001). The nelson exon map (SEQ. ID. NOS. 31-38) is not complete nor exactly the same as that sequenced in Figure 2B.

Figure 2 shows the cDNA nucleotide (Figure 2A, SEQ. ID. NO:39) and amino acid (Figure 2B, SEQ. ID. NO:40) sequence for CYP 27C1. Single letter amino acid code is used to indicate a 537 amino acid protein encoded by the above nucleotide sequence starting from the second ATG site.

Figure 3A is an agarose gel showing the identification of the 400bp CYP 27C1 band generated by PCR screening of the T84 colon, fetal brain, thymus, placenta and the NT-2 cDNA libraries using CYP 27C1 primers 210(SEQ. ID. NO:4) and 211(SEQ. ID. NO:5). Figure 3B identifies 27C1 expressing tissues using RACE-cDNA panels. RACE-cDNA samples representing various human tissues were obtained from Origene and used as templates for PCR-amplification with primers 210(SEQ. ID. NO:4) and 211 (SEQ. ID. NO:5). After agarose gel electrophoresis, the expected 0.4 kbp product was detected in colon and utereus tissue. The following tissues are represented on the gel 1 -brain, 2-heart, 3-spleen, 4-liver, 5, colon, 6- testis and 7-uterus.

Figure 4 shows human tissue expression of CYP 27C1. Figure 4A shows a RACE PCR products with primers ADP2(SEQ. ID. NO:49) and 216(SEQ. ID. NO:6). The gel shows a 600bp band produced in colon tissue following second round amplification. Figure 4B (i) is an RNA dot blot from 76 non diseased human tissues shows slightly elevated expression in fetal brain (a11), fetal kidney (c11), fetal liver (d11), fetal lung (g11), thalamus (c3), trachea (h7), and jejunum (d5). Figure 4B(ii) is the index for the dot blot of Figure 4B. Figure 4C shows expression of CYP 27C1 in Hep3B cells using RT-PCR with primers 210(SEQ. ID. NO:4) and 211 (SEQ. ID. NO:5). CYP 27A1 shows expression in Hep3B cells as indicated by the 880bp band and 1600bp band and GAPDH, the positive control, shows a gene specific 1000bp band (not marked) in both Hep3B and HepG2 cells. Figure 4D is a Northern blot analysis of CYP 27C1 expression in Hep3B and HepG2 cells. A ~5kb band is abundant in the Hep3B RNA lane following hybridization with CYP 27C1 probe. Figure 4E shows the abundance and placement of the 28S and 18S ribosomal bands in Hep3B and HepG2 total RNA prior to blotting of the gel is shown.

Figure 5 shows cloning of the 5' end of CYP 27C1. Pangene Inc. results following screening of a fetal brain library using primers 216(SEQ. ID. NO:6), 227(SEQ. ID. NO:8), and 248(SEQ. ID. NO:9) and digestion of mini-preps with EcoRI is shown in Figure 5A. The 350bp band indicated in clone A represents the internal EcoRI site in CYP 27C1 and was confirmed to be CYP 27C1 by sequencing Figure 5B shows the identification of an additional 300bp of CYP 27C1 from Hep3B and colon RNA towards the 5' end using RT-PCR with primers 303(SEQ. ID. NO:13)/224(SEQ. ID. NO:7 and cloning of the 5' end using primers 302(SEQ. ID. NO:12)/224(SEQ. ID. NO:7).

Figure 6 shows a sequence comparison of CYP 27C1(SEQ. ID. NO:40), CYP27A1(SEQ. ID. NO:41), CYP27B1(SEQ. ID. NO:42) and X laevis 27C7^"(SEQ. ID. NO:43). Shaded residues indicate amino that are identical. Dashes are used to optimize identity in the alignment between the two sequences. CYP 27A1 and CYP 27C1 show 36% identity in their amino acid sequences. Numbers on the right indicate the position of the amino acid residues. The amino acid sequence comparison between human CYP 27C1 and Xenopus laevis CYP 27C1 indicates that the sequences have 70% amino acid identity. Following approximately 80 amino acids in the sequence alignment most amino acids are identical or conservatively substituted. Numbers on the right keep tract of the residue count.

Figure 7A shows a Kyte-Doolittle hydropathy plot of the first 200 amino acids of the 5' end of CYP 27C1 and of CYP 27 A1. Figure 7B shows a Kyte-Doolittle hydropathy plot of the first 200 amino acids of the 5' end of CYP 27C1 and CYP26. Figure 8 shows construction of the Xenopus laevis/CYP 27C1 hybrid gene (Figure 8A). Figure 8B shows a band following PCR with primers 314(SEQ. ID. NO:20) and 311(SEQ. ID. NO:17) of approximately 1600bp.

Figure 9 shows in vitro transcription and translation of Xenopus laevis CYP 27C1 hybrid gene. Autoradiograph shows hybrid protein in lanes

4 and 5 at approximately 55kDa. P450RAI-1 in lanes 3 and 6 is approximately 50kDa in size. Lane 1 , the negative control, is clear and lane

2, the kit's positive control, shows the expected banding pattern.

Figure 10 is a gel showing RT-PCR analysis of CYP 27C1 transcriptions following induction of Hep3B and HepG2 cells with ETOH,

1 ,25(OH)₂D₃, 25(OH)D₃ and 1∞(OH)D₃. Faint bands are seen at approximately 950bp in Hep3B cells following RT-PCR on total RNA isolated from these induced cells. High levels of primer remain at the bottom of the gel. Figure 11 is a nucleotide (Figure 11 A, SEQ. ID. NO:44) and polypeptide(Figure 11 B, SEQ. ID. NO:45) sequence of the Xenopus laevis

CYP 27C1 hybrid gene.

Figure 12 are the results of an advanced blastn search of the expressed sequence tagged (EST) in the NCBI database using the default settings and the predicted CYP 27C1 sequence.

Figure 13 is an immunoblot illustrating the expression of his-tagged CYP27C1 from pFastBad-CYP27CH6 in insect cells.

Figure 14 A and 14 B are gels showing the results of RT-PCR analysis of clones for his-tagged CYP27C expression in V79 cells. Figure 15 are spectrophotometric graphs illustrating the expression of functional CYP27C1 (graph of fraction before CO binding (Figure 15 A) versus graph of fraction after CO binding (Figure 15 B)) in the mitochondrial fraction of insect cells.

Figure 16 is a RT-PCR analysis of CYP27C1 expression in various tumor cell lines: DU-145, VVTE, SKMES, SK-luci-6, PC3 and MCF-7. DETAILED DESCRIPTION OF THE INVENTION i) Definitions

The following definitions are provided to facilitate understanding of certain terms used in this application. The following standard abbreviations for the amino acid residues are used throughout the specification: A, Ala - alanine; C, Cys - cysteine; D, Asp- aspartic acid; E, Glu - glutamic acid; F, Phe - phenylalanine; G, Gly - glycine; H, His - histidine; I, lie - isoleucine; K, Lys - lysine; L, Leu - leucine; M, Met - methionine; N, Asn - asparagine; P, Pro - proline; Q, Gin - glutamine; R, Arg - arginine; S, Ser - serine; T, Thr - threonine; V, Val - valine; W, Trp- tryptophan; Y, Tyr - tyrosine; and p.Y., P.Tyr - phosphotyrosine.

In the present invention, "isolated" refers to material removed from its original environment [e.g., the natural environment if it is naturally occurring], and thus is altered "by the hand of man" from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be "isolated" because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. However, isolated polynucleotides do not include chromosomes in the present invention.

In the present invention a "secreted" protein refers to a protein capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as a protein released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a mature protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and ptoteolytic cleavage.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See for example, Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M..J. Gait ed. 1984); Nucleic Acid Hybridization B.D. Hames & S.J. Higgins eds. (1985); Transcription and Translation B.D. Hames & S.J. Higgins eds (1984); Animal Cell Culture R.I. Freshney, ed. (1986); Immobilized Cells and enzymes IRL Press, (1986); and B. Perbal, A Practical Guide to Molecular Cloning (1984). ii) CYP 27C1 Polynucleotides and Polypeptides The present invention provide a novel cytochrome P450 polypeptide,

CYP 27C1 , and polynucleotide encoding the same. Fragments and modifications (or variants) to the polypeptide and polynucleotide of the novel cytochrome are also encompassed within the scope of the present invention. The present inventors isolated human CYP 27C1 and the encoding polynucleotide. CYP 27C1 showed expression in a number of different tissues. It is in one embodiment isolated from human fetal brain, colon and NT-2 tissue and Hep3B cells. The nucleotide sequence is shown in Figure 2A (SEQ. ID. NO:39). It is 2566 nucleotides in length and contains an open reading frame encoding a polypeptide of 537 amino acid residues, the sequence of which is shown in Figure 2B(SEQ. ID. NO:40). Certain exons of CYP 27C1 were first identified on genomic clone RP11-30F3, a 188 88bp long polynucleotide sequence(SEQ. ID. NO:21) consisting of 40 unordered segments. The present inventors have identified the whole cDNA sequence and determined its function. The cytochrome's N-terminus is approximately 85% GC content. The sequence in the first exon also contains two potential start codons in the same reading frame 15 nucleotides apart. The second ATG is located in a more hydrophobic region of amino acids than the first. In addition, CYP 27C1 contains a highly conserved ferredoxin binding site similar to other vitamin D family mitochondrial hydroxylases which require this site when receiving electrons from NADPH-ferredoxin reductase. There is one difference in the first amino acid of the ferredoxin binding site between CYP 27C1 and other vitamin D mitochondrial cytochromes.

CYP 27C1 protein expression was noted in many tissues but elevated expression was observed in Hep3B cells, thalmus, trachea, jejunum, colon and fetal (brain , liver, lung), and brain tissue and NT-2 cells. These results suggest CYP 27C1 role in cell differentiation carcinogenic, tumour and embryonic cell lines (4, 7).

CYP 27C1 is anticipated to have application to general physiological processes including various conditions such as those related to vitamin D metabolism and 25 hydroxylase activity. Conditions such as cholestatic or paremchymal liver disease, premature infant vitamin D metabolism, obesity, sarcoidosia, tuberculosis, primary hypothyroidism and vitamin D dependent rickets type II may be related to CYP 27C1 activity. CYP 27C1 activity may also be related to certain cancerous conditions, or cholesterol, steroid, or lipid, a metabolic disorders.

CYP 27C1 has been shown to be about 36% identical to CYP 27A which is known to catalyze the first step in side chain oxidation of sterol intermediated in bile acid biosynthesis. The sterol storage disorder cerebrotendinous xanthomatosis (CX) is characterized by abnormal deposition of cholesterol and cholestanol in tissues like the Achilles tendon and nervous tissues. This disease is caused by mutations in the CYP 27A1 gene. As the formation of bile acids is the only way the body can eliminate cholesterol, if the pathway becomes blocked cholesterol can build up. The disease can be treated by giving an end product of bile acid synthesis, such as cholic acid, which acts as feeedback inhibitors to shut down the bile acid pathway. Thus CYP 27C1 may play a similar role in the metabolism of cholesterol.

The cytochrome P450s are heme-binding proteins that contain the putative family signature F(XX)G(XXX)C(X)G (X means any residue; conserved residues are in bold). (Nelson, D.R. , Methods in Molecular Biology, Vol. 107: Cytochrome P450 Protocols, Cytochrome P450 Nomenclature, pp. 15-24, Phillips, I.R. and Shephard, E.A., eds., Humana Press Inc., Totowa, NJ (1998)). The heme-binding signature in CYP 27C1 can be found at amino acids 476- 485 of Figure 2B and contains the motif FGHGVRSCIG (SEQ. ID. NO:47).

Heme-binding proteins, such as myoglobin, hemoglobin and cytochromes, play an important role in several cellular protcesses, such as respirtation and detoxification. For example, the capacity of myoglobin or hemoglobin to bind oxygen depends on the presence of a heme group. Heme consists of an organic part and an iron atom. The iron atome in heme alternates between a ferrous (+2) and a ferric(+3) state; however, only heme containing an iron atom in the +2 oxidation state binds oxygen. (For a review, see e.g., Stryer, Biochemistery (3^rd edition) W.H. Freeman and Co., New York, pp. 144 and 404-405 (1988).)

Cytochrome P450s play an important role in the detoxification of toxic substances (xenobiotics), such as phenobarbital, codeine and morphine, by oxidation. It is the ability of P450s to bind heme and oxygen that enables them to function as oxidative enzymes, (for a review, see e.g. Darnelle et al., Molecular Cell Biology (2^nd edition), W.H. Freemand and Co., New York, pp 397 and 981-982 (1990)). Thus peptides of CYP 27C1 containing the heme-binding motif or the oxygen binding domain and related activities and functions are also contemplated by the inventor.

The CYP 27C1 cDNA sequence, such as Figure 2A(SEQ. ID. NO:39), is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in CYP 27C1 cDNA sequence. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from the CYP 27C1 amino acid sequence, such as disclosed in Figure 2B(SEQ. ID. NO:40)., may be used to generate antibodies which bind specifically to CYP 27C1.

Nonetheless, DNA sequencings generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequences. In this case, the predicted amino acid sequences diverge from the actual amino acid sequences, even though the generated DNA sequences may be greater than 99.9% identical to the actual DNA sequence. For example, one base insertion or deletion in an open reading frame of over 1000 bases.

For those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence of CYP 27C1 as depicted in Figure 2A(SEQ. ID. NO:39) and the predicted translated amino acid sequence of Figure 2B, but also a sample of the plasmid DNA contained in the human clone Topo 2.1 CYP 27C1 , the methods of producing which are described herein. The nucleotide sequence of the CYP 27C1 clone can be determined by sequencing the clone in accordance with known methods. The predicted amino acid sequence of CYP 27C1 can be verified. Furthermore, the amino acid sequence of the protein encoded by the clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the human CYP 27C1 cDNA, collecting the protein, and determining its sequence. The present invention also relates to the CYP 27C1 gene and the gene corresponding to Figure 2A(SEQ. ID. NO:39). The CYP 27C1 gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the CYP 27C1 gene from appropriate sources of genomic materials.

Also provided in the present invention are species homologs of CYP 27C1. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homolog. The CYP 27C1 polypeptide shown in Figure 2B is about 70% identical to a Xenopus laevis protein. A human/Xenopus laevis hybrid CYP 27C1 polynucleotide sequence encoding a corresponding hybrid CYP 27C1 hybrid polypeptide have been made, cloned and sequenced by the present inventor. The polynucleotide sequence is shown in Figure 11A(SEQ. ID. NO:44) and the amino acid sequence is shown in Figure 11 B(SEQ. ID. NO:45). Both are encompassed within the scope of the present invention.

As used herein and encompassed within the scope of this invention, a CYP 27C1 "polynucleotide" refers to a molecule having the nucleic acid sequence as shown in Figure 2A(SEQ. ID. NO:39) or the coding region thereof. For example, CYP 27C1 polynucleotide can contain the nucleotide sequence of the full length cDNA sequence as well as fragments, epitopes, domains and variants of the nucleic acid. Furthermore, a CYP 27C1 "polypeptide" referred to herein refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined and preferably having the sequence of Figure 2B(SEQ. ID. NO:40). The CYP 27C1 polypeptide can comprise additional sequences such as sequence tags, such as a polyhistidine (e.g VDHHHHHH SEQ. ID. NO:55), preferably at the "C" terminus of the peptide. "CYP 27C1 polynucleotide" encompasses nucleic acid molecules encoding such polypeptides. "CYP 27C1 "hybrid as defined herein has an analogous definition as

CYP 27C1 polynucleotide except that it refers to a polynucleotide encoding for a CYP 27C1 hybrid polypeptide wherein part of the amino acid sequence is derived from one species and part from another species to form a novel CYP 27C1 polypeptide homolog. Preferably, the CYP 27C1 hybrid polypeptide encodes the human/Xetiopi/s laevis CYP 27C1 disclosed herein and preferably having the nucleotide sequence of Figure 11A(SEQ. ID. NO:44) or the coding region thereof. The CYP 27C1 hybrid polypeptide referred to herein refers to a molecule having the translated amino acid sequence generated from the CYP 27C1 "hybrid polynucleotide" as broadly defines and preferably having the sequence of Figure 11 B(SEQ. ID. NO:45). ln the present invention, the full length CYP 27C1 cDNA sequence was obtained by screening different cDNA tissue libraries including human colon, Hep 3B, NT2 and fetal tissue such as fetal brain

A CYP 27C1 "polynucleotide" or hybrid thereof also refers to isolated polynucleotides which encode the CYP 27C1 polypeptides or respective hybrid polypetide, as the case may be, and to polypeptides closely related thereto.

A CYP 27C1 "polynucleotide" also refers to isolated polynucleotides which encode the amino acid sequence in Figure 2B(SEQ. ID. NO:40), or a biochemically active fragment thereof. A CYP 27 C1 hybrid polynucleotide also refers to isolated polynucleotides which encode the amino acid sequence in Figure 11 B(SEQ. ID. NO:45).

A CYP 27C1 polynucleotide or hybrid thereof also encompasses those polynucleotides which differ from any of the polynucleotides of the invention in codon sequence due to the degeneracy of the genetic code such polynucleotides encode functionally equivalent polypeptides but differ in sequence from the above mentioned sequences due to degeneracy in the genetic code.

A CYP 27C1 polynucleotides also encompasses nucleic acid molecules encoding proteins having substantial sequence identify to CYP 27C1 (SEQ. ID. NO. 40)

A CYP 27C1 "polynucleotide" also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to polynucleotide sequences disclosed herein, such as those of Figure 2A (SEQ. ID. NO:39)and 11A(SEQ. ID. NO:44) , or the complement thereof. "Stringent hybridization conditions" refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5X SSC [750 mM NaCl, 75 m m sodium citrate], 50 mM sodium phosphate [pH 7.6], 5X Denhardt's solution, 10% dextran sulfate, and 20 ug/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 65°C.

Of course, a polynucleotide which hybridizes only to poly A+ sequences [such as any 3' terminal poly A+ tract of a cDNA] or to a complementary stretch of T [or U] residues, would not be included in the definition of "polynucleotide", since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly [A] stretch or the complement thereof [e.g., practically any double-stranded cDNA clone]. The CYP 27C1 polynucleotide or nucleic acid molecule can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. As such, in the sequences refered to herein "T" can also be "U". For example, CYP 27C1 polynucleotide or hybrid thereof can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double stranded regions. In addition, the CYP 27C1 polynucleotide or hybrid thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.

In addition, CYP 27C1 polynucleotide or hybrid thereof may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms. Modified forms also encompass analogs of the polynucleotide sequence of the invention, wherein the modification does not alter the utility of the sequences described herein. In one embodiment, the modified sequence or analog may have improved properties over unmodified sequence.

