AU776572B2 - Gene screening method using nuclear receptor - Google Patents

Description

S&F Ref: 493659D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Chugai Seiyaku Kabushiki Kaisha 5-1, Ukima 5-chome, Kita-ku, TOKYO 115-8543 Japan Shigeaki Kato, Ken-ichi Takeyama, Sachiko Kitanaka Spruson Ferguson St Martins Tower,Level 35 SEC 31 Market Street 104 Sydney NSW 2000 (CCN 3710000177) -Method fo Gene Screening wit e efNuclear Receptors Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c ry Specification Gene Screening Method Using Nuclear Receptor Technical Field This invention relates to a method for screening a compound using the nature of transcriptional regulatory factors, mainly nuclear receptors, and a method for determining the compound.

Specifically, it relates to a method for screening a gene encoding a polypeptide that converts a ligand precursor into a ligand, a polypeptide that converts a ligand precursor obtainable by the screening method into a ligand, a gene encoding the polypeptide, and a method for determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand. In addition, it relates to a method for screening a ligand that binds to a nuclear receptor, a ligand obtainable by the screening method, and a method for determining whether or not a test compound is a ligand that binds to a nuclear receptor. Furthermore, it relates to a method for screening a gene encoding a polypeptide that converts an inactive formlof a transcriptional regulatory factor into an active form.

Background Art

D

3 (1a,25(OH) 2 D3)(A. W. Norman, J. Roth, L. Orchi, Endocr. Rev. 3, 331 (1982); H. F. DeLuca, Adv. Exp. Med. Biol. 196, 361 (1986); M. R. Walters, Endocr. Rev. 13, 719 (1992)) is a hormone form of vitamin D and the most biologically active natural metabolite. This compound is generated by sequential hydroxylation. First, it is hydroxylated in the liver to generate 20 25-hydroxyvitamin D 3 (25(OH)D3), then subsequently hydroxylated in the kidney to generate 1a,25(OH) 2

D

3 Kawashima, S. Torikai, K. Kurokawa, Proc. Natl. Acad. Sci. USA 78, 1199 (1981); H. L. Henry et al., J. Cell. Biochem. 49, 4 (1992)). The transactivation effect of vitamin D receptor (VDR) is provoked by the binding of 1a,25(OH) 2

D

3 to VDR Beato, P. Herrlich, G. Schuts, Cell 83, 851 (1995); H. Darwish and H. F. DeLuca, Eukariotic Gene Exp. 3, 89 (1993); B. D. Lemon, J. D.

25 Fondell, L. P. Freedman, Mol. Cell. Biol. 17, 1923 (1997)). This regulates the transcription of a series of target genes involved in the major functions of vitamin D, such as calcium homeostasis, cell differenciation, and cell proliferation D. Bikle and S. Pillai, Endoc. Rev. 14, 3 (1992); R. Bouillon, W. H. Okamura, A. W. Norman, Endoc. Rev. 16, 200 (1995); M. T. Haussler et al., Recent Prog.

Horm. Res. 44, 263 (1988); P. J. Malloy et al., J. Clin. Invest. 86, 2071 (1990)). The importance of the hydroxylation of 25(OH)D3 in the kidney in the synthesis of active vitamin D has been shown, and it has been believed for a long time that the hydroxylation is done by 25(OH)D 3 -la hydroxylase (la(OH)-ase), which is localized especially at proximal renal tubules. The activity of la(OH)-ase is negatively regulated by its final product, la,25(OH) 2

D

3 Tanaka and H. F. DeLuca, Science 183, 1198 (1974); K. Ikeda, T. Shinki, A. Yamaguchi, H. F. DeLuca, K. Kurokawa, T. Suda, Proc. Natl.

Acad. Sci. USA 92, 6112 (1995); H. L. Henry, R. J. Midgett, A. W. Norman, J. Biol. Chem. 249, 7584 (1974)), and positively regulated by peptide hormones like calcitonin and PTH, which participate in calcium regulation Kawashima, S. Torikai, K. Kurokawa, Nature 291, 327 (1981); K. W. Colston, L.

M. Evans, L. Galauto, L. Macintyre, D. W. Moss, Biochem. J. 134, 817 (1973); D. R. Fraser and E.

Kodicek, Nature 241, 163 (1973); M. J. Beckman, J. A. Johnson, J. P. Goff, T. A. Reinhardt, D. C.

[R:\LIBC]08091 .doc:ais Beitz, R. L. Horst, Arch. Biochem. Biophys. 319, 535 (1995)). The complicated regulation of the la (OH)-ase activity by these hormones maintains the serum concentration of 1a,25(OH)2D3 at a certain level. The mutation of the la(OH)-ase gene may causes a genetic disease, vitamin D-dependent type I rickets Fraser, S. W. Kooh, H. P. Kind, M. F. Hollick, Y. Tanaka, H. F. DeLuca, N. Engl. J. Med.

289, 817 (1973); S. Balsan, inRickets, F. H. Glorieux, Ed. (Raven, New York, 1991), pp155-165), which also demonstrate the importance of the enzyme in vivo in the function of vitamin D. The biochemical analysis of partially purified la(OH)-ase protein strongly suggested that this enzyme belongs to P450 family Wakino et al., Gerontology 42, 67 (1996); Eva Axen, FEBS Lett. 375, 277 (1995); M. BurgosTrinidad, R. Ismaii, R. A. Ettinger, J. M. Prahl, H. F. DeLuca, J. Biol. Chem. 267, 3498 (1992); M. Warner et al., J. Biol. Chem. 257, 12995 (1982)). However, the biochemical characteristics of the enzyme and the molecular mechanism of the negative feedback by 1a,25(OH)2D3 are not well understood since the enzyme purification is difficult and cDNA has not been cloned yet. Thus, the cDNA cloning of the enzyme had been desired. Recently, the cloning of .the rat enzyme that hydroxylates the la position of vitamin D has been reported Bone Min. Res.

15 i Vol. 11 (supl) 117 (1996)).

Conventionally, methods depend on phosphorylation of intracellular signal transduction factors or ion channels of membrane receptors as criteria have mainly been employed to screen genes encoding polypeptides that act on a specific nuclear receptor directly or indirectly, including la (OH)ase mentioned above. Specifically, expression vectors into which a cDNA library or cDNA is inserted 20 are introduced into cells or haploid individuals, for example Xenopus oocytes, and then phosphorylation, cell growth and the change in the electric current has been monitored for the screening.

However, it has been very difficult to isolate genes by using these methods. Especially, highly sophisticated techniques are required for the expression cloning of an enzyme itself because the 25 indicators available for the detection are limited. Therefore, the development of a simple and efficient screening method has been desired.

Disclosure of the Invention An objective of the present invention is to provide a simple and efficient method for screening a gene encoding a polypeptide that converts a ligand precursor into a ligand, and a method for determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand. Another objective of the present invention is to provide a method for isolating a polypeptide that converts a ligand precursor into a ligand and a gene encoding it, using the screening method.

Furthermore, an objective of the invention is to provide a method for screening a ligand that binds to a nuclear receptor, a method for determining whether or not a test compound is a ligand for a nuclear receptor, and a method for screening a gene encoding a polypeptide that converts an inactive form of a transcriptional regulatory factor into an active form, based on the screening method and the determination method described above.

The present inventors investigated to achieve the above objectives and focused on the nature of nuclear receptors, which function as transcriptional regulatory factor by being bound by a ligand.

C08091 We successfully constructed the system in which a ligand is formed by the expression of a polypeptide that converts a ligand precursor into a ligand, and the ligand thus formed binds to a nuclear receptor to thereby induce the expression of a reporter gene located downstream of the target sequence. We searched a gene library using this system and succeeded in isolating a gene encoding a polypeptide capable of converting a ligand precursor into a ligand.

Specifically, the inventors constructed a vector comprising a gene encoding a fusion polypeptide of DNA binding domain of GAL4 and ligand-binding domain of vitamin D receptor and a vector in which the lacZ gene, a reporter, is located downstream of the binding sequence of the DNA binding domain of GAL4. These two vectors, and subsequently the cDNA library, were introduced into cells. Then the reporter activity was measured after adding the vitamin D precursor. Clones with the reporter activity were selected to examine whether or not they have the activities to convert the vitamin D precursor into vitamin D, thereby finding a clone that has the activity.