One example of a modification to prepare an analog within the scope of this invention is to replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine or thymidine) of the sequence shown in Figure 2A or 11A with a modified base such as such as xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8- thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecule shown in Figure 2A or 11 A. For example, the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates. A further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991 , 254, 1497). PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). The analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.

The CYP 27C1 polypeptide of the invention or hybrid thereof can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The CYP 27C1 polypeptides and hybrids may be modified by either natural processes, such as post-translational processing or by chemical modification techniques which are well known in the art. Such modifications are described in basic texts, research manuals and research literature. Modifications may occur anywhere in the CYP 27C1 polypeptide or hybrid peptide, including the peptide backbone, the amino acid side-chain and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given CYP 27C1 polypeptide or hybrid. In addition, a given CYP 27C1 or hybrid may contain many types of modification. The modifications may result from post-translational natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulphide bond formation, demethylation, formation of covalent cross-links, reduction of disulphide bonds into free cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination [See, for example, Proteins-Structure and Molecular Properties, 2nd Ed., T.E. Creighton, W.H. Freeman and Company, New York (1993); Post-Translational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, p 1-12 (1983); Seifter et al., Methods in Enzymology 182: 626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663: 48-62 (1992).]

A CYP 27C1 polypeptide or hybrid thereof exhibiting activity similar, but not necessarily identical to, an activity of a CYP 27C1 polypeptide or hybrid as the case may be, including mature forms, as measured by a given biological assay, with or without dose dependency. Where dose dependency exists, it need not be identical to the CYP 27C1 polypeptide or hybrid, but rather substantially similar to the dose-dependency in a given activity as compared to the CYP 27C1 polypeptide or hybrid. For example, the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity and most preferably, not more than about three-fold less activity relative to CYP 27C1 polypeptide.

CYP 27C1 polypeptide or hybrid thereof may include various structural forms of the primary protein that retain biological activity. For example, a polypeptide of the invention may be in the form of acidic or basic salts or in neutral form. The polypeptides of the invention may be in the form of a secreted protein (i.e. could include fusion proteins or solubulized forms of the proteins of the invention), including the mature form or may be part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues (such as in SEQ. ID. NO: ), or an additional sequence for stability during recombinant production.

CYP 27C1 polypeptides are preferably provided in an isolated form, and preferably are substantially purified, A recombinantly produced version of a CYP 27C1 polypeptide or hybrid, including the secreted polypeptide, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988). CYP 27C1 polypeptides also can be purified from natural or recombinant sources using antibodies of the invention raised against the polypeptides of the invention in methods well known in the art.

Production of Polynucleotides and Polypeptides of the Invention

The polynucleotides and polypeptides of the invention can be prepared in any suitable manner, such means being known to persons skilled in the art. Such methods include isolating naturally occuring polypeptides and polynucleotides, recombinantly or synthetically/chemically produced polynucleotides or polypeptides or a combination of these methods.

An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of the nucleic acid sequences as shown in Figures 2A(SEQ. ID. NO:39) or 11A (SEQ. ID. NO:44) and using this labelled nucleic acid probe to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library). For example, a genomic library isolated can be used to isolate a DNA encoding a novel protein of the invention by screening the library with the labelled probe using standard techniques. Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.

An isolated nucleic acid molecule of the invention that is DNA can also be isolated by selectively amplifying a nucleic acid encoding a novel protein of the invention using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleic acid sequence as shown in Figures 2A(SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44) for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. It will be appreciated that cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294 5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL). An isolated nucleic acid molecule of the invention which is RNA can be isolated by cloning a cDNA encoding a novel protein of the invention into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein of the invention. For example, a cDNA can be cloned downstream of a bacteriophage promoter, (e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.

A nucleic acid molecule of the invention may also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401 ,796 and 4,373,071).

Determination of whether a particular nucleic acid molecule encodes a novel protein of the invention may be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the activity of the protein using the methods as described herein. A cDNA having the activity of a novel protein of the invention so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing or by automated DNA sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.

The initiation codon and untranslated sequences of nucleic acid molecules of the invention may be determined using currently available computer software designed for the purpose, such as PC/Gene (IntelliGenetics Inc., Calif.). Regulatory elements can be identified using conventional techniques. The function of the elements can be confirmed by using these elements to express a reporter gene which is operatively linked to the elements. These constructs may be introduced into cultured cells using standard procedures. In addition to identifying regulatory elements in DNA, such constructs may also be used to identify proteins interacting with the elements, using techniques known in the art. The sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule. The term "antisense" nucleic acid molecule is a nucleotide sequence that is complementary to its target. Preferably, an antisense sequence is constructed by inverting a region preceding or targeting the initiation codon or an unconserved region. In another embodiment the antisense sequence targets all or part of the mRNA or cDNA of CYP 27C1 or hybrid thereof. In particular, the nucleic acid sequences contained in the nucleic acid molecules of the invention or a fragment thereof, preferably a nucleic acid sequence shown in Figures 2A(SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44) may be inverted relative to its normal presentation for transcription to produce antisense nucleic acid molecules. In one embodiment the antisense molecules can be used to inhibit CYP 27C1 expression and/or vitamin D and/or cholesterol metabolism.

The antisense nucleic acid molecules of the invention or a fragment thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.

The invention also provides nucleic acids encoding fusion proteins comprising a novel protein of the invention and a selected protein, or a selectable marker protein (see below). The proteins of the invention (including modifications, variations, truncations, insertions, analogs, fusion proteins, etc.) may be prepared using recombinant DNA methods. These proteins may be purified and/or isolated to various degrees using techniques known in the art. Accordingly, nucleic acid molecules of the present invention having a sequence that encodes a protein of the invention may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression "vectors suitable for transformation of a host cell", means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. "Operatively linked" is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.

The invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a nucleotide sequence comprising the nucleotides as shown in Figure 2A(SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44) or fragments thereof. Regulatory sequences operatively linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of selectable marker genes are genes encoding a protein which confers resistance to certain drugs, such as G418 and hygromycin. Examples of other markers which can be used are: green fluorescent protein (GFP), b-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as b-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest. The recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of a target recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The term "transformed host cell" is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The terms "transformed with", "transfected with",

"transformation" and "transfection" are intended to encompass introduction of nucleic acid (e.g. a vector) into a cell by one of many possible techniques known in the art. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium chloride mediated transformation. Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other such laboratory textbooks. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).

The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). iii) Polynucleotide and Polypeptide Variants

"Variant" refers to a polynucleotide or polypeptide differing from the CYP21C1 polynucleotide or polypeptide or respective hybrids, but retaining essential properties thereof. Typically, variants are overall closely similar and, in many regions, identical to the CYP 27C1 polynucleotide or polypeptide and hybrids. The invention includes homologs, analogs and isoforms of the polypeptides and as applicable polynucleoitdes of the invention. Insertions, deletions, mutations and substitutions are also intended to be encompassed within the scope of the invention.

It will be appreciated that the invention includes polynucleotides comprising nucleic acid sequences having substantial sequence homology a identity with the sequences of Figures 2A(SEQ. ID. NO:39) and 11A(SEQ. ID. NO:44). The term "sequences having substantial sequence homology" means those nucleic acid sequences that have slight or inconsequential sequence variations from these sequences, i.e., the sequences function in substantially the same manner to produce functionally equivalent proteins. The variations may be attributable to local mutations or structural modifications. Preferably such polynucleotides have at least 85, preferably 90 and most preferably 95% identity with the sequence of Figures 2A(SEQ. ID. NO:39). or 11A(SEQ. ID. NO:44). However, it should be noted that the invention is not limited thereto and includes polynucletide sequence having at least 50 %, 60% and 70% homology to the sequence of Figures 2A(SEQ. ID. NO:39) and 11A(SEQ. ID. NO:44).

By a polynucleotide having a nucleotide sequence at least, for example, 90% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polypeptide is identical to the reference sequence except that the polynucleotide sequence may include up to ten point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the CYP21C1 polypeptide. Therefore, to obtain a polynucleotide having a nucleotide sequence at least 90% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 10% of the total nucleotides in the reference sequence may be inserted into a reference sequence. The query sequence may be an entire sequence of CYP 27C1 or any fragment specified as described herein. Whether a particular nucleic acid molecule or polypeptide is at least

90%, 95%, 96%, 97%, 98% or 99% homologous to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence [a sequence of the present invention] and a subject sequence, also referred to as a global alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6: 237-245 (1990). In a sequence alignment, the query and the subject sequence are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix -Unitary, k-tuple=4, Mismatch Penalty=1 , Joining Penalty=30, Randomization Group Length=09, Cutoff Score=1 , Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the result. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specific parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated from the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence [number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence] so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. As another example, a 90 base subject sequence may be compared with a 100 base query sequence. This time the deletion may be an internal deletion so that there are no bases on the 5' or 3' end of the subject sequence which are not matched/aligned with the query. In such a case, the percent identity calculated by FASTDB is not manually corrected. Only bases 5' and 3' of the subject sequence which are not match/aligned with the query sequence are manually corrected.

By a polypeptide having an amino acid sequence at least, for example, 90% "identical" or homologous to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to ten amino acid alterations per each 100 amino acids of the query amino acid sequence. Therefore, to obtain a polypeptide having an amino acid sequence at least 90% identical to a query amino acid sequence, up to 10% of the amino acid residues in the subject sequence may be inserted, deleted or substituted with another amino acid. The alterations in the reference sequences may occur at the amino or carboxy terminal position of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequences encoded by clone RP11-30F3 can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence [a sequence of the present invention] and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6: 237-245 (1990). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, Mismatch Penalty=1 , Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1 , Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C- terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C- terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C- termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is match/aligned is determined by results of FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specific parameters, to arrive at a final percent identity score. Only residues to the N- and C- termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence [number of residues at the N- and C- termini not matched/ total number of residues in the query sequence] so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C- termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Only residues positions outside the N- and C- terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for.

CYP 27C1 variants may contain alterations in the coding regions, non-coding regions or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted or added in any combination are also preferred. CYP 27C1 polynucleotide variants can be produced for a variety of reasons including to optimize codon expression for a particular host.

Naturally occurring CYP 27C1 variants are called "allelic variants" and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis. . It will be appreciated that variant forms of polynucleotides of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of CYP 27C1 polypeptide. For example, one or more amino acids may be deleted from the N- terminus or C- terminus of the protein without substantial loss of biological function [see for example Ron et al., J. Biol. Chem. 268: 2984-2988 (1993); Dobeli et al. J. Biotechnology 7; 199- 216 (1988); and Gayle et al. J. Biol. Chem. 268: 22105-22111]. If deleting one or more amino acids from the N-terminus or C- terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N- terminus or the C-terminus. Whether a particular polypeptide lacking N- or C- terminal residues of a protein retains such immunological activities can readily be determined by routine methods described herein and know in the art. The invention further includes CYP 27C1 polypeptide variants which show substantial biological activity. Such variants include deletions, insertions, inversions, repeats and substitutions selected according to general rules known in the art [see for example Bowie, J.U. et al., Science 247: 1306-1310 (1990)].

One strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acids positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Therefore, positions tolerating amino acid substitutions could be modified while still maintaining biological activity of the protein.

Another strategy employs genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis can be used [see for example Cunningham and Wells, Science 244; 1081-1085 (1989)]. The resulting polypeptide may be tested for biological activity.

Besides conservative amino acid substitutions, this invention contemplates variants of CYP 27C1 including [1] substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or [2] substitution with one or more of the amino acid residues having a substituent group, or [3] fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide or [4] fusion of the polypeptide with additional amino acids, such as an IgGFc fusion region peptide, or a sequence facilitating purification. Such variants are deemed to be within the scope of those skilled in the art from teachings herein.

For example, CYP 27C1 polypeptide variants containing amino acid substitutions of charged amino acids with another charged or neutral amino acids my produce polypeptides with improved characteristics [see for example. Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); and Clevland et al., Crit. Rev.

Therapeutic Drug Carrier System 10: 307-377 9!993)]. iv) Polynucleotide and Polypeptide Fragments

In the present invention "polynucleotide fragment" refers to a short polynucleotide having a nucleic acid sequence of Figure 2B(SEQ. ID.

NO:39) or 11 B(SEQ. ID. NO:44). The short nucleotide fragments are preferably at least about 15nt and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length. For example, a fragment "at least 20 nt is length" is intended to include 20 or more contiguous bases from the cDNA sequences of Figure 2A(SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44). The nucleotide fragments may be useful as diagnostic probes and primers. In addition, larger fragments are also useful as diagnostic probes [for example. 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, etc... nucleotides.]

In one embodiment the polynucleotide fragments of the invention preferably hybridize to nucleic acid molecules of the invention (such as Figure 2A(SEQ. ID. NO:39) and 11A(SEQ. ID. NO:44) under hybridization conditions, preferably stringent hybridization conditions. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed: 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C. The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be at high stringency conditions, such as at about 65°C. Examples of representative polynucleotide fragments are 1-50, 51-

100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801- 850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-500, 1501-1550, 1551-1600 , etc... to the end of the cDNA contained in Figure 2A (SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44). In a preferred embodiment the polynucleotide fragment comprises or consists of all or at least a 15 nucleotide portion of nucleotides 898-1061 of Figure 2A(SEQ. ID. NO:39). In this context, "about" includes the particular ranges that may be larger or smaller by several nucleotides at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein.

In the present invention, a "polypeptide fragment" refers to a short amino acid sequence encoded by the cDNA Figure 2A(SEQ. ID. NO:39) or 11A(SEQ. ID. NO:44) as depicted in Figure 2B (SEQ. ID. No: 40). Protein fragments may be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. For example, polypeptide fragments may include fragments from about amino acid number 1-20, 21-40, 41-60 etc. to the end of the coding region. The polypeptide fragments may be about 20, 30, 40 , 50, 60, 70, 80, 90, etc. amino acids in length. "About" includes the ranges described herein and ranges larger or smaller by several amino acids, at either extremes or both extremes.

Preferred polypeptide fragments include the naissant and mature forms of CYP 27C1 and CYP 27 C1 hybrid. Furthermore, any combination of amino and carboxy terminus deletions are preferred. For example, the ability of shortened CYP 27C1 mutants to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by methods known in the art. It is not unlikely that a CYP 27C1 mutant with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as five amino acid residues may evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of CYP 27C1 or the hybrid. For example, the ability of the shortened CYP 27C1 mutant to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C- terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described in the art. It is not unlikely that a CYP 27C1 mutant with a large number of deleted C-terminal amino acid residues may retain some biological or immunological activities.

The invention also contemplates polypeptides having one or more amino acids deleted from both the amino and the carboxy termini of CYP 27C1 polypeptide.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences would be related to CYP 27C1 sequence and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Preferred fragments are those demonstrating some biological or biochemical activity, preferably functional fragments or obvious chemical equivalents to CYP 21 C1 , such fragments are those exhibiting activity similar, but not necessarily identical to CYP 27C1 polypeptide or polynucleotide. The activity may include an improved desired activity or a decreased undesired activity. Such fragments would also include, but is not necessarily limited to any polypeptide or polynucleotide fragments which are beneficial in the modulation or simulation of CYP 27C1 or CYP 27C1 expression, or in the identification or production of such agents. v) Epitopes and Antibodies

In the present invention, "epitope" refers to CYP 27C1 or corresponding hybrid polypeptide fragments having antigenic or immunogenic activity in an animal. In one embodiment embodiment the present invention provides a CYP 27C1 fragment comprising an epitope, as well as the polynucleotide encoding the said fragment. A region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope". In contrast, an "immunogenic epitope" is defined as a part of a protein that elicits an antibody response [see for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81 : 3998-4002 (1983)].

Fragments which function as epitopes may be produced by any conventional means, [see for example, Houghten, R.A., Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985)].

In the present invention, antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably, between about 15 to about 30 amino acids. Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specify binding the epitope [see for example, Wilson et al., Cell 37: 767-778 (1984); and Sutcliffe J.G. et al., science 218: 660-666].

Similarly, immunogenic epitopes can be used to induce T cells according to methods well known in the art [see for example, Chow, M. et al. Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, F. J. et al. J. Gen. Virol. 66: 2347-2354 (1985)]. The immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acides, without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide such as Western blotting. As used herein, the term "antibody" [Ab] or "monoclonal antibody" [mAb] is meant to include intact molecules as well as antibody fragments [for example Fab and F(ab')2 fragments] which are capable of specifically binding proteins. Such fragments lack the Fc fragment of intact antibody and are typically produced by proteolytic cleavage using enzymes such as papin (to produce Fab fragments) or pepsin (to produce F(ab') fragments). Fab and F(ab')2 fragments clear more rapidly from the circulation and may have less non-specific tissue binding than an intact antibody [see for example Wahl et al., J. Nucl. Med. 24: 316-325 (1983)]. Thus these fragments are preferred, as are the products of a Fab or other immunoglobulin expression library. This invention includes chimeric, single chain and humanized antibodies. In addition, target protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry. These methodologies are known in the art. For further references, see examples including Current Protocols in Immunology, John Wiley & Sons, New York; Kennett, R. et al, eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analysis, Plenum Press, New York (1980) and Campbell, A., "Monoclonal Antibody Technology" in Burden, R., et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984).

Antibodies generated against a target epitope can be obtained by direct injection of the epitope or polypeptide into an animal or by administrating the polypeptides to an animal, preferably a nonhuman. Such an antibody will then bind the polypeptide itself. With this method, a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can be used to isolate the polypeptide encoding the polypeptide from an expression library using the method described herein. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique [Kohler and Milstein, Nature 256: 495-497 (1975)], the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunol. Today 4: 72 (1983)], and the EBV- hybridoma technique to produce human monoclonal antibodies [Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. p 77- 96 (1985)].

Techniques described for the production of a single chain antibodies [U.S. Patent 4,946,778] can be adapted to produce single chain antibodies to immunogenic polypeptide products of interest.

The antibodies useful in the present invention may be prepared by any of a variety of methods known in the art. For example, cells expressing the target protein or an antigenic fragment thereof can be administered to an animal in order to induce the introduction of sera containing polyclonal antibodies. In another method, a preparation of target protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In a preferred method, antibodies used in the present invention are monoclonal antibodies [or target protein-binding fragments thereof]. Such monoclonal antibodies can be prepared using hybridoma technology known in the art. In general, such procedures involve immunizing an animal [preferably a mouse] with a target protein antigen or, preferably, with a target protein-expressing cell. Suitable cells can be recognized by their capacity to bind an anti-target protein antibody. Such cells may be cultured in any suitable tissue culture medium. The splenocytes of immunized mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention. Preferably SP20 myeloma cell line is used which is available from American Type Culture Collection, Mannassas, V After fusion, the resulting hybridoma cells are selectively maintained in HAT medium followed by cloning out by limited dilution as described in the art [see for example, Wands et al., Gastroenterology 80: 225-232 (1981)]. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the target protein antigen.

Alternatively, additional antibodies capable of binding to the target protein antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to second antibody. With this method, target-protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the target protein-specific antibody can be clocked by the target antigen. Such antibodies comprise anti-idiotypic antibodies to the target protein-specific antibody and can be used to immunize an animal to induce formation of further target protein-specific antibodies.

Suitable labels for the target protein-specific antibodies of the present invention are provided below. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

Examples of suitable radioactive labels include ³H, ¹¹¹ln, ¹²⁵l, ¹³¹l, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ∞Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd etc.. Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o- phthaldehyde label and a fluorescamine label. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label and an aequorin label. Examples of nuclear magnetic resonance contrasting agents include heavy metal nuclei such as Gd, Mn and iron.

Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al., Cin. Chem. Acta. 70; 1-31 (1976) and Schurs et al., Clin. Cem. Acta 82: 1-40 (1970). Coupling techniques mentioned in the latter are gluteraldehyde method, the periodate method, the dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are incorporated by reference herein.

For in vivo use of antibodies in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described herein. Methods for producing chimeric antibodies are known in the art. ( For example, Morrison, Science 229:1202 (1985); Oi et al. BioTechniques 4:214 91986); cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al, WO 8601533; Robinson et al, WO 8702671 ; Boulianne er a/, Nature 312:643 (1984); Neuberger et al, Nature 314: 268 (1985).) vi) Disease States Diagnosis and Prognosis

The nucleic acid molecules, CYP 27C1 Related Proteins, and antibodies of the invention may be used in the prognostic and diagnostic evaluation of disorders involving a CYP 27C1 Related Protein (e.g. cancer or vitamin D, cholesterol, steroid or other lipid metabolic disorders), and the identification of subjects with a predisposition to such disorders. It is believed that certain conditions may cause mammals to express significantly altered levels of CYP 27C1 protein and mRNA levels encoding

CYP 27C1 protein when compared to a corresponding "standard" mammal i.e., a mammal of the same species not having the condition. Further irrespective of CYP 27C1 , levels modulators of CYP 27C1 expression and/or activity may be useful in the treatment of certain conditions.

Cytochrome P450s have been associated with a number of pathways and have been implicated in a number of medical conditions. In addition to conditions which may be associated with cytochrome P450s in general, CYP 27C1 is expressed in many different cell types but has elevated expression in Hep3B, NT-2, colon, fetal tissues and other tumour cell lines (see examples), suggesting a role in cellular differentiation of carcinogenic, tumor and embryonic cell lines. This the polypeptides and polynucleotides of this invention or modulators thereof may be useful in diagnosing and treating conditions related to cellular differentiation, such as cancer or developmental disorders.

In a particular embodiment of the invention, the nucleic acid molecules, CYP 27C1 Related Proteins, and antibodies of the invention may be used in the diagnosis and staging of CYP 27C1 related disorders.

Increased and decreased levels of CYP 27C1 Related Proteins may be associated with different forms of cancer on other conditions and may be an indicator of prognosis. Methods for detecting nucleic acid molecules and CYP 27C1 Related

Proteins of the invention, can be used to monitor disorders involving a CYP

27C1 Related Protein by detecting CYP 27C1 Related Proteins and nucleic acid molecules encoding CYP 27C1 Related Proteins. The applications of the present invention also include methods for the identification of compounds that modulate the biological activity of CYP 27C1 Related

Proteins. The compounds, antibodies etc. may be used for the treatment of disorders involving a CYP 27C1 Related Protein. It would also be apparent to one skilled in the art that the methods described herein may be used to study the developmental expression of CYP 27C1 Related Proteins and, accordingly, will provide further insight into the role of CYP 27C1 Related

Proteins. Further, P450c27 (CYP27A1), which has similarities to CYP 27C1 has been associated with the degradation of the side chain of C27 steroids in the hepatic bile acid biosynthesis pathway, which begins with7α- hydroxylation of cholesterol in liver. However, recognition that in humans P450c27 is a widely or ubiquitously expressed mitochondrial P450, and that there are alternative pathways of bile acid synthesis which begin with 27-hydroxylation of cholesterol catalyzed by P450c27, suggests the need to reevaluate the role of this enzyme and its catalytic properties. 27- Hydroxycholesterol was thought to be the only product formed upon reaction of P450c27 with cholesterol. However, the present study demonstrates that recombinant human P450c27 is also able to further oxidize 27-hydroxycholesterol giving first an aldehyde and then 3b-hydroxy- 5-cholestenoic acid. Kinetic data indicate that in a reconstituted system, after 27-hydroxycholesterol is formed from cholesterol, it is released from the P450 and then competes with cholesterol for reentry the enzyme active site for further oxidation. Under subsaturating substrate concentrations, the efficiencies of oxidation of 27-hydroxycholesterol and 3b-hydroxy-5- cholestenal to the acid by human P450c27 are greater than the efficiency of hydroxylation of cholesterol to 27-hydroxycho-lesteroI indicating that the first hydroxylation step in the overall conversion of cholesterol into 3b-hydroxy-5- cholestenoic acid is rate-limiting. Interestingly, 3b-hy-droxy-5-cholestenoic acid was found to be further metabolized by the recombinant human P450c27, giving two monohydroxylated products with the hydroxy group introduced at different positions on the steroid nucleus. This CYP 27C1 may also play a role in cholesterol/bile acid metabolism and associated conditions and thus the polypeptides and polynucleotides of this invention and/or modulators of CYP 27C1 activity may be useful in diagnosing and/or treating such conditions.

Further, CYP 27C1 or the polypeptides, polynucleotides of this invention and/or modulators of CYP 27C1 activity may be useful in treating disorders or conditions involving the vitamin D pathway, such as those noted herein. For example, low levels of circulating 25(OH)D can contribute to cholestatic or paremchymal liver disease. Premature infants have also been found to be unable to metabolize vitamin D to 25(OH)D, and low levels of 25(OH)D have been found in people with obesity problems, hyperhoshatemic tumoral calcinosis, sarcoidosis, tuberculosis, primary hyperparathyroidism and vitamin D dependent rickets type II.

Where a diagnosis has already been made according to conventional methods, the present invention is useful as a prognostic indicator whereby patients exhibiting altered CYP 27C1 gene expression will experience a worse clinical outcome relative to patients expressing the gene at a normal level.

By "assaying the expression of the gene encoding the CYP 27C1 polypeptide" is intended qualitatively and quantitatively measuring or estimating the level of CYP 27C1 protein or the level of the mRNA encoding the CYP 27C1 protein in a first biological sample either directly [e.g., by determining or estimating absolute protein level or mRNA level] or relatively [e.g. by comparing to the CYP 27C1 protein level or mRNA level in a second biological sample].

Preferably, the CYP 27C1 protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard CYP 27C1 protein level or RNA level, the standard being taken from a second biological sample obtained from an individual not having the condition. As will be appreciated in the art, once a standard CYP 27C1 protein level or mRNA level is known, it can be used repeatedly as a standard for comparison. By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture or other source which contains CYP21C1 protein or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits, mice, and humans. Particularly preferred are humans. Total cellular RNA can be isolated by methods well known in the art [see for example, Chmczynski and ZSacchi, Anal. Biochem. 162: 156-159 (1987)]. Levels of mRNA encoding the CYP 27C1 protein are then assayed using an appropriate method. These include Northern blot analysis [Harada et al., Cell 63: 303-312 (1990)], S1 nuclease mapping [Fujita et al., Cell 49: 357-367 (1987)], the polymerase chain reaction [PCR], reverse transcription in combination with the polymerase chain reaction [RT-PCR] [Mariko et al., Technique 2: 295-301 (1990)] and reverse transcription in combination with the ligase chain reaction [RT-LCR]. Assaying CYP 27C1 protein levels in a biological sample can occur using antibody-based techniques. For example, CYP 27C1 protein expression in tissue can be studied with classical immunohistological methods known in the art [for example, Jalkanen, M., et al., J. Cell Biol. 105: 3087-3096 (1987)]. Other antibody-based methods used for detecting CYP 27C1 protein gene expression including immunoassays, such as enzyme linked immunosorbent assay [ELISA] and the radioimmunoassay [RIA].

Suitable labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotope, such as iodine [¹²⁵l, ¹²¹l], carbon [¹⁴C], sulfer [³⁵S], tritium [³H], indium [¹¹²ln] and technetium [^99mTc] and fluorescent labels, such as fluorescein and rhodamine and biotin.

Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into antibody by labelling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labelled an appropriate detectable imaging moiety, such as a radioisotope [for example, ¹³¹l, ^112ln, ^99mTC], a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced [for example, parenterally, subcutaneously, or intraperitoneally] into mammals. It will be understood in the art that the size of the subject and the imaging system used will determine the quality of imaging moiety needed to produce diagnostic images. In vivo tumour imaging is described in S.W. Burchiel et al., "Immunopharmackinetics of Radiolabeled Antibodies and Their Fragments" [see also, Chapter 13 in Tumour Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and BA Rhodes, eds., Masson Publishing Inc. (1982)]. Diagnostic Methods A variety of methods can be employed for the diagnostic and prognostic evaluation of disorders involving a CYP 27C1 Related Protein, and the identification of subjects with a predisposition to such disorders. Such methods may, for example, utilize nucleic acid molecules of the invention, and fragments thereof, and antibodies directed against CYP 27C1 Related Proteins, including peptide fragments. In particular, the nucleic acids and antibodies may be used, for example, for: (1) the detection of the presence of CYP 27C1 mutations, or the detection of either over- or under- expression of CYP 27C1 mRNA relative to a non-disorder state or the qualitative or quantitative detection of alternatively spliced forms of CYP 27C1 transcripts which may correlate with certain conditions or susceptibility toward such conditions; and (2) the detection of either an over- or an under-abundance of CYP 27C1 Related Proteins relative to a non- disorder state or the presence of a modified (e.g., less than full length) CYP 27C1 Protein which correlates with a disorder state, or a progression toward a disorder state.

The methods described herein may be performed by utilizing prepackaged diagnostic kits comprising at least one specific CYP 27C1 nucleic acid or antibody described herein, which may be conveniently used, e.g., in clinical settings, to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a disorder.

Nucleic acid-based detection techniques are described, below. Peptide detection techniques are described, below. The samples that may be analyzed using the methods of the invention include those which are known or suspected to express CYP 27C1 or contain CYP 27C1 Related Proteins. The samples may be derived from a patient or a cell culture, and include but are not limited to biological fluids, tissue extracts, freshly harvested cells, and lysates of cells which have been incubated in cell cultures.

Oligonucleotides or longer fragments derived from any of the nucleic acid molecules of the invention may be used as targets in a microarray. The microarray can be used to simultaneously monitor the expression levels of large numbers of genes and to identify genetic variants, mutations, and polymorphisms. The information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents. The preparation, use, and analysis of microarrays are well known to a person skilled in the art. (See, for example, Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, et al. (1996) Proc. Natl. Acad. Sci. 93:10614- 10619; Baldeschweiler et al. (1995), PCT Application WO95/251116; Shalon, D. et al. (I 995) PCT application WO95/35505; Heller, R. A et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Methods for Detecting Nucleic Acid Molecules of the Invention

The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences of the invention in samples. Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding at least 5 sequential amino acids from regions of the CYP 27C1 Protein, preferably they comprise 15 to 30 nucleotides. A nucleotide probe may be labeled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as ³²P, ³H, ¹⁴C or the like. Other detectable substances which may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleic acid probes may be used to detect genes, preferably in human cells, that encode CYP 27C1 Related Proteins. The nucleotide probes may also be useful in the diagnosis of disorders involving a CYP 27C1 Related Protein; in monitoring the progression of such disorders; or monitoring a therapeutic treatment.

The probe may be used in hybridization techniques to detect genes that encode CYP 27C1 Related Proteins. The technique generally involves contacting and incubating nucleic acids (e.g. recombinant DNA molecules, cloned genes) obtained from a sample from a patient or other cellular source with a probe of the present invention under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.

The detection of nucleic acid molecules of the invention may involve the amplification of specific gene sequences using an amplification method such as PCR, followed by the analysis of the amplified molecules using techniques known to those skilled in the art. Suitable primers can be routinely designed by one of skill in the art.

Genomic DNA may be used in hybridization or amplification assays of biological samples to detect abnormalities involving CYP 27C1 structure, including point mutations, insertions, deletions, and chromosomal rearrangements. For example, direct sequencing, single stranded conformational polymorphism analyses, heteroduplex analysis, denaturing gradient gel electrophoresis, chemical mismatch cleavage, and oligonucleotide hybridization may be utilized. Genotyping techniques known to one skilled in the art can be used to type polymorphisms that are in close proximity to the mutations in a CYP 27C1 gene. The polymorphisms may be used to identify individuals in families that are likely to carry mutations. If a polymorphism exhibits linkage disequalibrium with mutations in a CYP 27C1 gene, it can also be used to screen for individuals in the general population likely to carry mutations. Polymorphisms which may be used include restriction fragment length polymorphisms (RFLPs), single-base polymorphisms, and simple sequence repeat polymorphisms (SSLPs). A probe of the invention may be used to directly identify RFLPs. A probe or primer of the invention can additionally be used to isolate genomic clones such as YACs, BACs, PACs, cosmids, phage or plasmids. The DNA in the clones can be screened for SSLPs using hybridization or sequencing procedures. Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of CYP 27C1 expression. For example, RNA may be isolated from a cell type or tissue known to express CYP 27C1 and tested utilizing the hybridization (e.g. standard Northern analyses) or PCR techniques referred to herein. The techniques may be used to detect differences in transcript size which may be due to normal or abnormal alternative splicing. The techniques may be used to detect quantitative differences between levels of full length and/or alternatively splice transcripts detected in normal individuals relative to those individuals exhibiting symptoms of a disorder involving a CYP 27C1 Related Protein.

The primers and probes may be used in the above described methods in situ i.e directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections. Methods for Detecting CYP 27C1 Related Proteins Antibodies specifically reactive with a CYP 27C1 Related Protein, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect CYP 27C1 Related Proteins in various samples (e.g. biological materials). They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of CYP 27C1 Related Protein expression, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of a CYP 27C1 Related Protein. Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on disorders involving a CYP 27C1 Related Protein, and other conditions. In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies. The antibodies of the invention may also be used in vitro to determine the level of CYP 27C1 expression in cells genetically engineered to produce a CYP 27C1 Related Protein.

The antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of a CYP 27C1 Related Protein and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests. The antibodies may be used to detect and quantify CYP 27C1 Related Proteins in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.

In particular, the antibodies of the invention may be used in immunohistochemical analyses, for example, at the cellular and sub-subcellular

level, to detect a CYP 27C1 Related Protein, to localize it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.

Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect a CYP 27C1

Related Protein. Generally, an antibody of the invention may be labeled with a detectable substance and a CYP 27C1 Related Protein may be localised in tissues and cells based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g., ³ H, ¹⁴C, ³⁵S, ¹²⁵l, ¹³¹l), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy. The antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip). Indirect methods may also be employed in which the primary antigen- antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against CYP 27C1 Related Protein. By way of example, if the antibody having specificity a CYP 27C1 Related Protein is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labeled with a detectable substance as described herein.

Where a radioactive label is used as a detectable substance, a CYP 27C1 Related Protein may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains. ln an embodiment, the invention contemplates a method for monitoring the progression of a CYP 27C1 disorder in an individual, comprising:

(a) contacting an amount of an antibody which binds to a CYP 27C1 Related Protein, with a sample from the individual so as to form a binary complex comprising the antibody and CYP 27C1 Related Protein in the sample;

(b) determining or detecting the presence or amount of complex formation in the sample; (c) repeating steps (a) and (b) at a point later in time; and

(d) comparing the result of step (b) with the result of step (c), wherein a difference in the amount of complex formation is indicative of the progression of the cancer in said individual.

The amount of complexes may also be compared to a value representative of the amount of the complexes from an individual not at risk of, or afflicted with, a CYP 27C1 condition. This method can also be used to distinct CYP 27C1. Methods for Identifying or Evaluating Substances/Compounds

The methods described herein are designed to identify substances that modulate the biological activity of a CYP 27C1 Related Protein including substances that bind to CYP 27C1 Related Proteins, or bind to other proteins that interact with a CYP 27C1 Related Protein, to compounds that interfere with, or enhance the interaction of a CYP 27C1 Related Protein and substances that bind to the CYP 27C1 Related Protein or other proteins that interact with a CYP 27C1 Related Protein. Methods are also utilized that identify compounds that bind to CYP 27C1 regulatory sequences.

The substances and compounds identified using the methods of the invention include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or

L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)2, and Fab expression library fragments, and epitope-binding fragments thereof)], and small organic or inorganic molecules. The substance or compound may be an endogenous physiological compound or it may be a natural or synthetic compound.

Substances which modulate a CYP 27C1 Related Protein can be identified based on their ability to bind to a CYP 27C1 Related Protein. Therefore, the invention also provides methods for identifying substances which bind to a CYP 27C1 Related Protein. Substances identified using the methods of the invention may be isolated, cloned and sequenced using conventional techniques. A substance that associates with a polypeptide of the invention may be an agonist or antagonist of the biological or immunological activity of a polypeptide of the invention.

The term "agonist", refers to a molecule that increases the amount of, or prolongs the duration of, the activity of the protein. The term "antagonist" refers to a molecule which decreases the biological or immunological activity of the protein. Agonists and antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that associate with a protein of the invention.

Substances which can bind with a CYP 27C1 Related Protein may be identified by reacting a CYP 27C1 Related Protein with a test substance which potentially binds to a CYP 27C1 Related Protein, under conditions which permit the formation of substance-CYP 27C1 Related Protein complexes and removing and/or detecting the complexes. The complexes can be detected by assaying for substance-CYP 27C1 Related Protein complexes, for free substance, or for non-complexed CYP 27C1 Related Protein. Conditions which permit the formation of substance-CYP 27C1 Related Protein complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein. The substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against CYP 27C1 Related Protein or the substance, or labeled CYP 27C1 Related Protein, or a labeled substance may be utilized. The antibodies, proteins, or substances may be labeled with a detectable substance as described above. A CYP 27C1 Related Protein, or the substance used in the method of the invention may be insolubilized. For example, a CYP 27C1 Related Protein, or substance may be bound to a suitable carrier such as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.