Also, the inventors found that this system, which takes the advantage of the transcriptional regulatory function of a nuclear receptor, makes it possible to screen a ligand that binds to a nuclear receptor and to examine whether or not a test compound is a ligand that binds to the nuclear receptor. Specifically, for example, a library of test •3 compounds can be used in place of a ligand precursor and a gene library comprising the 20 gene encoding a polypeptide that converts a precursor into a ligand in the system described o above. When a test compound functions as a ligand, the nuclear receptor promotes the transcription of the reporter gene. Thus, compounds that function as ligands can be .:screened from the library simply by detecting the reporter activity as an index.

Furthermore, the inventors found that the system utilizing the transcriptional 0 25 regulatory function of a nuclear receptor can be employed to screen genes that encode polypeptides capable of converting an inactive form of a wide range of transcriptional S. regulatory factors into an active form. In other words, the inventors found that the system o in which the transcriptional regulatory function can be used to isolate factors involved in activation of various transcriptional regulatory factors, which have inactive and active forms, such as transcriptional regulatory factors activated by phosphorylation as well as nuclear receptors activated by the binding of ligands.

[R:\LIBFF] 17891 spec.doc:GCC 3a According to a first embodiment of the invention, there is provided a method for screening a gene encoding a polypeptide that converts a ligand precursor into a ligand, the method comprising introducing a test gene into a cell comprising a vector carrying a gene encoding a nuclear receptor and a vector carrying the binding sequence of the nuclear receptor and a reporter gene located downstream of said binding sequence, contacting a ligand precursor to the cell into which the test gene is introduced, detecting the reporter activity, and isolating the test gene from the cell which showed the reporter activity.

According to a second embodiment of the invention, there is provided a method for determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand, the method comprising introducing a test gene into a cell comprising a vector carrying a gene encoding a nuclear receptor and a vector carrying the binding sequence of the nuclear receptor and a reporter gene located downstream of said binding sequence, contacting a ligand precursor to the cell into which the test gene is introduced, and detecting the reporter activity.

According to a third embodiment of the invention, there is provided a method for 20 screening a gene encoding a polypeptide that converts an inactive form of vitamin D 3 into S: an active form, the method comprising introducing a test gene into a cell comprising a vector carrying a gene encoding "a nuclear vitamin D receptor and a vector carrying the binding sequence of the vitamin D oo receptor and a reporter gene located downstream of said binding sequence, S" 25 contacting an inactive form of vitamin D 3 to the cell into which the test gene is introduced, detecting the reporter activity, and :j isolating the test gene from the cell that shows the reporter activity.

According to a fourth embodiment of the invention, there is provided a method for determining whether or not a test gene encodes a polypeptide that converts an inactive form of vitamin D 3 into an active form, the method comprising [R:\LIBFF] 17891 spec.doc:GCC 3b introducing a test gene into a cell comprising a vector carrying a gene encoding a nuclear vitamin D receptor and a vector carrying the binding sequence of the vitamin D receptor and a reporter gene located downstream of said binding sequence, contacting an inactive form of vitamin D 3 with the cell into which the test gene is introduced, and detecting the reporter activity.

According to a fifth embodiment of the invention, there is provided an isolated gene encoding a polypeptide that converts a ligand precursor into a ligand comprising the sequence of SEQ ID NO:3 or 4.

According to a sixth embodiment of the invention, there is provided an isolated gene encoding a polypeptide that converts an inactive form of vitamin D 3 into an active form by hydroxylating position la comprising the sequence of SEQ ID NO:3 or 4.

According to a seventh embodiment of the invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or its derivative comprising said sequence in which one or more amino acids are substituted, deleted, or added, and having activity to convert an inactive form of vitamin D 3 into an active form.

According to an eighth embodiment of the invention, there is provided an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or its derivative comprising said sequence in which one or more amino acids are substituted, deleted, or 20 added, and having activity to convert an inactive form of vitamin D 3 into an active form.

According to a ninth embodiment of the invention, there is provided an isolated polypeptide encoded by a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 3, wherein the polypeptide has activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position la.

S 25 According to a tenth embodiment of the invention, there is provided an isolated polypeptide encoded by a DNA that hybridizes with the nucleotide sequence of SEQ ID NO: 4, wherein the polypeptide has activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position Ica.

According to an eleventh embodiment of the invention, there is provided an isolated DNA encoding the polypeptides in accordance with any one of the seventh to tenth embodiments of the present invention.

According to a twelfth embodiment of the invention, there is provided an isolated DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3 and encoding a polypeptide having activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position 1 a.

[R:\LIBFF] 17891 spec.doc:GCC According to a thirteenth embodiment of the invention, there is provided an isolated DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 4 and encoding a polypeptide having activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position lca.

According to a fourteenth embodiment of the invention, there is provided a vector comprising the DNA in accordance with any one of the eleventh to thirteenth embodiments of the present invention.

According to a fifteenth embodiment of the invention, there is provided a transformant expressively retaining the DNA in accordance with any one of the eleventh to thirteenth embodiments of the present invention.

According to a sixteenth embodiment of the invention, there is provided a method for producing the polypeptide in accordance with any one of the seventh to tenth embodiments of the present invention, the method comprising culturing the transformant DNA in accordance with the fifteenth embodiment of the present invention.

According to a seventeenth embodiment of the invention, there is provided an isolated antibody that specifically binds to the polypeptide in accordance with any one of S: the seventh to tenth embodiments of the present invention.

S•According to an eighteenth embodiment of the invention, there is provided a method S: for screening a gene encoding a polypeptide that converts an inactive form of S" 20 transcriptional regulatory factor into an active form, the method comprising introducing a test gene into cells into which a vector comprising a gene encoding an inactive form of transcriptional regulatory factor and a vector comprising the binding sequence of said inactive transcriptional regulatory factor and a reporter gene located downstream thereof are introduced, 25 detecting the reporter activity, and isolating the test gene from the cells showing the reporter activity.

More specifically, the disclosure herein relates to: 1. a cell comprising a vector carrying a gene encoding a nuclear receptor and a vector carrying the binding sequence of the nuclear receptor and a reporter gene located downstream of said binding sequence; 2. the cell of 1, wherein the nuclear receptor is a vitamin D receptor; 3. a cell comprising a vector carrying a gene encoding a fusion polypeptide comprising DNA binding domain of a nuclear receptor and ligand-binding domain of a nuclear receptor, and a vector carrying the binding sequence of the DNA binding domain of the nuclear receptor and a reporter gene located downstream of the binding sequence; [R:\LIBFF] 17891 spec.doc:GCC 3d 4. the cell of 3, wherein the DNA binding domain of the nuclear receptor is originated from GAL4; the cell of 3, wherein the ligand-binding domain of the nuclear receptor is originated from vitamin D receptor; 56. a method for screening a ligand that binds to a nuclear receptor, the method comprising 0 0 0**@0 :0.0.0 .0* 0: [R:\LIBFF]I 7891 spec-doc:GCC contacting a test compound with the cell of any one of 1 to detecting the reporter activity, and selecting the test compound which elicited the reporter activity in the cell; 7 a method for determining whether or not a test compound is a ligand that binds to a nuclear receptor, the method comprising, contacting a test compound with any one of the cell of 1 to 5, and detecting the reporter activity; 8 a method for screening a gene encoding a polypeptide that converts a ligand precursor into a ligand, the method comprising introducing a test gene into any one of the cell of 1 to contacting a ligand precursor to the cell into which the test gene is introduced, detecting the reporter activity, and isolating the test gene from the cell which showed the reporter activity; 9 a method for determining whether or not a test gene encoding a polypeptide that converts a 15 ligand precursor into a ligand, the method comprising introducing a test gene into any one of the cell of 1 to contacting a ligand precursor to the cell into which the test gene is introduced, and detecting the reporter activity; a method for screening a gene encoding a polypeptide that converts an inactive form of vitamin S- 20 D 3 into an active form, the method comprising introducing a test gene into the cell of 2 or contacting an inactive form of vitamin D 3 to the cell into which the test gene is introduced, detecting the reporter activity, and isolating the test gene from the cell that shows the reporter activity; 11 a method for determining whether or not a test gene encodes a polypeptide that converts an S• inactive form of vitamin D 3 into an active form, the method comprising introducing a test gene into the cell of 2 or contacting an inactive form of vitamin D 3 with the cell into which the test gene is introduced, and detecting the reporter activity; 12 a ligand that binds to a nuclear receptor, which is obtainable by the method of 6; 13 a gene encoding a polypeptide that converts a ligand precursor into a ligand, which is obtainable by the method of 8.