The invention also contemplates a method for evaluating a compound for its ability to modulate the biological activity of a CYP 27C1 Related Protein of the invention, by assaying for an agonist or antagonist (i.e. enhancer or inhibitor) of the binding of a CYP 27C1 Related Protein with a substance which binds with a CYP 27C1 Related Protein. The basic method for evaluating if a compound is an agonist or antagonist of the binding of a CYP 27C1 Related Protein and a substance that binds to the protein, is to prepare a reaction mixture containing the CYP 27C1 Related Protein and the substance under conditions which permit the formation of substance-CYP 27C1 Related Protein complexes, in the presence of a test compound. The test compound may be initially added to the mixture, or may be added subsequent to the addition of the CYP 27C1 Related Protein and substance. Control reaction mixtures without the test compound or with a placebo are also prepared. The formation of complexes is detected and the formation of complexes in the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of the CYP 27C1 Related Protein and substance. The reactions may be carried out in the liquid phase or the CYP 27C1 Related Protein, substance, or test compound may be immobilized as described herein. The ability of a compound to modulate the biological activity of a CYP 27C1 Related Protein of the invention may be tested by determining the biological effects on cells. It will be understood that the agonists and antagonists i.e. inhibitors and enhancers that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non- competitive antagonist binding sites or allosteric sites. The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of CYP 27C1 Related Protein with a substance which is capable of binding to the CYP 27C1 Related Protein. Thus, the invention may be used to assay for a compound that competes for the same binding site of a CYP 27C1 Related Protein. The invention also contemplates methods for identifying compounds that bind to proteins that interact with a CYP 27C1 Related Protein. Protein- protein interactions may be identified using conventional methods such as co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns. Methods may also be employed that result in the simultaneous identification of genes which encode proteins interacting with a CYP 27C1 Related Protein. These methods include probing expression libraries with labeled CYP 27C1 Related Protein.

Two-hybrid systems may also be used to detect protein interactions in vivo. Generally, plasmids are constructed that encode two hybrid proteins. A first hybrid protein consists of the DNA-binding domain of a transcription activator protein fused to a CYP 27C1 Related Protein, and the second hybrid protein consists of the transcription activator protein's activator domain fused to an unknown protein encoded by a cDNA which has been recombined into the plasmid as part of a cDNA library. The plasmids are transformed into a strain of yeast (e.g. S. cerevisiae) that contains a reporter gene (e.g. lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the transcription activator's binding site. The hybrid proteins alone cannot activate the transcription of the reporter gene. However, interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. It will be appreciated that fusion proteins may be used in the above- described methods. In particular, CYP 27C1 Related Proteins fused to a glutathione-S-transferase may be used in the methods.

A modulator of a CYP 27C1 Related Protein of the invention may also be identified based on its ability to inhibit or enhance catalytic activity of the protein.

The reagents suitable for applying the methods of the invention to evaluate compounds that modulate a CYP 27C1 Related Protein may be packaged into convenient kits providing the necessary materials packaged into suitable containers. The kits may also include suitable supports useful in performing the methods of the invention. vii) Fusion proteins

Any CYP 27C1 polypeptide (any polypeptide of the invention - hybrid, variations, fragments, etc..) may be used to generate fusion proteins. For example, the CYP 27C1 polypeptide, when fused to a second polypeptide, can be used as an antigenic tag. Antibodies raised against the CYP 27C1 polypeptide can be used to indirectly detect the second protein by binding to the CYP 27C1 polypeptide. Examples of domains that can be fused to CYP 27C1 include heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences. In certain preferred embodiments, CYP 27C1 fusion polypeptides may be constructed which include additional N-terminal and/or C-terminal amino acid residues. In particular, any N-terminally or C-terminally deleted CYP 27C1 polypeptide disclosed herein may be altered by inclusion of additional amino acid reisdues at the N-terminus to produce a CYP 27 C1 fusion polypeptide, In addition, CYP 27 C1 fusion polypeptides are contemplated which include additional N-terminal and/or C-terminal amino acid residues fused to a CYP 27 C1 polypeptides comprising any combination of N- and C-terminal deletions set forth above.

In addition, fusion proteins may be engineered to improve characteristics of the CYP 27C1 polypeptide. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the CYP 27C1 polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Furthermore, peptide moieties may be added to the CYP 27C1 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the CYP 27C1 polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

CYP 27C1 polypeptides, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins [IgG], resulting in chimeric polypeptides. These proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394, 827; Traunecker eta , Nature 331: 84-86 (1988). Fusion proteins having disulphide-linked dimeric structures [due to the IgG] can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone [see for example, Fountoulakis et al., J. Biochem. 270: 3958-3964 (1995)].

Similarly, EP-A-O 469 533 (Canadian counterpart 2045869) disclosed fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In addition, in drug discovery, human proteins, such as hlL-5, have been fused with FC portion for the purpose of high-throughout screening assays to identify antagonists of hlL-5 [see for example, Bennet, D. et al., J. Molecular Recognition 8: 52-58 (1995); K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)].

Moreover, the CYP 27C1 polypeptides of the invention can be fused to other proteins, e.g. NADPH cytochrome P450 reductase, NADPH ferredoxin reductase, other flavoproteins or ferrodixins or other proteins or co-factors which may function as a cytochrome CYP 27C1 reductase or faciliatate such an activity to create a multiprotein fusion complex. Such a multiprotien fusion complex may function as an enzymatically active covalently linked CYP 27C1 -reductase complex. A multiprotein complex can be cynthesized by the means of chemical crosslinking or assembled via novel intramolecular interactions, e.g., by the use of specific antibodies stablizing the complex.

CYP 27C1 polypeptide can be fused to hydrophillic molecules, including but not limited to polyethylene glycol and modified oligosaccharide and polysaccharides, whereby the hydrophobic moieties are used to stabilize CYP 27C1 interactions with other proteins, natural membranes, or artificial membranes, or to create new interactions with other proteins, natural membranes or artificial membranes. Fusion of the CYP 27C1 polypeptide to hydrophillic molecules can also be used to change its solubility.

CYP 27C1 polypeptide variants which contain non-standard amino acids or additional chemical modifications which have use in purification, stabilization or identification of the resulting modified CYP 27C1 protein, or influence its other properties such as enzymatic activity or interaction with other proteins, membranes, solid supports or chromatographic resin are contemplated. This includes, but is not limited to, biotinylated derivatives or fusions of CYP 27C1 polypeptides.

Also, modification of the CYP 27C1 polynucleotide sequences include those where relevant regions of the CYP 27C1 gene or polypeptide are inserted into another gene sequence to create a chimeric protein with a desired activity (enzymatic or otherwise). Such chimeric proteins can be obtained by, for example, replacing regions of other cytochrome P450 genes or polypeptides with a relevant CYP 27C1 regions whereby such a modifcation confers a new functional property to the resulting chimeric protein, including but not limited to new specificity, changed enzymatic kinetics, new or changed interactions with reductase or other relevant molecules or membranes, changed solubility and changed stability.

Moreover, the CYP 27C1 polypeptide can be used to marker sequences, such as a peptide which facilitates purification of CYP 27C1. In preferred embodiments, the marker amino acid sequence is a hexa- histidine peptide [His-tag], such as the tag provided in a pQE vector [QIAGEN, In., 9259 Eton Avenue, Chatsworth, CA, 91311], among others, many of which are commercially available, [for example see Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989)], for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponding to an epitope derived from the influenza haemagglutonin protein [see for example, Wilson et al., Cell 37: 767 (1984)]. Any of these fusions can be engineered using the CYP 27C1 polynucleotides or the CYP 27C1 polypeptides of this invention. viii) Vectors. Host Cells and Protein Production The present invention also relates to vectors containing the polynucleotides of the invention, preferably the polynucleotide encoding CYP 27C1 , host cells and to the production of the polypeptides of the invention, preferably the CYP 27C1 polypeptide, by recombinant techniques. For example, the vector may be a phage, plasmid, viral, or retroviral. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

Introduction of constructs into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).

CYP 27C1 can be recovered and purified from recombinant cell cultures by methods well-known in the art including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ["HPLC"] may be employed for purification.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary and immortalized host cells of vertebrate origin, particularly mammalia, origin, that have been engineered to delete or replace endogenous genetic material and / or to include genetic material [e.g. heterologous polynucleotide sequences] that is operably associated with CYP 27C1 polynucleotide of the invention, and which activates, alters, and/or amplifies endogenous CYP 27C1 polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions [e.g. promoter and/or enhancer] and endogenous CYP 27C1 polynucleotide sequences via homologous recombinations [see for example Koller et al., Proc. Natl. Acad. Sci. 86: 8932-8935 (1989); and Zijilstra et al., Nature 342: 435-438 (1989)]. ix) Uses of CYP 27C1 Polynucleotide The CYP 27C1 polynucleotides referred to herein can be used in numerous ways as agents. The following describes some examples using techniques know in the art. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data [repeat polymorphisms], are presently available.

Briefly, sequences can be mapped to chromosomes by PCR primers [preferably 5-25bp] from the sequence shown in Figure 2A. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human CYP 27C1 gene corresponding to the given sequence (preferably the sequence of Figure 2A) will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping of the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycle. Moreover, sublocalization of the CYP 27C1 polynucleotide may be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, precreening with labelled flow-sorted chromosomes and preselection by hybridization to construct chromosomes specific-cDNA libraries. Precise chromosomal location of CYP 27C1 polynucleotides can also be achieved using fluorescent in situ hybridization [FISH] of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred [see for example. Verma et al., "Human Chromosomes: a Manual of Basic Techniques", Pergamon Press, New York (1988)].

For chromosome mapping, the CYP 27C1 polynucleotide can be used individually [to mark a single chromosome or a single site on that chromosome] or in panel [for marking multiple sites and/or multiple chromosomes]. Preferred polynucleotides corresponding to the noncoding regions of the cDNAs becomes the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping. Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Therefore, once coinheritance is established, differences in the CYP 27C1 polynucleotide and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosomes spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the CYP 27C1 polynucleotide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis. The presence of a polymorphism can also be indicative of a disease or a predisposition to a disease. Therefore, a method of diagnosis of a CYP 27C1 -related disease or predisposition to a CYP 27C1 related disease, by identifying a polymorphism in CYP 27C1 gene, is also contemplated by this invention. In addition, a diagnostic kit for identification of polymorphisms in the CYP 27C1 gene by screening the CYP 27C1 gene from human for polymorphisms is also an embodiment of the present invention.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using CYP 27C1 polynucleotides. Any of these alterations [altered expression, chromosomal rearrangement or mutation] can be used as a diagnostic or prognostic marker. ln addition, CYP 27C1 polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polypeptide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription or to the mRNA itself [see for example, Dervan et al., Svience 251 : 1360 (1991); Okano, J. Neurochem. 56; 560 (1991)]. Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotide in an effort to treat disease.

CYP 27C1 polynucleotides are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the gene defect. CYP 27C1 offers a means of targeting such genetic defects in a highly accurate manner. Thus, for example, cells removed from a patient can be engineered with a CYP 27C1 polynucleotide [DNA or RNA] encoding a CYP 27C1 polypeptide ex vivo, with the engineered cells then being infused back into a patent to be treated with the polypeptide. Such methods are well-known in the art. For example, cells can be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding CYP 27C1.

Another goal of gene therapy is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. The CYP 27C1 polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism [RFLP] for identification of its person. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to yield unique bands for identifying personnel. The CYP 27C1 polynucleotides can be used as additional DNA markers for RFLP. The CYP 27C1 polynucleotides can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences.

Forensic biology also benefits from using DNA-based identification techniques as described herein. DNA sequences taken from very small biological samples such as tissues e.g., hair, or skin or body fluids such as blood or saliva can be amplified using PCR [see for example Erlich, H. PCR Technology, Freeman and Co. (1992)]. Similarly, CYP 27C1 polynucleotide can be used as polymorphic markers for forensic purposes.

The invention provides a diagnostic method of a disorder, which involves: [1] assaying CYP 27C1 gene expression level in a biological sample from the individual, such as a tissue or cell sample of an individual; [2] comparing the CYP 27C1 gene expression level with a standard CYP 27C1 gene expression level, whereby an increase or decrease in the assayed CYP 27C1 gene expression level compared to the standard expression level is indicative of the disorder.

In the very least, the CYP 27C1 polynucleotide can be used as a molecular weight marker on Southern gels, as diagnostic probes for the presence of a specific mRNA in a cell type, as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a "gene chip" or other support, or raise anti-DNA antibodies using DNA immunization techniques and as an antigen to elicit an immune response. x) Uses of CYP 27C1 Polypeptides

CYP 27C1 polypeptide can be used in a number of ways including the following examples.

CYP 27C1 polypeptide can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissue can be studied with classical immunohistological techniques [see for example, Jalkanen, M. et al,., J. Cell Biol. 105: 3087- 3096 (1987)]. Other antibody-based methods used for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine [¹²⁵l, ¹²¹l], carbon [¹⁴C], sulfer [³⁵S], tritium [³H], indium [¹¹²ln] and technetium [^99mTc] and fluorescent labels, such as fluorescein and rhodamine and biotin. Moreover, the CYP 27C1 polypeptides of the invention can be used to treat disease. For example, patients can be administered CYP 27C1 polypeptides in an effort to replace absent or decreased levels of the CYP 27C1 polypeptide, to supplement absent or decreased levels of a different polypeptide or molecule, to inhibit the activity of a polypeptide to activate the activity of a polypeptide to reduce the activity of a membrane bound receptor by competing with it for free ligand, or to bring about a desired response.

Antibodies directed to CYP 27C1 polypeptide may be used to treat disease. As described in detail in the "Epitopes and Antibodies" section herein the polypeptides of the present invention can be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting CYP 27C1 protein expression from a recombinant cell, as a way of assessing transformation of the host cell, or as antagonists capable of inhibiting CYP 27C1 protein function. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor). Further, such polypeptides can be used, in the yeast two-hybrid system to "capture" CYP 27C1 protein binding proteins which are also candidate agonist and antagonist according to the present invention. The yeast two hybrid system is described in Fields and Song, Nature 340:245-246 (1989). Small molecules that are specific substrates or metabolites of CYP

27C1 protein can also be used in the diagnosis or analysis of disease state involving CYP 27C1 or to monitor progress of therapy. CYP 27C1 or its derivatives, CYP 27C1 fusions, complexes and chimeric proteins can also be used in the analysis of individual chemicals or complex mixtures of chemicals including, but not limited to, the screening for improved or changed small molecules. These molecules may have use in development of new therapeutic agents or new diagnostic methods for CYP 27C1 -related disorders.

CYP 27C1 or its derivatives, CYP 27C1 fusions, complexes and chimeric proteins in an isolated state or as a part of complex mixtures can also be used to synthesize or modify small molecules. These molecules can in turn be used as therapeutic or diagnostic agents. Furthermore, these molecules can be used in the development of additional new molecules for therapeutic or diagnostic use.

CYP 27C1 or its derivatives, CYP 27C1 homologs, chimeras and protein fusions can be expressed in natural host cells or organisms, or in experimentally created cells or organisms for the purpose of producing, analyzing or modifying therapeutically and diagnostically important small molecules.

CYP 27C1 or its derivatives and CYP 27C1 fusions can be expressed in cells or organisms to modify the normal or diseased function and state of such hosts. In particular, this encompasses, but is not limited to, the use of CYP 27C1 polypeptides and derivatives for gene-therapy of humans or animals. CYP 27C1 polypeptides can also be used in experimental animals to reproduce physiological states, which are useful in the study and analysis of human disease, health or development. CYP 27C1 polypeptides or derivatives and CYP 27C1 fusions can be expressed in natural host cells or organisms or in experimentally created cells or organisms and used in the extraction, conversion, localization or bioremediation of small molecules in natural or artificial environments. This use includes, but is not limited to, the removal or neutralization of environmental or industrial pollutants by cultivating transgenic or genetically modified plants or microorganisms in water or soil, or by assembling so-called bioreactors that host such organisms. At the very least, CYP 27C1 polypeptide may be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. xi) Heme-binding. Oxygen-binding and Detoxification All cytochrome P450s are heme-binding proteins that contain the putative family signature, F[XX]G[XXX]C[X]G [X= any residue; conserved residues are in bold].

Heme-binding proteins, such as the cytochromes P450s, play an important role in the detoxification of toxic substances or xenobiotics. For example, toxic substances can be detoxified by oxidation. Cytochrome P450s can function as oxidative enzymes to detoxify toxic substances, such as phenobarbital, codeine and morphine. The capacity of cytochrome P450s to bind oxygen depends on the presence of a heme group and the oxygen-binding domain. Thus, the ability of P450s to bind heme and molecular oxygen enables them to detoxify toxic substances by oxidation.

Thus CYP 27C1 polypeptides are also useful as oxidative enzymes to detoxify toxic substances or xenobiotics, such as phenobarbital, codeine and morphine. xii) Antagonist. Agonist and Antisense Methods This invention further provides methods for screening compounds to identify agonists and antagonists to the CYP 27C1 polypeptides of the present invention. An agonist is a compound that has similar biological function, or enhances the function, of the polypeptide, while antagonists block such functions. Examples of potential CYP 27C1 antagonists include antibodies, drugs, small molecules or in some cases, oligonucleotides, which bind to the polypeptides.

Antisense constructs prepared using antisense technology are also potential antagonists. Therefore, the present invention is further directed to inhibiting CYP 27C1 in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the [mature] polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the polypeptides [for example, antisense- Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CPR Press, Boca Raton, FL (1988)]. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription [triple-helix, see Lee et al., Nucl. Acids Res. 6: 3073 (1979); Cooney et al., Science 241 : 456 (1988); and Dervan et al., Science 251 : 1360 (1991)], thereby preventing transcription and the production of the CYP 27C1 polypeptides.

Another potential CYP 27C1 antagonist is a peptide derivative of the polypeptides which are naturally or synthetically modified analogs of the polypeptides that have lost biological function yet still recognize CYP 27C1 substrate(s). Examples of peptide derivatives include, but are not limited to, small peptides or peptide-like molecules.

The antagonist may be employed to treat disorders which are either CYP 27C1 -induced or enhanced or modulated, for example, vitamin D metabolic disorders, such as, cholestatic or paramchymal liver disease, premature infant vitamin D metabolism, obesity, sarcoidosia, tuberculosis, primary hypothyroidism and vitamin D dependent rickets type II or cholesterol metabolic disorders, or cancers. Instead of inhibiting CYP 27 C1 activity at the nucleic acid level, CYP

27C1 activity can be directly inhibited by binding to an agent, such as a suitable small molecule or drug. The present invention thus includes a method of screening drugs for their effect on activity [i.e., as a modulator, preferably an inhibitor] of CYP 27C1 polypeptide. In particular, modulators of CYP 27C1 activity, such as drugs or peptides, can be identified in a biological assay by expressing CYP 27C1 in a cell, adding a substrate and detecting activity of CYP 27C1 polypeptide on the substrate in the presence or absence of a modulator. Thus, the CYP 27C1 protein can be exposed to a prospective inhibitor or modulating drug and the effect on protein activity can be determined. Prospective drugs can be tested for inhibition of the activity of other P450 cytochromes, which are desired not to be inhibited. In this way, drugs that are selectively inhibit CYP 27C1 over other P450s can be identified.

Polynucleotides that encode CYP 27C1 protein or that encode proteins having a biological activity similar to that of a CYP 27C1 protein, can be used to generate either transgenic animals or "knock-out" animals. These animals are useful in the development and screening of therapeutically useful reagents. A transgenic animal [e.g. a mouse] is an animal having cells that contain a transgene, which was introduced into the animal or an ancestor of the animal at prenatal, e.g. an embryonic stage. A transgene is a DNA molecule that has integrated into the genome of a cell from which a transgenic animal develops.

In one embodiment, a human CYP 27C1 cDNA, comprising the nucleotide sequence herein (figure 2A(SEQ. ID. NO:39)), or an appropriate variant, fragment or subsequence thereof, can be used to generate transgenic animals that contain cells which express human CYP 27C1 protein. Methods for generating transgenic animals, such as rats, hamsters, rabbits, sheep and pigs, and particularly mice, have become conventional in the art [see for example U.S. Patent No. 4,736,866 and 4,870,009].

In a preferred embodiment, plasmids containing recombinant molecules of the present invention are microinjected into mouse embryos. In particular , the plasmids of the present invention are microinjected into the male pronuclei of fertilized one-cell mouse embryos, the injected embryos at the 2-4 cell stage are transferred to pseudo-pregnant foster females, and the embryos in the foster females are allowed to develop to term, [see Hogan et al., A Laboratory Manual, Cold Spring Harbour, N.Y. Cold Spring Harbour (1986)] Alternatively, an embryonal stem cell line can be transfected with an expression vector comprising a polynucleotide encoding a protein having CYP 27C1 activity, and cells containing the polynucleotide can be used to form aggregation chimeras with embryos from a suitable recipient mouse stain. The chimeric embryos can be implanted into a suitable pseudopregnant female mouse of the appropriate strain and the embryo brought to term. Progeny harbouring the transfected DNA in their germ cells can be used to breed uniformly transgenic mice.

Transgenic animals that include a copy of a CYP 27C1 transgene introduced into the germ line of the animal by an embryonic stage can also be used to examine the effect of increased CYP 27C1 expression in various tissues.

Conversely, "knock-out" animals that have a defective or altered CYP 27C1 gene can be constructed [see for example Lemoine and Cooper, Gene Therapy, Human Molecular Genetics Series, BIOS Scientific Publishers, Oxford, U.K. (1996)]. Knock-out animals can be made that cannot express a functional CYP 27C1 polypeptide. For example, a portion of the murine homlog of CYP 27 C1 DNA (e.g. an exon) can be deleted or replaced with another gene, such as a gene encoding a selectable marker, that can be used to monitor integration. The altered CYP 27C1 DNA can then be transfected into an embryonal stem cell line where it will homologously recombine with the endogenous CYP 27C1 gene in certain cells. Clones containing the altered gene can be selected. Cells containing the altered gene are injected into a blastocyst of an animal, such as a mouse, to form aggregation chimeras and chimeric embryos are implanted as described above for trangenic animals. Transmission of the altered gene into the germline of a resultant animal can be confirmed using standard techniques and the animal can be used to breed animals having an altered CYP 27C1 gene in every cell. Such a knock-out animal may be used, for example, to test the effectiveness of an agent in the absence of a CYP 27C1 protein, if lack of CYP 27C1 expression does not result in lethality. The knock-out animal can also be used to monitor the development of any conditions. xiii) Pharmaceutical Compositions

CYP 27C1 may play a role in a number of diseases, such as those associated with cancer, cholesterol or vitamin D metabolism as previously noted herein. However, CYP 27C1 is not limited to being associated with such conditions only. In particular CYP 27C1 may play a role in cell differentiation disorders, and apoptoic disorders such as cancer. As such, the invention comprises methods for modulating or simulating CYP 27C1 activity or CYP 27C1 expression, preferably for treating or preventing a CYP 27C1 related condition. The invention further comprises uses of the modulating (any change or controlling effect on CYP 27C1 activity or expression) or simulating agents disclosed herein for the preparation of a medicament for treating or preventing a condition associated with CYP 27C1 expression or activity. In another embodiment the invention provides a use of the modulating or simulating agents for the treatment or prevention of a CYP 27C1 related condition.

Accordingly, the present invention provides a method of treating or preventing a disease associated with CYP 27C1 expression or activity comprising administering an agent that modulates or simulates CYP 27C1 expression or activity to an animal in need thereof, such as in an animal with cancer, a vitamin D deficiency disorder or a cholesterol metabolic disorder (e.g. build up of cholesterol in the body).

In a preferred embodiment, preferably such agents stimulate or simulate CYP 27C1 activity. Examples of agents that activate or simulate CYP 27C1 activity would include without limitations, CYP 27C1 , the gene encoding for CYP 27C1 with suitable promoters, such promoters preferably being tissue specific promoters and therapeutically effective fragments of the nucleic acid and amino acid sequences of the invention. Further, such agent may include small molecules or drugs identified to have such effect. In another embodiment, preferably the CYP 27C1 , if administered is solubilized. In another embodiment, the CYP 27C1 polypeptide of the invention can be co-administered preferably with co-factors such as with the suitable NADPH reductase and preferably with ferrodixin or flavoprotein as the case may be. In another embodiment, the CYP 27C1 polypeptide of the invention can be co-administered with the subtrate, ie. in the case of vitamin D deficiency, co-administered with Vitamin D, The substrate and co-factors could both be administered with the CYP 27C1 polypeptide, or can potentially be effective alone or together.

Further, there may be diseases or conditions in which inhibition of CYP 27C1 may be required. Accordingly, the invention provides a method for treating or preventing a disease or condition associated with CYP 27C1 expression or activity, (either any expression or activity or elevated expression or activity) by administering to a patient in need thereof an agent which inhibits or supresses CYP 27C1 expression or activity.

Examples of agents that inhibit CYP 27C1 include antisense nucleic acid molecules, antibodies and transdominant inhibitors, as described herein. They can further include subtrate competitive molecules, such as certan mutated CYP 27C1.

Agents that modulate (either alone or with another agent as explained above) CYP 27C1 expression or activity can be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. As used herein "biologically compatible form suitable for administration in vivo" means a form of the substance to be administered in which therapeutic effects outweigh any toxic effects. The substances may be administered to animals in need thereof. Animals, as used herein refers to any animal susceptible to a disease associated with CYP 27C1 expression preferably dogs, cats, mice, horses and humans.

The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (and potential side effects), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. Administration of an "effective amount" of pharmaceutical compositions of the present invention is defined as an amount of the pharmaceutical composition, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as disease state, age, sex, and weight of the recipient, and the ability of the substance to elicit a desired response in the recipient. Dosage regima may be adjusted to provide an optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. Subject to therapeutic discretion, preferably dosages of administration of active compound will be in the range of about 1 μg/kg/day to 10mg/kg/day of patient body weight and most preferably at least 0.01 mg/kg/day, and most preferably for humans between between about 0.01 and 1 mg/kg/day. An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, topical, intratumoral etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound, prior to reaching the desired site of delivery. It can also be formulated into a sustained release composition.

The compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

The compositions are indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment (e.g. chemotherapy or radiotherapy). For example, the compositions may be used in combination with anti-proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-inflammatories. In particular, the compounds may be used in combination with anti-viral and/or anti- proliferative agents. The compositions of the invention may be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.

Recombinant nucleic acid molecules comprising a sense, an antisense sequence or oligonucleotide fragment thereof, may be directly introduced into cells or tissues in vivo using delivery vehicles known in the art such as retroviral vectors, adenoviral vectors and DNA virus vectors(see for example Sanbrook et al. (supra) and Ausubel et al (supra). They may also be introduced into cells in vivo using physical techniques known in the art such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules may also be delivered in the form of an aerosol or by lavage.

The utility of the substances, antibodies, sense and antisense nucleic acid molecules, and compositions of the invention may be confirmed in animal experimental model systems. Suitable animal model systems which can be used to determine activity may include, but is not limited to CYP 27C1 or knock-out transgenic animals.

The nucleic acid molecules comprising full length cDNA sequences and/or their regulatory elements enable a skilled artisan to use sequences encoding a protein of the invention as an investigative tool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) regulation of gene function. Such technology is well known in the art, and sense or antisense oligomers, or larger fragments, can be designed from various locations along the coding or control regions.

Genes encoding a protein of the invention can be turned off by transfecting a cell or tissue with vectors which express high levels of a desired CYP 27C1 -encoding fragment. Such constructs can inundate cells with untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases.

Modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA or PNA, to the regulatory regions of a gene encoding a protein of the invention, i.e., the promoters, enhancers, and introns. Preferably, oligonucleotides are derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence. The antisense molecules may also be designed so that they block translation of mRNA by preventing the transcript from binding to ribosomes. Inhibition may also be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Therapeutic advances using triplex DNA were reviewed by Gee J E et al (In: Huber B E and B I Carr (1994) Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco N.Y.).

Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. The invention therefore contemplates engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a protein of the invention.

Specific ribozyme cleavage sites within any potential RNA target may initially be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once the sites are identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be determined by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Methods for introducing vectors into cells or tissues include those methods discussed herein and which are suitable for in vivo, in vitro and ex vivo therapy. For ex vivo therapy, vectors may be introduced into stem cells obtained from a patient and clonally propagated for autologous transplant into the same patient (See U.S. Pat. Nos. 5,399,493 and 5,437,994). Delivery by transfection and by liposome are well known in the art.

An antibody against a CYP 27C1 Related Protein may be conjugated to chemotherapeutic drugs, toxins, immunological response modifiers, hematogenous agents, enzymes, and radioisotopes and used in the prevention and treatment of cancer (e.g. thyroid, prostate, colon, kidney, testicular cancer). For example, an antibody against a CYP 27C1 Related Protein may be conjugated to toxic moieties including but not limited to ricin A, diphtheria toxin, abrin, modeccin, or bacterial toxins from Pseudomonas or Shigella. Toxins and their derivatives have been reported to form conjugates with antibodies specific to particular target tissues, such as cancer or tumor cells in order to obtain specifically targeted cellular toxicity (Moolten F.L. et al, Immun. Rev. 62:47-72, 1982, and Bernhard,, M.I. Cancer Res. 43:4420, 1983).

Conjugates can be prepared by standard means known in the art. A number of bifunctional linking agents (e.g. heterobifunctional linkers such as N-succinimidyl-3-(2-pyridyldithio)propionate) are available commercially from Pierce Chemically Company, Rockford, III.

Administration of the antibodies or immunotoxins for therapeutic use may be by an intravenous route, although with proper formulation additional routes of administration such as intraperitoneal, oral, or transdermal administration may also be used.

A CYP 27C1 Related Protein may be conjugated to chemotherapeutic drugs, toxins, immunological response modifiers, enzymes, and radioisotopes using methods known in the art.

Vaccines can be derived from a CYP 27C1 Related Protein, peptides derived therefrom, or chemically produced synthetic peptides, or any combination of these molecules, or fusion proteins or peptides thereof. The proteins, peptides, etc. can be synthesized or prepared recombinantly or otherwise biologically, to comprise one or more amino acid sequences corresponding to one or more epitopes of a tumor associated protein. Epitopes of a tumor associated protein will be understood to include the possibility that in some instances amino acid sequence variations of a naturally occurring protein or polypeptide may be antigenic and confer protective immunity against cancer or anti-tumorigenic effects. Sequence variations may include without limitation, amino acid substitutions, extensions, deletions, truncations, interpolations, and combinations thereof. Such variations fall within the scope of the invention provided the protein containing them is immunogenic and antibodies against such polypeptide cross-react with naturally occurring CYP 27C1 Related Protein to a sufficient extent to provide protective immunity and/or anti-tumorigenic activity when administered as a vaccine. The proteins, peptides etc, can be incorporated into vaccines capable of inducing an immune response using methods known in the art. Techniques for enhancing the antigenicity of the proteins, peptides, etc. are known in the art and include incorporation into a multimeric structure, binding to a highly immunogenic protein carrier, for example, keyhole limpet hemocyanin (KLH), or diptheria toxoid, and administration in combination with adjuvants or any other enhancer of immune response.

Vaccines may be combined with physiologically acceptable media, including immunologically acceptable diluents and carriers as well as commonly employed adjuvants such as Freund's Complete Adjuvant, saponin, alum, and the like. It will be further appreciated that anti-idiotype antibodies to antibodies to CYP 27C1 Related Proteins described herein are also useful as vaccines and can be similarly formulated.

The administration of a vaccine in accordance with the invention, ma be generally applicable to the prevention or treatment of related disorder.

The activity of the proteins, substances, compounds, antibodies, nucleic acid molecules, agents, and compositions of the invention may be confirmed in animal experimental model systems. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₅₀ ( the dose therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics. The therapeutic index is the dose ratio of therapeutic to toxic effects and it can be expressed as the ED₅o/LD₅₀ ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred.

The following non-limiting examples are illustrative of the present invention:

EXAMPLES MATERIALS AND METHODS In vitro Cell Culture

Hep3B and HepG2 human hepatoma cells were maintained in

Minimal Essential Medium (MEM, Gibco BRL Grand Island, NY) containing

Earle Salts and L-glutamine, 2.2g/L of sodium bicarbonate, 10%FBS, and antibiotics (penicillin 50units/ml and streptomycin 50ug/ml). Cells were maintained in the presence of 5% CO₂ at 37°C. Hep3B and HepG2 cells were grown to 80% confluency and induced with 25(OH)D₃, 1 ,25(OH)₂D₃,

1α(OH)D₃, and the bile acids, cholic, taurocholic, and chenodioxycholic acid to a final concentration of 2.5x10^"5M for 18hrs. Ethanol was added to one plate of Hep3B and HepG2 cells as a control. Cells were collected and frozen at -70°C until further use.

RNA Isolation Total RNA was isolated from HepG2 and Hep3B cultured cells using the TRIzol reagent according to the manufacturer's instructions (Gibco BRL). Treatment with the DNA-free kit according to the manufacturer's protocol completed the isolation (Ambion, Inc. Austin, TX). RT-PCR: RT-PCR was done on Hep3B and HepG2 total RNA and on colon mRNA (Origene Technologies, Inc. Rockville, MD) using a sense and antisense gene specific primer according to the manufacturer's recommendations in the Advantage One-Step RT-PCR Kit (Clontech Laboratories, Inc., Palo Alto, CA). To control for contamination of the reaction components a reaction without RNA was performed along with the positive mouse control and a reaction with the primers for GAPDH as a second positive control (Clontech Laboratories, Inc.).

RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM- RACE) was performed on colon mRNA and Hep3B total RNA according to the manufacturer's protocol (Ambion, Inc.). In each reaction 10mg of total RNA and 500ng of mRNA was used. RT-PCR and RLM Race products were analysed on a 1.2% agarose gel with ethidium bromide. PCR Analysis of cDNA Libraries/Pools The following human cDNA libraries were screened by PCR: T84 colon, fetal brain, NT-2, thymus, and placenta. 0.5mg of template cDNA was used per reaction. The following components were mixed on ice to perform PCR reactions (final concentrations): 1xPCR buffer (Qiagen Mississauga, ON), 0.2mM dNTPs, 1mM MgCI2, 5pmol/mL sense primer, 5pmol/mL antisense primer, distilled water to 49.5mL, and then 0.5mL Taq DNA polymerase (5U/mL; Qiagen). Initial denaturation temperature was 99°C for 2 minutes followed by 35 cycles of 99°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes. The final product elongation was completed by incubation at 72°C for 5 minutes and a hold at 4°C. Independent control reactions were performed with no template cDNA. A Sure-RACE (Rapid Amplification of cDNA Ends) kit was used to screen libraries using 5' RACE PCR. This kit included a 48 well multi- tissue RACE panel with 24 different cDNA tissue libraries, and was done according to the manufacturer's protocol (Origene, Inc.). Adapter primer 1 (ADP1), 5'-CGGAATCGTCACTCAGCG-3' (SEQ. ID. NO:48), and a downstream gene specific primer were used in the first round of amplification followed by adapter primer 2 (ADP2), 5'- AGCGCGTGAATCAGATCG-3'(SEQ. ID. NO:49), and a nested downstream second gene specific primer for the second round. A change to the manufacturer's protocol was an elongation temperature of 68°C.

Three gene specific primers, an upstream, downstream and nested primer, were sent to Pangene Inc. to be used for the isolation of CYP 27C1 from a fetal brain cDNA library.

Rapid screening of an arrayed fetal brain library was done according to the manufacturer's protocol (Origene, Inc.). Upstream and downstream gene specific primers were used for screening along with the vector primer 5'GCAGAGCTCGTTTAGTGAACC3' (SEQ. ID. NO:50) of the library. All PCR reactions were analysed on a 1.2% agarose gel stained with ethidium bromide.

Hybridization Studies

End labeling of primers was done using γ-[³²P]ATP and T4 polynucleotide kinase (15). For random priming α-[ P]dATP was used with the Prime-A-Gene Labeling Kit according to the manufacturer's protocol (Promega Corp., Madison, WI ).

Southern Alkali Transfer of PCR products: PCR products were fractionated on a 1.2% agarose gel and blotted onto a nitrocellulose membrane (Micron Separations, Inc. Westboro, MA) overnight using 0.4M NaOH as the transfer buffer. The blot was dried at 80°C for 2 hours, prehybridized for 2 hours with Express-Hyb hybridization buffer at 42°C, and hybridized using a gene specific internal oligonucleotide at 42°C for 2 hours (Clontech Laboratories, Inc.).

Northern blot analysis: 10mg of RNA was electrophoresed on a denaturing 1 % formaldehyde agarose gel. RNA was transferred using 20x

SSPE buffer onto a Hybond-N+ membrane (Amersham Pharmacia Biotech

Ltd. Buckinghamshire, England). The membrane was rinsed with 2x SSPE, dried at 80°C in a vacuum oven, and then cross-linked for 45 seconds on a UV box. Pre-hybridization and hybridization were done in Express-Hyb hybridization buffer at 42°C for 1 and 2 hours, respectively (Clontech Laboratories, Inc.). Multiple Tissue Expression (MTE): A 76 human tissue poly A+ RNA dot blot (Clontech Laboratories, Inc.) was probed with a gene specific PCR product for CYP 27C1. Hybridization was done according to the manufacturer's directions. Northern blots were washed with SSPE while Southern blots and MTE's were washed with SSC. All blots were washed with 2x SSPE/SSC, 1 % SDS for δminutes at room temperature, 0.1x SSPE/SSC, 0.5% SDS for 15minut.es at 42°C, and 0.1x SSPE/SSC, 0.1% SDS at 50°C for 15 minutes. The blots were then exposed at -70°C for 24 hours to Kodak X-Omat AR film (Eastman Kodak Co. Rochester, NY). cDNA Isolation, Subcloning, and Sequencing PCR bands were gel purified from LMP-agarose gel (15). The gel purified bands were cloned into the Topo 2.1 vector using the Topo TA Cloning Kit and transformed into Top 10 cells according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). Ligations not involving the Topo 2.1 vector were performed overnight at 37°C in 10mL reactions with 1mL of T4 DNA ligase, 1mL T4 DNA ligase buffer (10x). Transformations were plated on LB ampicillin plates (100ug/ml) with 40uL of X-gal (20mg/mL) and grown at 37°C overnight. White colonies were picked and grown up in 5mL LB Broth with 5uL Amp (10mg/mL) at 37°C and 225 rpm. Plasmid DNA was isolated from those bacterial cultures using the Qiagen Mini-Prep Kit (Qiagen, Inc.) and samples were digested with EcoRI for 2 hours. Digests were analyzed on a 1 % agarose gel stained with ethidium bromide.