14 a gene encoding a polypeptide that converts an inactive form of vitamin D 3 into an active form, which is obtainable by the method of 15 a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or its derivative comprising said sequence in which one or more amino acids are substituted, deleted, or added, and having activity to convert an inactive form of vitamin D 3 into an active form; 16 a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or its derivative comprising said sequence in which one or more amino acids are substituted, deleted, or added, and having activity to convert an inactive form of vitamin D 3 into an active form; C08091 17 a polypeptide encoded by a DNA that hybridises with a DNA having the nucleotide sequence of SEQ ID NO: 3, wherein the polypeptide has activity to convert an inactive form of vitamin D 3 into an active form; 18 a polypeptide encoded by a DNA that hybridises with the nucleotide sequence of SEQ ID NO: 4, wherein the polypeptide has activity to convert an inactive form of vitamin D 3 into an active form; 19 a DNA encoding any one of the polypeptide of 15 to 18; a DNA hybridising with a DNA having the nucleotide sequence of SEQ ID NO: 3 and encoding a polypeptide having activity to convert an inactive form of vitamin D 3 into an active form; 21 a DNA hybridising with a DNA having the nucleotide sequence of SEQ ID NO: 4 and encoding a polypeptide having activity to convert an inactive form of vitamin D 3 into an active form; 22 a vector comprising any one of the DNA of 19 to 21; 23 a transformant expressively retaining any one of the DNA of 19 to 21; 24 a method for producing any one of the polypeptide of 15 to 18, the method comprising culturing the transformant of 23; 25 an antibody that binds to any one of the polypeptide of 15 to 18; 26 a method for screening a gene encoding a polypeptide that converts an inactive form of transcriptional regulatory factor into an active form, the method comprising introducing a test gene into cells into which a vector comprising a gene encoding an inactive form of transcriptional regulatory factor and a vector comprising the binding sequence of said inactive S: 20 transcriptional regulatory factor and a reporter gene located downstream thereof are introduced, detecting the reporter activity, and isolating the test gene from the cells showing the reporter activity; 27 a method of 26, wherein the inactive transcriptional regulatory factor is a complex of nonphosphorylated NFKB and IKB, non-phosphorylated HSTF, or non-phosphorylated AP1.

25 The term "ligand" used herein means a compound that binds to a nuclear receptor and regulates the transcriptional activating ability of a target gene of the nuclear receptor. It includes not only naturally-occurring compounds but also synthetic compounds.

The term "nuclear receptor" used herein means a factor that binds to an upstream site of a promoter of a target gene and ligand-dependently regulates transcription.

The "polypeptide that converts a ligand precursor into a ligand" includes a polypeptide that acts directly on a ligand precursor to convert it into a ligand. It also includes a polypeptide that indirectly converts a ligand precursor into a ligand, for example, a polypeptide activating a polypeptide that directly acts on a ligand precursor to convert it into a ligand.

The "transcriptional regulatory factor" used herein means a factor that binds to an upstream site of a promoter of a target gene and regulates transcription of the target gene. The above-described nuclear receptor is included in the transcriptional regulatory factor of the invention.

The "polypeptide that converts an inactive form of transcriptional regulatory factor into an active form" used herein includes not only a polypeptide that acts directly on an inactive form of transcriptional regulatory factor to convert it into an active form but also a polypeptide that indirectly converts an inactive form to an active form. When an inactive form of transcriptional regulatory factor C08091 is converted into an active form by phosphorylation, the transcriptional regulatory factor of the invention includes a polypeptide that activates a polypeptide phosphorylating the inactive form and indirectly converts the inactive form into the active form as well as a polypeptide directly involved in the phosphorylation.

The first aspect of the present invention relates to a method for screening a gene encoding a polypeptide that converts a ligand precursor into a ligand, and a method for determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand. In these methods, a vector carrying a gene encoding a nuclear receptor (expression unit and a vector carrying the binding sequence of the nuclear receptor and a reporter gene located downstream thereof (expression unit 2) are introduced into cells. Then, a test gene is introduced into the cells.

The "gene encoding a nuclear receptor" in the expression unit 1 is not particularly limited and any nuclear receptor gene can be used. For example, when orphan receptors such as PPAR, LXR, FXR, MB67, ONR, NUR, COUP, TR2, HNF4, ROR, Rev-erb, ERR, Ftz-F1, TIx and GCNF (Tanpakusitsu Kakusan Koso (Protein, Nucleic Acid, Enzyme) Vol. 41 No. 8 p1265-1272 (1996)) are S 15 used as the nuclear receptor in the below-mentioned screening of unknown ligands that bind to .nuclear receptors or determination whether or not a test'compound is a ligand binding to a nuclear receptor, the naturally-occurring or synthesized ligand can be detected and isolated. Furthermore, nuclear receptors for which the ligand and ligand precursor are known, such as VDR (vitamin D receptor), ER, AR, GR, MR (Tanpakusitsu Kakusan Koso (Protein, Nucleic Acid, Enzyme) Vol. 41 No.

20 8 p1265-1272 (1996)) are preferably used in the below-mentioned screening of genes encoding polypeptides that convert a ligand precursor into a ligand or the determination whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand. However, nuclear receptors used in the present invention are not limited thereto.

In the present invention, the nuclear receptor gene can be used alone, and a fusion polypeptide i 25 gene comprising the DNA binding domain of a nuclear receptor and the ligand-binding domain of another nuclear receptor can also be used. For example, the DNA binding domain of GAL4 is preferably used as the DNA binding domain because it enhances the expression of the reporter gene downstream thereof.

The "binding sequence of a nuclear receptor" in the expression unit 2 varies depending on the nuclear receptor. In most nuclear receptors, sequences comprising "AGGTCA" are usually used. In the case of a dimeric nuclear receptor, the binding sequence is preferably composed of two repetition of the sequence. The repetitive sequences include the direct-repeat type, in which the two sequences are aligned in the same direction, and the palindrome type, in which the sequences are directed to the center (Tanpakusitsu Kakusan Koso (Protein, Nucleic Acid, Enzyme) Vol. 41 No. 8 p1265-1272 (1996)). A spacer sequence usually exists between the repetition sequences, which can determine the specificity of the nuclear receptor Umesono et al., Cell Vol. 65, p1255-1266 (1991)).

A reporter gene located downstream of a nuclear receptor is not particularly limited. Preferable reporter genes are, for example, lacZ, CAT, and luciferase. Resistant genes to toxins or antibiotics, C08091 such as ampicillin resistant gene, tetracycline resistant gene, kanamycin resistant gene, can also be used to select cells by applying the corresponding toxin or antibiotic.

The binding sequence of a nuclear receptor and the reporter gene are not necessarily connected directly. Some sequences that alter the strength of the promoter, for example, the promoter region of p-globin, can be inserted between the binding sequence and the reporter gene.

Animal cells are preferable for introducing these expression units. Cells with high transformation efficiency such as COS-1 cells and HeLa cells are particularly preferable. Vectors for animal cells such as "pcDNA3" (Invitrogen) are preferred to construct expression units. Vectors can be introduced into host cells by a known method such as calcium phosphate method, lipofection method, electroporation method and the like.

A test gene is introduced into cells thus prepared. A test gene is not particularly limited, and any genes whose capability of converting a ligand precursor into a ligand is detected can be used.

S.Genes are screened from cells or cDNA libraries prepared from mRNA isolated from tissues or the like, which are expected to express an objective gene. For example, a gene encoding a polypeptide that converts vitamin D precursor into active vitamin D can be screened from a cDNA library derived from kidney or the like. In this case, a vector expressing adrenodoxin (ADX) and an vector expressing S. adrenodoxin reductase (ADR) are preferably introduced into cells together with a test gene so as to efficiently generate active vitamin D. A test gene can be inserted into an appropriate vector and S. introduced into cells. For example, preferable vectors are 'pcDNA3' (Invitrogen) mentioned above or 20 the like.