Sequencing: Purified PCR products or PCR products in Topo 2.1 vector were sent to Cortec Inc. (Kingston, ON) for sequencing. Sequencing assembly and alignments were done using OMIGA (Oxford Molecular, CA) and Lasergene (DNASTAR Inc. Madison, WI) software. BLAST searches were then performed on sequences using the National Center for Biotechnology Information database (NCBI) (www.ncbi.nlm.nih.gov).

Protein Analysis

Single Tube Protocol 3 (STP3) was used to perform in vitro transcription and translation with ³⁵S according to the manufacturer's protocol (Novagen Inc. Madison, WI). P450RAI-1 was used as a positive control. A 10% SDS PAGE protein gel was run with the translated reactions, dried for 2 hours on Watman filter paper, and audioradiography was performed for 45 minutes at room temperature using Kodak X-Omat AR film (Eastman Kodak Co.).

Primers

All primers used in PCR, RT-PCR, sequencing, and hybridization reactions are listed in Table 1 (SEQ. ID. NOS: 1-20) and were synthesized at Cortec Inc. (Kingston, ON). Example 1 : Identification and Confirmation of CYP 27C1

Dr. David Nelson predicted a partial CYP 27C1 peptide on his web page, http://drnelson.utmem.edu/P450lect.html and http://drnelson.utmem.edu/whatsnew.html

(see Figure 1C) and noted its similarity to human CYP27A1. However, it was never fully identified, nor actually isolated, cloned or sequenced. CYP 27C1 is located in a genomic clone RP11-30F3 (accession number ACO27142, SEQ. ID. NO:21) as illustrated in Figures 1A (SEQ. ID. NO:21) and 1 B (SEQ. ID. NO:22-25 and 27-30). RP11-30F3 is 188888bp long consisting of 40 unordered segments. The cDNA sequence was originally pieced together but was missing the 163 (SEQ. ID. NO:26) nucleotides between 898 and 1061bp. The present inventors have now identified the full length polynucleotide sequence for the CYP 27C1 cDNA which is shown in Figure 2A (SEQ. ID. NO:39). The amino acid sequence is shown in Figure 2B (SEQ. ID. NO:40).

To determine whether the cDNA sequence could produce a true cytochrome, primers 210 (SEQ. ID. NO:4) and 211(SEQ. ID. NO:5) were used to amplify the cDNA, including the heme region from 1106bp to 1512bp. Primers 187(SEQ. ID. NO:1), 188(SEQ. ID. NO:2), and 189(SEQ. ID. NO:3) were designed to amplify the 5' end at the first start codon of the sequence. PCR done in T84 colon, fetal brain, thymus, placenta, and NT-2 libraries produced the 400bp band shown in Figure 3A from primers 210(SEQ. ID. NO:4)/211(SEQ. ID. NO:5) in the fetal brain and NT-2 library, and a non-specific banding pattern with the 5' end primers (figure not shown). Digests of the 400bp band in the Topo 2.1 vector using Sacl showed the expected 155bp band for one orientation and a 340bp band for the opposite orientation of the fragment in the vector. Sequencing at Cortec Inc. with the M13 forward and reverse primers confirmed the identity of the 400bp band as the proposed CYP 27C1 gene. The sequencing showed a conserved heme region with the signature sequence of FXXGXXXCXG (amino acid 476-485(SEQ. ID. NO:47)), and 18 residues from the cysteine, LQXF (amino acid 501-504(SEQ. ID. NO:51)). Similar protocols were used to screen CYP 27C1 expression in human tissue from the brain, heart, spleen, liver colon, testis and uterus. As can be seen in Figure 3B, CYP 27C1 expression can be seen in tissue from the colon and uterus. Example 2 - Tissue Expression Patterns of CYP 27C1

The 24 tissues of the 5'Sure RACE panel, which contained fetal brain, were screened. A very specific 600bp band made by primers 216(SEQ. ID. NO:6) and ADP2(SEQ. ID. NO:49) in colon cDNA following round two of RACE PCR is displayed in Figure 4A. EcoRI digests of the 600bp band in the Topo 2.1 vector showed the expected banding pattern: a 350bp band, a 260bp band, and a 50 bp band, plus the cut vector. Sequencing at Cortec using the M13 forward and reverse primers showed identical sequence to the predicted cDNA sequence. The sequencing also discovered new information on exon 5(SEQ. ID. NO:26). It revealed the 163 nucleotides from 898bp to 1061bp not present in the human clone RP11-30F3(SEQ. ID. NO:21). In an attempt to clone the 5' end of CYP 27C1 RACE PCR in colon cDNA was tried again. Second round RACE PCR with primers 224(SEQ. ID. NO:7) and ADP2(SEQ. ID. NO:49) produced a 120bp product in colon tissue that hybridized to the internal oligonucleotide probe of 227(SEQ. ID. NO:8) in a Southern Blot (figure not shown), but gave no further sequence results to the 5' end. These results illustrated the limited use of RACE PCR for cloning the sequence of CYP 27C1 within the first 500 nucleotides of the 5' end.

The multiple tissue expression dot blot with 76 different human non- diseased tissues produced ubiquitous expression levels in all tissues when probed with the α-[³²P]dATP labeled 400bp piece from PCR in the NT-2 library with primers 210(SEQ. ID. NO:4)/211(SEQ. ID. NO:5) (figure not shown). The blot did show levels of expression in the control dots. In Figure 4B a different dot blot was probed with the 600bp random labeled PCR product from colon tissue and again produced ubiquitous expression in all tissues with slightly elevated levels in thalamus, fetal kidney, fetal brain, fetal liver, fetal lung, trachea, and jejunum.

Semi-quantitative human tissue expression experiments were also conducted. Human Rapid Scan cDNA panel (Origene) was used in PCR- amplification with CYP27C-specific primers (1 ,000 pg, 100 pg and 10 pg samples) or beta-actin primers (1 pg samples) and Hot Start Taq polymerase (Qiagen). All reactions were incubated at 95°C for 15 min. and then subjected to 35 cycles composed of 30 sec. at 99°C, 30 sec. at 65°C (CYP27C) or 72°C (beta-actin) and 1 min. at 72°C, followed by 5 min. at 72°C.

The following CYP27C primers were used: Forward primer: 5'-CCAGAAGTGCAGCAGACGGTGTAC-3' (SEQ. ID. NO:4)

Reverse primer: 5'-CTGGATCACGACGAGGTGAATCTC-3'(SEQ. ID. NO:5) 10 ul of each reaction were fractionated on 1% agarose gel and visualized under UV after staining with ethidium bromide.

Semi-quantitative expression in the tissue samples was obtained.

The results are shown in Table 2. As in the table, expression was observed in the brain, heart, kidney, lung, muscle, stomach, testis, adrenal gland, ovary, prostate, skin, and fetal brain, and especially in the testis, adrenal gland and fetal brain. ln Figure 4C, RT-PCR done in Hep3B and HepG2 total RNA with primers 210(SEQ. ID. NO:4) and 211 (SEQ. ID. NO:5) detected expression of the CYP 27C1 gene, as indicated by the 400bp band, in Hep3B but not in HepG2 cells. Primers 253(SEQ. ID. NO:14), 254(SEQ. ID. NO:15) and 256(SEQ. ID. NO: 16) for CYP 27A1, and primers 115and 116 for GAPDH were also used and showed expression of CYP 27A1 in Hep3B cells and GAPDH in both Hep3B and HepG2 cells. The expression pattern of CYP 27C1 in Hep3B cells was confirmed by Northern blot analysis (Figure 4D and 4E). It showed a very intense, dark band only in Hep3B total RNA, consistent with RT-PCR results, and not in HepG2 total RNA following hybridization with a random labeled 500bp PCR probe from primers 227(SEQ. ID. NO:8) and 248(SEQ. ID. NO:9). The transcript size was estimated to be 4.5-5kb. Example 3 - Cloning the 5' end of CYP 27C1 Pangene Inc. found four clones (A,B1 ,B2,C) in the fetal brain cDNA library using gene specific primers 216(SEQ. ID. NO:6), 248 (SEQ. ID. NO:9) and 227(SEQ. ID. NO:8). This gave the banding pattern shown in Figure 5A when each clone was digested with EcoRI. Clone A contained the expected 350bp band produced by the EcoRI digest. Sequencing of all of the clones showed only clone A contained the CYP 27C1 gene. Alignment to the predicted sequence in Lasergene showed two missing nucleotides at 1305 and 1306bp that were present in the partial clones obtained from the NT-2 library and in the predicted genomic sequence. Clone A did show identical sequence to the 505-1512bp region already cloned by PCR, added an additional 1069bp to the 3' end, and 18bp to the 5' end. However, no intact 5' end was present.

To isolate the 5' end of the CYP 27C1 gene RT-PCR was attempted using Hep3B total RNA and colon mRNA with primer sets 303(SEQ. ID. NO:13) and 224(SEQ. ID. NO:7), and 302(SEQ. ID. NO:12) and 224(SEQ. ID. NO:7). This resulted in the 300bp band in Figure 5B, which when sequenced aligned precisely in Lasergene to the predicted CYP 27C1 cDNA sequence at the 5' end for 295bp to 578bp of the cDNA sequence. The RT-PCR results from primers 302(SEQ. ID. NO: 12) and 224(SEQ. ID. NO:7) produced the smeared banding pattern in Figure 5B and following Southern blot analysis, shot gun cloning of the lane, digests, and sequencing of the suspected band, it was confirmed to be the 5' end of CYP 27C1.

In order to clone the remaining nucleotides of the 5' end RLM RACE and rapid screening of a fetal brain library were also tried. These techniques did not produce any products that contained the CYP 27C1 gene. Example 4 - Sequence Analysis and Alignments

Alignment of the predicted and partially cloned sequences of CYP 27A(SEQ. ID. NO:41), CYP 27B1(SEQ. ID. NO:42) and Xenopus laevis CYP 27C1(SEQ. ID. NO:43) in OMIGA is shown in Figure 6. The alignment produced a 36% sequence identity between CYP 27C1(SEQ. ID. NO:40) and CYP 27A1(SEQ. ID. NO:41) and a 38% sequence identity as calculated between CYP 27C1(SEQ. ID. NO:40) and CYP 27B1 (SEQ. ID. NO:42). A 70% sequence identity was calculated between CYP 27C1(SEQ. ID. NO:40) and Xenopus laevis CYP 27C1(SEQ. ID. NO:43). A Kyte-Doolittle hydropathy plot of CYP 27A1 , CYP 27C1 , and CYP 27B1 sequences was done to predict the microsomal or mitochondrial nature of the 5' ends of these cytochromes. Figure 7A illustrates that CYP 27C1 is twice as hydrophobic as CYP 27A1 and four times as hydrophobic as CYP 27B1 (figure not shown). Figure 7B shows a Kyte-Doolittle hydropathy plot with CYP 27C1 and a known microsomal P450, CYP 26. The microsomal cytochrome is two times more hydrophobic then CYP 27C1. Example 5 - CYP 27C1 homologs from different species

An advanced blastn search of the expressed sequence tagged (EST) in the NCBI database using the default settings and the predicted CYP 27C1 sequence as a query was done and the results are shown in Figure 12. The results showed identity of CYP 27C1 to Bos taurus cDNA (accession number BE723057) and Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA clone (accession number AA423993). A further tblastx search of the EST database in NCBI showed homology of CYP 27C1 to Xenopus laevis cDNA clones with accession numbers BE669236 (EST 70) and AW637606 (EST 71). The alignment oi Xenopus laevis EST 70 and CYP 27C1(SEQ. ID. NO:40) over the first 310 amino acid region showed 70% sequence identity (Figure 6). The EST's were ordered, received, and sequenced. Example 6 - Construction of Xenopus laevis/CYP 27C1 Hybrid Gene

Two clones of CYP 27C1 : Pangene clone A and the 300bp piece from Hep3B cells were joined by PCR using primer 311 (SEQ. ID. NO: 17) to introduce a Notl site on the 3' end and primer 313(SEQ. ID. NO: 19) to introduce an Xhol site on the 5' end. EST 70 from Xenopus laevis was also amplified using primer 312(SEQ. ID. NO:18) to produce an Xhol site on the 3' end and primer 314(SEQ. ID. NO:20) to introduce an Nhel site on the 5' end. The products were ligated following digestion with Xhol and re- amplified (Figure 8B). The band in Figure 8B was inserted into the expression vector, pcDNA 3.1 hygromycin using the Nhel and Notl sites. Example 7 - Xenopus laevis/CYP 27C1 Hybrid Protein

In vitro transcription and translation on the hybrid gene produced a 55kDa protein (Figure 9). P450RAI-1 showed a band at 50kDA. The negative control lane was clear and the positive control lane showed the expected banding pattern. The nuceotide sequence of the hybrid clone is shown in Figure 11A(SEQ. ID. NO:44). The amino acid sequence of the hybrid is shown in Figure 11 B(SEQ. ID. NO:45). Example 8 - Induction Studies of CYP 27C1 In order to predict the metabolic pathway that this novel cytochrome

P450 is involved in and whether or not it is regulated, induction studies introducing vitamin D compounds and bile acid compounds to Hep3B and HepG2 cells were done. Figure 10 shows that following an 18 hour induction of Hep3B and HepG2 cells with 25(OH)D₃, 1,25(OH)₂D₃, and 1α(OH)D₃ , Hep3B cells consistently produced identical levels of CYP 27C1 mRNA transcripts after amplification by RT-PCR reactions using primers 227 (SEQ. ID. NO:8) and 210 (SEQ. ID. NO:4) The expression of CYP 27C1 was less abundant than that produced in Hep3B cells in figure 4C. Induction with the three bile acids did not produce any product following RT-PCR reactions in the treated or controlled cells (figure not shown). A novel human cytochrome P450 in the CYP 27 family that is involved in the vitamin D metabolic pathway has been cloned, sequenced, and examined for its tissue expression patterns. This cytochrome, CYP 27C1 , shows 36% and 38% amino acid identity to two mitochondrial vitamin D P450s: CYP 27A1 and CYP 27B1 respectively. CYP 27C1 belongs to its own sub-family because it does not show greater than 55% sequence identity to another known cytochrome (1).

To determine whether the first exon at the 5' end of the predicted sequence is correct (2), this region from 1-294bp(SEQ. ID. NO:57), which was originally questionable, has now been cloned following many efforts using different PCR and RT-PCR strategies. Past research has found that the sequence near a cytochrome's N-terminus is generally very GC rich. This property results in the formation of secondary structures that makes PCR through this region more difficult (16). The predicted sequence in the first exon is no exception to the previous findings as it has an approximate 85% GC content. The sequence in the first exon also contains two potential start codons in the same reading frame 15 nucleotides apart. The Kyte Doolittle hydropathy plot in Figure 7A indicates that the second ATG is located in a more hydrophobic region of amino acids then the first, and thus may be the start codon if the P450 is the microsomal 25- hydroxylase. Correct sequencing of the first exon at the 5' end of the predicted sequence can be indicative of whether CYP 27C1 is a microsomal or mitochondrial cytochrome. Gonzalez et al. (17) reported that microsomal P450s contain hydrophobic N-terminal amino acid residues whereas mitochondrial cytochromes are less hydrophobic at the N- terminal protein sequence. Scientists studying the vitamin D pathway have identified vitamin D metabolizing activity in the microsomes of rat, pig, and human liver extracts but have never cloned the P450 involved with certainty (4,6,9,14). Even if the predicted sequence is correct for CYP 27C1 in the first exon, it may not be hydrophobic enough to be considered the microsomal 25-hydroxylase in liver. In Figure 7B a true microsomal cytochrome P450, CYP 26 is shown and is more hydrophobic at its N- terminal region than CYP 27C1 (18). CYP 27C1 does contain a highly conserved ferredoxin binding site similar to other vitamin D family mitochondrial hydroxylases which require this site when receiving electrons from NADPH-ferredoxin reductase (4,5). Microsomal cytochromes require NADPH cytochrome P450 reductase and a flavoprotein for activity, not ferredoxin (5). There is however one difference in the first amino acid of the ferredoxin binding site between CYP 27C1 and the other vitamin D mitochondrial cytochromes.

In Figure 3, CYP 27C1 tissue expression is highest in NT-2 (human embryonic carcinoma) cells, colon, fetal brain, and Hep3B (heptocellular carcinoma) cells. In the MTE in Figure 4B, expression levels of CYP 27C1 appeared slightly elevated in several fetal tissues: fetal kidney, fetal brain, fetal liver, and fetal lung. The elevated expression of the gene in fetal tissue was significant because for example, 82ng of RNA/dot in the fetal brain sample represented a much higher level of expression than that in liver, which contained 491ng/dot of RNA. CYP 27C1 may be located in the NT-2 cell line which differentiates after treatment with retinoic acid, in fetal tissues, and in Hep 3B cells since the active form of vitamin D, 1 ,25(OH)₂D₃, is known to play a role in cellular differentiation such as that in promyeloctye and cancer cells (4). Evidence has shown that vitamin D is not necessary for embryogenesis and thus the development of major organ systems, but may still be involved in differentiation at the cellular level (4). The abundance of CYP 27C1 in colon may correspond to the 25 hydroxylase activity found in rat intestinal cells which functionally perform hydroxylations on dietary vitamin D (7). The expression of CYP 27C1 in Hep3B and not in HepG2 cells following Northern Blot and RT-PCR analysis is interesting. In a previous study, Northern analysis of Hep3B RNA showed no CYP 27A1 transcripts even though the cell line was capable of performing 25-hydroxylations on 1a-OH-D₃ (9,14,19). The cell line also has been shown to 25-hydroxylate vitamin D₂ compounds while CYP 27A1 prefers 25-hydroxylation on 1α- hydroxylated vitamin D compounds (9). These pieces of evidence support the possibility that another P450 such as CYP 27C1 may play an important role in the vitamin D pathway (9). Contrary to other study's Northern analysis results is the RT-PCR results in Figure 4C, performed in triplicate, which show the presence of CYP 27A1 and CYP 27C1 in Hep3B cells. The bands were not sequenced and the high sensitivity of RT-PCR or the stage of growth of the cells may have contributed to this expression pattern. If these bands are real, the presence of CYP 27A1 and CYP 27C1 in Hep3B cells may be relevant to results obtained by Strugnell et al. (20) who found concentration dependent differences in the metabolism of 1α-OH-D₃ and 1 -OH-D₂ in Hep3B cells. These differences in metabolism such as 24- hydroxylation versus 25-hydroxylation of 1α-OH-D₂ at greater than 100nM concentration could be explained by the presence of CYP 27A1 at low levels in Hep3B cells. CYP 27A1 can normally 24, 25, and 26(27)- hydroxylate 1 -OH-D₃ in HepG2 cells and perhaps 1α-OH-D₂ when the substrate is present at high levels in Hep3B cells (20). If CYP 27A1 is not found in Hep3B cells as previous studies have proposed, another important and yet to be identified cytochrome may be present and work in Hep3B cells when high concentrations of vitamin D compounds are present.