Next, cells into which a test gene is introduced are contacted with a ligand precursor. As the ligand precursor, the one that acts on a nuclear receptor expressed by the expression unit 1 mentioned above is usually used. Examples of the ligand precursor include, without limitation, hydroxyvitamin D 3 a precursor of VDR ligand (active vitamin D, la,25(OH)2D3); testosterone, a .i 25 precursor of ER ligand (estrogen) and AR ligand (dihydroxytestosterone); 11-deoxycortisol, a precursor of GR ligand (cortisol), corticosterone, a precursor of MR ligand (aldosterone), etc. The contact of the ligand precursor with the cells can be performed by adding the ligand precursor to the culture medium of the cells, or a similar method.

The reporter activity is then detected. If a test gene that is introduced into cells encodes a polypeptide that converts a ligand precursor into a ligand, the ligand generates from the ligand precursor contacted with the cells, and binds to the nuclear receptor to make a ligand- nuclear receptor complex, which then binds to its target sequence to express the reporter gene. If the test gene does not encode a polypeptide that converts a ligand precursor into a ligand, the ligand is not produced from the ligand precursor and thus the reporter gene is not expressed. In this way, detecting the reporter activity enables judging whether or not the test gene encodes a polypeptide that converts a ligand precursor into a ligand. The reporter activity can be detected by a method well known in the art using criteria such as staining, fluorescence, or cell viability, depending on the reporter gene.

C08091 8 When a gene library or the like is used instead of a single gene, cells are selected by the reporter activity to isolate the test gene. The test gene can be extracted from cells by, for example, the method described in H. S. Tong et al., Joumal of Bone and Mineral Research Vol. 9, 577-584 (1994). The primary structure of the gene extracted can be determined by a known method such as dideoxy method.

The cells into which expression units 1 and 2 are introduced can be used for screening genes encoding polypeptides capable of converting a ligand precursor into a ligand or determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand. Furthermore, the cells can be used for screening ligands that bind to a nuclear receptor or determining whether or not a test compound is a ligand that binds to a nuclear receptor. Specifically, a candidate for a ligand that acts on a nuclear receptor (a single test compound or a library of test compounds) is used instead of a ligand precursor and a candidate for a gene encoding a polypeptide that converts the ligand .i precursor into the ligand (a single candidate gene, gene libraries, etc.). When a test compound functions as a ligand, a complex of a nuclear receptor and the test compound (ligand) activates the s15 reporter located downstream of the target sequence and thus whether or not the test compound function as a ligand can be judged. Furthermore, compounds that function as ligands can be screened from plural compounds by detecting the reporter activity.

The inventors screened genes encoding polypeptides capable of converting the vitamin D precursor into active vitamin D as an example of the screening of genes encoding enzymes capable of converting a ligand precursor into a ligand, and obtained a desired gene. The present invention also relates to a polypeptide that converts the vitamin D precursor into active vitamin D and a gene encoding it.

Polypeptides derived from mouse and human that convert the vitamin D precursor into active vitamin D, which are encompassed by the polypeptides of the present invention, are shown in SEQ ID 25 NO: 1 and SEQ ID NO: 2, respectively. Vitamin D is first hydroxylated in the liver to generate 3 then hydroxylated in the kidney to generate la,25(OH) 2

D

3 The polypeptide of the present invention converts 25(OH)D3 into la,25(OH) 2

D

3 by hydroxylation, namely hydroxylates the la position of vitamin D (la(OH)-ase).

The polypeptide of the present invention can be a naturally-occurring protein. Alternatively, it can be prepared as a recombinant polypeptide by gene recombination techniques. Both are included in the polypeptide of the present invention. A naturally-occurring protein can be isolated by methods well known in the art, for example, from kidney cell extract by affinity chromatography using an antibody binding to the polypeptide of the present invention as described below. On the other hand, a recombinant protein can be prepared by culturing cells transformed with a DNA encoding the polypeptide of the present invention as described below.