A Xenopus laevis EST which showed high homology to the cloned sequence for CYP 27C1 and the predicted 5' end of the gene, was joined to the 1300bp cloned CYP 27C1 at the 5' end to produce a chimeric gene. Since the catalytic site of cytochromes exists closer to the COOH terminus, the Xenopus laevis 5' prime end on the human clone may not affect the cytochrome's activity during functional analysis (4). Amphibians such as the Xenopus laevis represent an intermediate stage in the evolution of endocrine mineral metabolism systems such as that of calcium, which is controlled by vitamin D status (21). In the aquatic environment these amphibians siphon calcium from the water, but when on land depend on vitamin D to increase blood calcium (21). The different requirements for calcium and thus a vitamin D endocrine system in Xenopus laevis may result in differences in the function and expression of the protein for the chimeric gene. However, it has been discovered that the sequence of the vitamin D receptor (VDR) is well conserved between Xenopus laevis and mammals (21). This information could postulate further that proteins involved in the Xenopus laevis vitamin D metabolic system may have conserved functions to that of the human enzymes. In vitro transcription and translation of the chimeric gene showed production of a protein approximately 55kDa in size. Without sequencing, the construction and repair of this gene appeared to be completed successfully because a mutation or gap in the sequence would have introduced stop codons causing a shortened protein, which was not the case. Valuable knowledge may be learned from the full length clone of human CYP 27C1 , the chimeric clone of Xenopus laevis and CYP 27C1 , and the full length Xenopus laevis CYP 27C1 such as its substrates, regulation, and applications to vitamin D or bile acid biosynthesis diseases. The clones can be put into an expression vector and transfected into cells such as COS-1 , V79, HepG2, or HeLa after which analysis of substrate and metabolites will be done by High Performance Liquid Chromatography (HPLC).

The regulation mechanisms of the 25-hydroxylases in the mitochondrial and microsomal membranes is not clear (5,7,22). Evidence shows that cytochromes are often not constitutively expressed but require compounds for induction (17). For example, heme biosynthesis is induced by phenobarbital and TCDD, which subsequently allows for P450 biosynthesis (17). Mainly ubiquitous expression was found on the MTE for any of the normal human tissues, which does not exclude the possibility that CYP 27C1 may be induced in response to environmental factors or during disease. The present study showed that induction of Hep3B and HepG2 cells with 25(OH)D₃, 1 ,25(OH)₂D₃, and 1α(OH)D₃ produced no increase or decrease in the level of CYP 27C1 expression following RT- PCR analysis. These results are similar to a study by Armbrecht et al. (5) which showed little regulation in microsomal 25-hydroxylase enzyme activity but different from Reinholz and DeLuca (22) who suspected that since the mitochondrial 25-hydroxylase is not regulated by vitamin D levels the microsomal enzyme is responsible for regulating the hepatic production of 25(OH)D₃. Hep3B and HepG2 cells can be induced with 1 α(OH)D₂ or vitamin D₂ since previous findings already discussed, indicated that a novel cytochrome is responsible for hydroxylating these compounds (9,20). The rate limiting step in the acidic bile acid biosynthesis pathway is performed by CYP 27A1 and based on this knowledge, the induction of the bile acids: cholic, taurocholic, and chenodioxycholic acid were done in Hep3B and HepG2 cells to determine whether CYP 27C1 is also involved in the oxidation of cholesterol molecules (14). CYP 27A1 is regulated by bile acid concentrations through down regulation at the transcriptional level (14,23,24). The results did not show any expression of CYP 27C1 in any of the bile acid induced or ethanol controls of Hep3B or HepG2 cells. Degradation of the total RNA isolated or the stage of cell growth may have contributed to this outcome. Cyclosporin A, an immunosuppressive drug, inhibits 27-hydroxylation in the bile acid synthesis pathway resulting in a decrease in chenodeoxycholic acid (25). The full length human clone of CYP 27C1 may play a role in the pathway. Interestingly cerebrotendinous xanthomatosis (CX) results from a mutation in the 27-hydroxylase gene and causes reduced levels of bile acid production but does not result in vitamin D deficiency type diseases (24-26). This is further evidence that a second P450 is involved in the 25-hydroxylase step of vitamin D metabolism, such as CYP 27C1.

Providing CYP 27C1 is involved in the vitamin D pathway as the second 25-hydroxylase, this cytochrome may prove to be a crucial factor in diseases common in the vitamin D pathway. Low levels of circulating 25(OH)D can contribute to cholestatic or paremchymal liver disease. Premature infants have also been found to be unable to metabolize vitamin D to 25(OH)D, and low levels of 25(OH)D have been found in people with obesity problems, hyperhoshatemic turmoral calcinosis, sarcoidosis, tuberculosis, primary hyperparathyroidism, and vitamin D dependent rickets type II (27,28). As indicated by the expression studies in various cancer cell lines CYP 27C1 may also play a role in various cancers. EXAMPLE 9 - Expression of CYP27C in insect and mammalian cells Generation of untagged CYP27C expression constructs

For expression in mammalian cells, CYP27C RT-PCR fragments were cloned into pcDNA3.1-Hygro expression vector (Invitrogen) with artificial Nhel and Notl sites added in front of start codon and after stop codon, respectively. Identity of that expression clone was confirmed by sequencing.

Nhel-Notl fragment containing CYP27C cDNA was then subcloned into pVL1393 baculovirus transfer vector (BD Pharmingen) and identity of the construct was confirmed by restriction enzyme analysis. Recombinant baculovirus was then generated by co-transfection of Sf9 cells with the above CYP27C transfer vector and linearized baculovirus genomic DNA using Baculo-Gold co-transfection kit (BD Pharmingen) under conditions recommended by manufacturer. Viral stock was then expanded and titrated by end-point dilution.

Expression of CYP27C1 in mammalian cells

To express human CYP27C in mammalian cells, hamster V79 cells were transfected with pcDNA-CYP27C construct in the presence of Fugene- 6 reagent (Roche) under recommended conditions. Cells expressing recombinant CYP27C were selected with hygromycin B (250ug/ml in DMEM medium) and cloned by end-point dilution. Expression of recombinant human CYP27C1 was confirmed in individual clone by RT-PCR.

Expression of CYP27C1 in insect cells

Recombinant baculovirus was used for expression of human CYP27C1 in insect cells. Sf9 cells (0.5 x 10⁶ cells/ml) in TNM-FH medium (BD Pharmingen) were infected at MOI about 2. Medium was supplemented with hemin, ferric citrate and d-aminolevulinic acid and cells were maintained at 27°C for 3 days. Afterwards cells were collected by centrifugation and membrane fractions were prepared as described for his- tagged CYP27C below.

Generation of his-tagged CYP27C

His-tagged version of CYP27C was generated based on assembled full-length CYP27C in pcDNA3.1. First, the C-terminal portion of the gene was amplified by PCR. The forward primer used is 5' - ATACAGTACCAAATGGACCGAGGC - 3' (SEQ. ID. NO:53) and the reverse primer used is 5' - TGGCTCGAGCTTTCTGTTAACAAATCGCACG - 3'(SEQ. ID. NO:54). The reverse primer removes the stop codon and has an Xhol site (underlined). The 700 bp PCR fragment was digested with Bg/ll and Xho\ and the 610 bp Bgr/ll - Xho\ fragment was cloned into the tagging vector pBS-H6 that had been digested with BamHI and Sa/I. The fidelity of the PCR fragment was confirmed by sequencing. This resulted in the addition of 8 amino acids, VDHHHHHH(SEQ. ID. NO:55), at the C-terminus of the CYP27C (to the C-terminus of (SEQ. ID. NO:56)). Then the resulting vector was digested with Sa/I and Kpnl and the 600 bp Sa/I - Kpnl fragment was recovered. Plasmid pcDNA3.1-CYP27C was digested with Nhel and Sa/I and the 1050 bp N/?el - Sa/I fragment was also recovered. The two gene fragments were ligated together to the backbone of pBS-H6, which had been digested with he\ and Kpπl, to give pBS-CYP27CH6. The presence of the full-length gene was confirmed by restriction digestion.

Expression of his-tagged CYP27C in insect cells

Plasmid pBS-CYP27CH6 was digested with NΛel and Kpnl and the 1.65 kb gene fragment was recovered and ligated to the donor plasmid pFastBad that had been digested with Spel and Kpnl. The resulting recombinant donor plasmid was used to generate baculovirus encoding CYP27CH6, according to the suggestions of the supplier (Gibco Life Technology). The expression of his-tagged CYP27C1 was confirmed by immunoblotting, as shown in Figure 13.

In Figure 13, whole cell lysates of Sf9 insect cells infected with two viruses encoding CYP27C were loaded onto the gel and then screened using a primary antibody: monoclonal Penta-His antibody (QIAGEN) used at

0.1 ng/ml and a secondary antibody: HRP conjugated goat anti mouse Ig used at 1 :10,000.

Expression of his-tagged CYP27C in V79 cells

The 1.65 kb NΛel - Kpnl gene fragment described above was also ligated to pcDNA3.1-Hygro that had been digested with the same enzymes. The plasmid with the proper insert was maxi-prepared and used to transfect V79 cells using FuGENE reagent as recommended by the supplier. The cells were selected for two weeks in the presence of 100 μg/ml hygromycin before being single-cell cloned in 96-well plate to isolate the expression clones. The cells in 96-well plate were expanded into 6-well plate and used for RNA isolation. RT-PCR analysis was performed (See Figure 14A and

14B) and positive clones (#4, 5, 7, and 10) were expanded and stocks prepared.

EXAMPLE 10 - Subcellular localization of CYP27C expressed in insect cells

Infection and subcellular fraction preparation

Sf9 cells were seeded in roller bottles at 0.8 million cells/ml in the presence of 2 μg/ml hemin chloride, 100 μM δ-amino-levulinic acid, and 100 μM ferric citrate. Baculovirus encoding CYP27C was added at multiplicity of infection of 2. The cells were collected 66-68 hours after infection and the mitochondrial and microsomal fractions were prepared as described below.

However other methods known in the art could be used.

The mitochondrial fraction was prepared as follows. Cells were washed once with cold MT buffer (50 mM Tris-CI, pH 7.4, 0.25 M sucrose, and 10 mM KCl), suspended in MT buffer, and then transferred to 50 ml tubes. The cells were then pelleted and the supernatant discarded.

Protease inhibiting tablet was disolved in MT buffer (one tablet/10 ml of MT buffer). CaCI₂ was added to 0.25 mM. The pelleted cells were then suspended by vortex in the above buffer (~ 10 ml per 150 million Sf9 cells). The cells were transferred into a chamber of pre-cooled nitrogen bomb. The cover was closed (clamped tightly) and the cells were equilibrated for 10 min under pressure of 300 psi. The cells were then slowly released from the bomb into the 50 ml tube. EGTA was added to 1 mM final concentration. The cells were then centrifuged at 800 g for 10 minutes at 4 °C. The supernatant was transferred to a Beckman 50 ml tube for JA20 rotor and centrifuged at 10,000 g for 10 minutes at 4 °C. The supernatant was kept for preparation of microsomes (See below). The pellet was washed once with 20 ml of MT buffer and centrifuged again at 10,000 g for 10 min. The supernatant was discarded and the tube carefully drained by inverting the tube on paper tower for a minute. 1 ml of MT buffer was added The mitochondrial fraction (pellet) was homogenized by pipetting up and down (avoid bubbles). Mitochondrial fractions can be combined if needed. The protein concentration was determined and then diluted to a concentration of 5 mg/ml. 180 μl was taken for the CO binding assay and the rest aliquoted in cryogenic vials. Samples were stored in -70 °C freezer until needed.

To prepare the microsomal fractions, the same supernatant, noted above, was transferred to a Beckman tube for SW28 rotor. The volume was brought to over 4/5 of the capacity of the tube with MT buffer and then centrifuged at 100,000g for 60 minutes at 4 °C. The supernatant was then discarded. The tube was carefully drained by inverting the tube on paper for a minute. 1 ml of MT buffer was added to the tube. The microsomes (pellet) were then centrifuged with a needle and syringe (avoid bubbles). Microsome preparations can be combined if needed. The protein concentration of the microsomal fraction was then determined and diluted to 5 mg/ml. 180 μl of the sample was taken for CO binding assay and the rest aliquoted in cryogenic vials. Samples were stored in -70 °C freezer until needed.

Spectrometric determination of subcellular localization of CYP27C The subcellular localization of CYP27C expressed in insect cells was determined by difference spectrometry using protocols known in the art. The results indicated that CYP27C is present in the mitochondrial fraction where CO binding is observed(see Figure 15A and 15B), but not the microsomal fraction (data not shown).

EXAMPLE 11 - Expression of CYP27C in Tumor Cell Lines

Total RNA was prepared from control or calcitriol-treated ( 10nM, 18 hrs) cells using NucleoSpin RNA II Purification kit (Clontech) and mRNA was isolated with NucleoTrap mRNA Mini kit (Clontech). MRNA was dissolved in 20ul of RNase-free water and stored at -80°C.

1 ul of mRNA was converted into cDNA using Thermoscript RT-PCR kit (Invitrogen) and one tenth of the product was used for the amplification of GAPDH or CYP27C target sequences with corresponding primers and Hot Start Taq polymerase (Qiagen). For PCR, samples were incubated at 95∞C for 15 min., followed by 40 cycles composed of 30 sec. at 99°°C, 30 sec. at 65∞C and 1 min. at 72°°C. Then samples were incubated for 5 min. at 72 ∞C and cooled to 4 °°C. 10 ul of each sample were fractionated on a 1 % agarose, IX TAE gel, stained in ethidium bromide solution and bands were visualized under UV. Human GAPDH specific primers yielded 0.95 kbp PCR fragment and human CYP27C specific primers produced 0.45 kbp PCR band.

The following CYP27C primers were used : Forward primer: 5'-CCAGAAGTGCAGCAGACGGTGTAC-3' (SEQ. ID. NO:4) Reverse primer: 5'-CTGGATCACGACGAGGTGAATCTC-3' (SEQ. ID. NO:5)

Figure16 shows the expression of human CYP27C in tumor cell lines (DU-145 (prostate), WTE(lung), SKMES (lung), SK-luci-6 (lung), PC3 (rostate) and MCF-7 (breast). Only 0.45kb RT-PCR band specific for CYP27C is shown. Sets of control (c) and calcitriol-treated (+) samples contained similar amount of mRNA as measured by RT-PCR with primers specific for GAPDH with exception of WTE (significantly less in "+" sample), SK-luci-6 (less in "+" sample) and MCF-7 (less in "c" sample). Table 3 summarizes the expression results.

EXAMPLE 12 - CYP27C Enzymatic Assay From the Isolated Mitochondrial Fraction The general method is one example of a sillastate screening assay, where CYP 27C1 is incubated with a [*] substate a test compound and the products monitored/deducted by coronctography and/or mass spechometry methods. The assay can also be used to detect modulators of CYP 27C1 activity, wherein CYP 27C1 substate and the potential modulators are incubated under conditions that promote CYP 27C1 any substate interaction. The products are then monitored for any modulation in activity. Materials CYP27C expressing mitochondria

48-Well plate

MT buffer (100 mM potassium phosphate, pH 7.4 and 250 mM sucrose)

DTT

EDTA Sodium isocitrate

NADPH

Isocitrate dehydrogenase

10% Acetic acid

Methanol Dichloromethane

Saturated KCl

Procedure

1. For substrate screening, CYP27C expressing mitochondria (150 μg protein) is mixed with testing compounds (50 to 100 μM final concentration) in 48-well plate in a total volume of 200 μl containing 100 mM potassium phosphate, pH 7.4, 250 mM sucrose, 1 mM DTT, 1 mM EDTA, 1 mM NADPH, 5 mM D, L-trisodium isocitrate, and 0.2 units isocitrate dehydrogenase. 2. The above is then incubated at 37 °C for 30 to 60 min.

3. 5 μl 10% acetic acid and 0.5 ml methanol is added to each well and then transferred to 1.5 ml centrifuge tubes. 4 0.25 ml dichloromethane is added and mixed by vortexing for 30 s. 0.25 ml dichloromethane and then 0.25 ml saturated KCl. The contents are then spun at 3,000 rpm for 10 min. The aqueous phase (top) is aspired and the organic phase (bottom) vacume dried.

4. The resulting dried products is then dissolve in 150 μl mobile phase (LC/MS) and spun at 10,000 rpm for 5 min. 140 μl of sample is then carefully transferred to the insert for LC/MS analysis.

]_ LC/MS analysis

INSTRUMENTATION*

Name Specification Company

HPLC System Reverse Phase Waters

Mass spectrometer + APCI source Micromass

HPLC Column Nova-Pack Waters

DD Water Cytochroma

Methanol HPLC grade

Acetonitrile HPLC grade

* Equivalent equipment may be substituted.

PREPARATION OF REAGENTS^*

Note: Solutions should be protected from direct light during preparation and storage.

Standard Solution "A". 1uα/ul

Cholesterol 10 mg in 1 ml Ethyl Acetate 1 ug/ul

25 OH Cholesterol 6 mg in 6 ml Ethyl Acetate + 3 ml DMSO (0.6 ug/ul)

7 Beta OH Cholesterol 1.4 mg in 0.4 ml Ethyl Acetate + 1 ml DMSO (1 ug/ul)

22 R OH Cholesterol 1 mg in 0.5 ml Ethyl Acetate + 0.5 ml DMSO (1 ug/ul)

22 S OH Cholesterol 2 mg in 1 ml Ethyl Acetate + 1 ml DMSO (1 ug/ul) 20 Alpha OH Cholesterol 3 mg in 2 ml Ethyl Acetate + 1 ml DMSO (1 ug/ul)

Solution "B". 1 ng/ul

Pipette 1.0 uL of 1 ug/ul Standard Stock Solution, into 1 mL test tube. Add 999.0 uL 70:30 Methanol: Acetonitrile. Store in 1.5 ml Eppendorf tubes at 0 to -20°C. ANALYTICAL PROCEDURE

Samples

Once the samples have been dried in a speed_vac Add 110 ul of 70:30 Methanol: Acetonitrile, vortex and centrifuge for 5 min. Transfer 100 ul to an insert in HPLC vail

Chromatographic Conditions

Note: The following conditions are guidelines and may vary with specific need or other conditions known in the art

Analytical column: Nova-Pak C₁₈, 5 μm, 3.9 x 150 mm Flow rate: 1.OmL/min

Detection: Micromass Ultima and UV spectrometer.

Injection volume: 10 μL (may vary depending upon the limit of detection)

Run time: 60minut.es

Mobile Phase

Mobile phase A: Water Mobile phase B: Acetonitrile Mobile phase C: Methanol

Linear Gradient Table

Mass Spectrometer Setting Ion Mode: positive Atmospheric pressure chemical ionization (+APCI) Corona (uA) 10.35 Cone (V) 38 Hex 1 (V) 0.0 Aperture (V) 0.8 Hex 2 (V) 0.9

Source Temperature (oC) 140 Desolvation Temperature (oC) 170 Cone Gas Flow (L/Hr) 86

Desolvation Gas Flow (L/Hr) 467 Nebulizer Gas Low (L/Hr) Max

Analyzer in MS Mode LM 1 11.9

LM 2 12.1

Ion energy 1 1.0

Entrance -17

Collision -1 Exit -22

LM 2 12.5

Ion Energy 2 0.7

Multiplier (V) 500 Pressure Gauges OFF

For MS MS and MRM the Collision energy should be set to 29 eV and the collision cell pressure should be set to 5 x 10-4 torr. Analyzer in MS MS Mode

LM 1 11.9

LM 2 12.1

Ion energy 1 1.0

Entrance -47 Collision -29

Exit -46

LM 2 12.5

Ion Energy 2 0.7 Multiplier (V) 500

The above method is described using mitochondria, but intact mammal ion cells or insect cells. The substrate used in the present example is cholesterol, but the protocol can be modified for other substrates. Further, once he substrate is identified, the method can be used to identify modulators of CYP 27C1 activity where the disappearance of the substrate and formation of the products are monitored in the presence and absence of potential modulators.

Mass Spectrometer optimization A Stock solution of 1 ug/ul of the standard is infused with a syringe pump into the HPLC Flow with a solvent composition of 90:10 Acetonitrile: Methanol .The HPLC flow was set to 1000 ul/min. The Total signal of the analyte m/z was optimized for full scan and msms scan.

Procedure

Analyze supematants^* obtained from standards, QC's and samples on HPLC system using C18 column (4 μm, 4.2 x 150 mm) with UV detection at a wavelength of 207 nm and the mass spectrometer. Unknown Cholesterols concentration in the samples is determined by comparison of their peak areas with those of authentic standards and QC's samples.

Dilute with mobile phase if needed.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

TABLE 1

SEQ. ID. NO:

10

1 1

1₂

1₃

1

15

16

17

19

₂0

^J primers for CYP 27 A 1 sequence (nucleotide sequence not shown) ^b primers tor Xenopus laevis EST sequence (nucleotide sequence not shown)

TABLE 2

Semi-guantitative estimation of CYP27C expression in human tissues.

Unequivocal detection of CYP27C PCR band (0.45 kbp) is indicated by "+" while the presence of very faint band of the same size is marked with "±". No CYP27C PCR product was detected in samples marked with "-". Majority of tissues showed amplification of human beta-actin target (0.65 kbp band) with only 1 pg of cDNA. TABLE 3 Summary of CYP27C1 expression in human tumor cell lines.

"-" CYP27C RT-PCR band not detected

"±" CYP27C RT-PCR band detectable

"+" CYP27C RT-PCR band unequivocally detected

"++" CYP27C RT-PCR band enhanced after calcitriol treatment

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

1. Shannon, M. (1997) Pediatric Emergency Care 13, 350-353

2. Nelson, D.R. (2000) http://drnelson.utmem.eduJP450lect.html: Cytochrome P450s in humans, p. 1-8. 3. Beckman, M., and DeLuca, H. (1997) Methods in Enzymol. 282,

200-223.

4. Jones, G., Strugnell, S., and DeLuca, H. (1998) Physiol. Rev. 78, 1193-1231.

5. Armbrecht, H.J., Okuda, K., Wongsurawat, N., Nemani, R., Chen, M., and Boltz, M. (1992) J. Steroid Biochem. Molec. Biol. 43, 1073-1081.

6. Postlind, H., Axen, E., Bergman, T., and Wikvall, K. (1997) Biochem. Biophys. Res. Commun. 241 , 491-497.

7. Tucker, G., Gagnon, R., and Haussler, M. (1973) Arch. Biochem. Biophys. 155, 47-57. 8. Vlahcevic, Z.R., Sanjeev, K.J., Heuman, D.M., Stravitz, R.T.,

Hylemon, P.B., Acadhani., N.G., and Pandak, W.M. (1996) Am. J. Physiol.

270, G646-G652.

9. Guo, Y.-D., Strugnell, S., Back, D.W., and Jones, G. (1993) Proc.

Natl. Acad. Sci. 90, 8668-8672. 10. Jones, G., Ramshaw, H., Zhang, A., Cook, R. Byford, V., White, J., and

Petkovich, M. (1998) Endocrinology 140, 3303-3310.

11. Bell, N.H., (1998) J. Bone Miner. Res. 13, 350- 352.

12. Fukushima, M., Susuki, Y., Tohira, Y., Nishii, Y., Suzuki, M., Sasaki, S., and Suda, T. (1976) FEBS Lett. 65, 211-214.

13. Guo, Y.-D., and Jones, G. (1994) J. Bone Miner. Res. 9, S289.

14. Feldman, D., Glorieux, F.H., and Pike, W. (1997). Vitamin D. (San Diego, CA), pp. 41-55.

15. Sambrook, J., Fritsh, E.F., and Maniantis, T. (1989) Molecular Cloning: A laboratory manual 2nd Edition. Cold Springs Harbour

Laboratory Press, pp. 6.3, 10.51. 16. O'Leary, K., and Kasper, C. (2000) Arch. Biochem. Biophys. 379, 97-108.

17. Gonzales, F.J. (1989) Pharmacol. Rev. 40, 243-288.

18. White, JA, Ramshaw, H., Taimi, M., Stangle, W., Zhang, A, Everingham, S., Creighton, S., Tarn, S.-P., Jones, G., and Petkovich, M.

(2000) Proc. Natl.

Acad. Sci. 97, 6403-6408.

19. Guo, Y.-D., Stugnell, S., and Jones, G. (1991) J. Bone Miner. Res. 6, S120. 20. Strugnell, S., Byford, V., Makin, H., Moriarty, R., Gilardi, R., LeVan,

L,

Knutson, J., Bishop, C, and Jones, G. (1995) Biochem. J. 310, 233-241.

21. Chun Li, Y., Bergwitz, C, Juppner, H., and Demay, M.B. (1997)

Endocrinology 138, 2347-2353. 22. Reinholz, G., and DeLuca, H.F. (1998) Arch. Biochem. Biophys.

355, 77-83.

23. Castillo-Olivares, A, and Gil, G. (2000) Nucleic Acids Res. 28, 3587-3593.

24. Hasler, J. (1999) Molecular Aspects of Medicine 20, 1-137. 25. Twisk, J., De Wit, E., and Princen, H. (1995) Biochem J. 305, 505-

511.

26. Clayton, P., Casteels, M., Mieli-Vergani, G., and Lawson, A. (1995) Pediatr Res. 37, 424-431. 27. Long, R.G., Skinner, R.K., and Meinhard, E. (1976) Gut 17, 824-

827. 28. Bell, N.H. (1985) J. Clin. Invest. 76, 1-6.

Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 90% identical to a sequence selected from the group consisting of :

(a) a polynucleotide encoding a polypeptide comprising amino acid sequence of Figure 2B or 11 ;

(b) a polynucleotide consisting of the nucleotide sequence of Figures 2A or 11A; (c) a polynucleotide of Figures 2A or 11 A wherein can also be U;

(d) a polynucleotide having a nucleic acid sequence complementary to (a), (b) or (c);

(e) a polynucleotide having a nucleic acid sequence which differs from any of the nucleic acid molecules of (a) to (c) in codon due to the degeneracy of the genetic code;

(f) a polynucleotide that is a variant, allelic variant or encodes a homolog or species homolog of any one of the polynucleotides of (a) to (c), or (e); and

(g) a polynucleotide comprising a fragment of the nucleic acid sequence of (a), (b), (c) ,(d), (e) or (f) that is at least 15 bases and which will hybridize to (a), (b), (c), (d), (e) or (f) under stringent hybridization conditions wherein the polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

2. The isolated nucleic acid molecule of claim 1 encoding a cytochrome P450, CYP27C1 protein.

3. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a mature form of the CYP 27C1 protein.

4. The isolated nucleic acid molecule of claim 1 , wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as CYP 27C1 identified in Figure 2A or 11 A.

5. The isolated nucleic acid molecule of claim 1 , wherein the polynucleotide fragment comprises the entire nucleotide sequence identified as CYP 27C1 encoded by the cDNA of Figure 2A or 11A.

6. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C- terminus or the N-terminus.

7. The isolated nucleic acid molecule of claim 4, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C- terminus or the N-terminus.

8. The isolated nucleic acid molecule of claim 4 further comprising a sequence that encodes a polyhistidine, VDHHHHHH peptide at the C- terminus of CYP 27C1.

9. A recombinant vector comprising an isolated nucleic acid molecule of any one of claim 1 - 8.

10. The recombinant vector of claim 9 wherein the isolated nucleic acid molecule is in a vector selected from the group of vectors consisting of Top 2.1, pcDNA 3.1 Hygro expression vector, pVL1393 baculovirus transfer vector or pBS.

11. A method of making a recombinant host cell comprising an isolated nucleic acid molecule of claim 1 wherein the host cell is transfected or transformed with a recombinant vector of claim 8 or 9.

12. A recombinant host cell produced by the method of claim 11.

13. The recombinant host cell of claim 12 comprising vector sequences.

14. The host cell of claim 12 wherein the vector is TOP 2.1 and the host cell is Top 10.

15. The host cell of claim 12, wherein the vector is pcDNA 3.1 Hygro expression vector and the host cell is a mammalian host cell.

16. The host cell of claim 15 , wherein the cell is a hamster V79 cell.

17. The host cell of claim 12, wherein the vector is pVL1393 and the cell is an insect cell.

18. The host cell of claim 17, wherein the insect cell is SF9.

19. An isolated polypeptide comprising an amino acid sequence at least 95% homologous to a sequence selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of Figure 2B or 11B;

(b) a polypeptide encoded by any of the polynucleotides selected from the group consisting of: (i) a polynucleotide encoding a polypeptide comprising amino acid sequence of Figure 2B or 11; (ii) a polynucleotide consisting of the nucleotide sequence of

Figures 2A or 11 A; (ii) a polynucleotide of Figures 2A or 11A wherein can also be U; (iii) a polynucleotide having a nucleic acid sequence which differs from any of the nucleic acid molecules of (h) to (j) in codon due to the degeneracy of the genetic code;

(iv) a polynucleotide that is a variant, allelic variant or encodes i a species homologue of any one of the polynucleotides of

(a) to (c),

(c) a mature form, variant, allelic variant or species homologue to any of the polypeptides of (a) or (b);

(d) a fragment of any of the polypeptides of (a) to (c).

20. An isolated antibody that binds specifically to the isolated polypeptide of claim 19 or immunogenic portion or epitope thereof.

21. A recombinant host cell that expresses the isolated polypeptide of claim 19.

22. The recombinant host cell of claim 21 , wherein the recombinant host cell is that of claim 14, 16 or 18.

23. A method of making an isolated polypeptide comprising:

[a] culturing the recombinant host cell of claim 21 or 22 under conditions such that said polypeptide is expressed; and

[b] recovering said polypeptide.

24. The polypeptide produced by claim 23.

25. The gene corresponding to the cDNA sequence of Figure 2A or 11A.

26. A pharmaceutical composition comprising the isolated polypeptide of claim 19, and/or optionally a modulator of CYP 27C1 activity in combination with a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, further comprising an adjuvant.

28. A method for preventing, treating or ameliorating a medical condition related to CYP 27C1 expression which comprises administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 19 or of the polynucleotide of claim 1 and/or a modulator of CYP 27C1.

29. A method of identifying CYP 27C1 activity in a biological assay, wherein the method comprises:

[a] expressing CYP 27C1 or the Xenopus laevis Cyp27C1 hybrid in a cell;

[b] isolating the biological fraction; [c] detecting an activity in a biological assay; and

[d] identifying the protein in the supernatant having the activity.

30. The product produced by the method of claim 30.

31. A method of diagnosis of a CYP 27C1-related disease or condition or a predisposition to a CYP 27C1 -related disease or condition comprising detecting a polymorphism in a CYP 27C1 gene, wherein detection of said polymorphism is indicative of the occurrence of said disease, condition or a predisposition thereto.

32. The method of claim 31 , wherein the polymorphism is detected by sequencing the CYP 27C1 peptide produced in said organism, or the coding nucleic acid molecule and comparing said sequence to that of known sequence to determine whether a polymorphism exists.

33. The method of claim 32, wherein the sequence is compared to those of individuals with a known disease state, to determine whether the sequence is indicative of said disease state or susceptibility to such a disease state.

34. A diagnostic kit for identification of polymorphisms in the CYP 27C1 gene, comprising screening the CYP 27C1 gene from a human for polymorphisms, wherein detection of said polymorphisms is indicative of the occurrence of a CYP 27C1 -related disease, condition or a predisposition thereto.

35. The method of claim 34 wherein the disease is related to vitamin D or vitamin D metabolite deficiency; a cholesterol, steroid or lipid metabolic disorder or cancer.

36. The method of claim 35 wherein disease related to vitamin D or its metabolites are selected from the group consisting of:

(i) in the parathyroid - hyper- and hypo-parathyroidism, Osudohypo-parathyroidism, Scondary hyperparathyroidism;

(ii) in the pancreas - diabetes; (iii) in the thyroid - medullary carcinoma;

(iv) (in the skin - psoriasis;

(v) in the lung - sarcoidosis and tuberculosis;

(vi) in the kidney - chronic renal disease, hypophosphtatemic VDRR, vitamin D dependent rickets; (vii) in the bone - anitconvulsant treatment, fibrogenisis imperfecta ossium, osteitits fibrosa cystica, osteomalacia, osteporosis, osteopenia, osteosclerosis, renal osteodytrophy, rickets;

(viii) in the intestine - glucocorticoid antagonism, idopathic hypercalcemia, malabsorption syndrome, steatorrhea, tropical sprue.

37. A method of treating a disease or condition related to vitamin D or vitamin D metabolite deficiency in a patient comprising administering to the patient in need thereof, an effective amount of a therapeutically effective polypeptide of claim 19 and/or an agonist thereof.

38. A method according to claim 37 wherein the polypeptide consists of the amino acid sequence of Figure 2B or a biologically active fragment or analog thereof.

39. The method of claim 35 wherein the disease is related to cholesterol metabolism.

40. The method of claim 39 wherein the disease is cerebrotendinous xanthomatosis.

41. A method of treating a disease or condition related to cholesterol metabolism in a patient comprising administering to the patient in need thereof, an effective amount of an effective amount of a therapeutically effective polypeptide of claim 19 and/or an agonist thereof.

42. A method of identifying modulators of CYP 27C1 activity in a biological assay, wherein the method comprises:

[a] expressing CYP 27C1 in a cell;

[b] adding a substrate; and [c] detecting activity of CYP 27C1 on said substrate in the presence or absence of a modulator.

43. A method of identifying a modulator of CYP 27C1 activity comprising:

[a] incubating CYP 27C1 or a cell expressing CYP 27C1 with a test compound under conditions that promote CYP 27C1 expression or activity;

[b] detecting the activity or expression, as the case may be, of CYP 27C1 in the presence of said test compound, a decrease in said activity or expression being indicative that the test compound is an inhibitor of CYP 27C1 expresssion or activity, while an increase in said expression or activity is indicative that the test compound is a CYP 27C1 agonist.

44. A method of identifying a substate of CYP 27C1 comprising:

[a] incubating CYP 27C1 with a test substrate under conditions that promote CYP 27C1/substate complex formation or interaction;

[b] determining CYP 27C1 /substrate complex formation or interaction.

45. The method of claim 44, wherein the incubation step further comprises a known modulator of CYP 27C1.

46. The method of claim 44 or 45 wherein step [b] can be determined by comparing the effect on CYP 27C1 in the absence and presence of the test substrate.

47. A method for identifying a substance which associates with a protein as claimed in claim 19 comprising

(a) reacting the protein with at least one substance which potentially can associate with the protein, under conditions which permit the association between the substance and protein, and

(b) removing or detecting protein associated with the substance, wherein detection of associated protein and substance indicates the substance associates with the protein.

48. A method for evaluating a compound for its ability to modulate the biological activity of a protein as claimed in claim 19 comprising providing the protein with a substance which associates with the protein and a test compound under conditions which permit the formation of complexes between the substance and protein, and removing and/or detecting complexes.

49. A method for identifying inhibitors of a CYP 27C1 Protein interaction, comprising

(a) providing a reaction mixture including the CYP 27C1 Related Protein and a substance that binds to the CYP 27C1 Related Protein, or at least a portion of each which interact;

(b) contacting the reaction mixture with one or more test compounds;

(c) identifying compounds which inhibit the interaction of the CYP 27C1 Related Protein and substance.

50. A method for detecting a nucleic acid molecule encoding a protein comprising an amino acid sequence of SEQ. ID. NO. 40 in a biological sample comprising the steps of:

(a) hybridizing a nucleic acid molecule of claim 1 to nucleic acids of the biological sample, thereby forming a hybridization complex; and

(b) detecting the hybridization complex wherein the presence of the hybridization complex correlates with the presence of a nucleic acid molecule encoding the protein in the biological sample.

51. A method as claimed in claim 50 wherein nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.

52. A composition comprising one or more of a nucleic acid molecule or protein claimed in any of the preceding claims, or a substance or compound identified using a method as claimed in any of the preceding claims, and a pharmaceutically acceptable carrier, excipient or diluent.

53. Use of one or more of a nucleic acid molecule or protein claimed in any of the preceding claims, or a substance or compound identified using a method as claimed in any of the preceding claims in the preparation of a pharmaceutical composition for treating a condition mediated by a protein as claimed in claim 19, or a nucleic acid molecule as claimed in claim 1.

54. A method of conducting a drug discovery business comprising:

(a) providing one or more assay systems for identifying agents by their ability to inhibit or potentiate the interaction of a CYP 27C1 Related Protein and a substance that binds to the CYP 27C1

Related Protein;

(b) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and (c) formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.

55. A vaccine for stimulating or enhancing in a subject to whom the vaccine is administered production of antibodies directed against a protein as claimed in claim 19.

56. A method for stimulating or enhancing in a subject production of antibodies directed against a protein as claimed in claim 19.

57. A method as claimed in claim 56 comprising administering to the subject a vaccine as claimed in claim 55 in a dose effective for stimulating or enhancing production of the antibodies.