In addition, those skilled in the art can prepare polypeptides with substantially the same biological activity as the polypeptide set forth in SEQ ID NO: 1 (or SEQ ID NO: 2) by substituting amino acid(s) of the polypeptide or the like known method. The mutation of amino acids can occur spontaneously. The polypeptide of the present invention also includes the mutants of the polypeptide C08091 set forth in SEQ ID NO: 1 (or SEQ ID NO: 2) whose amino acid(s) are modified by substitution, deletion or addition, and which possesses the activity to convert the inactive form of vitamin D3 into the active form. The known method of modifying an amino acid sequence is, for example, the method described in the literature, "Shin Saiboukougaku Jikken Protocol, Ed. Department of Oncology, The Institute of Medical Science, The University of Tokyo, p2 4 1-2 4 Mutations can be introduced by using commercially available 'QuickChange Site-Directed Mutagenesis Kit' (Stratagene).

It is a routine for those skilled in the art to prepare probes based on the entire or the partial nucleotide sequence of SEQ ID NO: 3 encoding the mouse polypeptide or SEQ ID NO: 4 encoding the human polypeptide, isolate DNAs with high homology with the probes from other species, and obtain polypeptides having the activities substantially equivalent to those of the polypeptide of the present invention using a known method such as hybridization technique Ebihara et al., Molecular and Cellular Biology, Vol. 9, 577-584 (1994)) or polymerase chain reaction technique Kitanaka et al., Journal of Clinical Endocrinology and Metabolism, Vol. 82, 4054-4058 (1997)). Therefore, the polypeptides of the present invention include those encoded by DNAs that hybridize with the DNA 1i having the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and having the activity to convert an inactive form of vitamin D3 into an active form. Animal species used for isolating DNAs hybridizing with the DNA having the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4 include rat, monkey, etc. DNAs encoding polypeptides with biological activities substantially equivalent to those of the polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO: 2 usually have high homology with the DNA set 20 forth in SEQ ID NO: 3 or SEQ ID NO: 4. The "high homology" means sequence identity of 70% or more, preferably 80% or more, and more preferably 90% or more. The homology of sequences can be calculated, for example, by the method described in Proc. Natl. Acad. Sci. USA 80, 726 (1983). An example of a method to isolate DNAs with high homology is as follows. The nucleotide sequence of SEQ ID NO: 3 (or SEQ ID NO: 4, which encodes the human polypeptide) is radiolabeled with 32P and 25 used as a template to screen a cDNA library of a desired species. The hybridization conditions are usually 10% formamide, 5x SSPE, 1x Denhardt's solution, 1x salmon sperm DNA, preferably formamide, 5x SSPE, lx Denhardt's solution, lx salmon sperm DNA, and more preferably formamide, 5x SSPE, 1x Denhardt's solution, 1x salmon sperm DNA.

Another aspect of the present invention relates to a DNA encoding the polypeptide of the present invention described above. The DNA of the present invention can be cDNA, genomic DNA, or synthetic DNA. It can be used not only to isolate a polypeptide with activities substantially equivalent to those of the polypeptide of the present invention from other species, but also to produce the polypeptide of the present invention as a recombinant polypeptide. Specifically, the DNA encoding the polypeptide of the present invention, for example, the DNA set forth in SEQ ID NO: 3 or SEQ ID NO: 4, is inserted into an appropriate vector, which are introduced into appropriate cells. The transformant cells are cultured to express the polypeptide, and the recombinant polypeptide is purified from the culture.

The cells used to produce the recombinant polypeptide include, for example, Escherichia coli and mammalian cells. The vectors used for expressing the recombinant polypeptide in the cells vary C08091 depending on host cells. For example, pGEX (Pharmacia) and pET (Novagen) are suitably used for E. col, and pcDNA3 (Invitrogen) is used suitably for animal cells. These vectors can be introduced into the host cells by heat-shock, for example. The recombinant polypeptide can easily be purified from the transformant by glutathione-Sepharose affinity chromatography when pGEX (Pharmacia) is used, and by nickel-agarose affinity chromatography when pET (Novagen) is used.

Those skilled in the art can readily raise antibodies that bind to the polypeptide of the invention using the polypeptide prepared as described above. The polyclonal antibodies of present invention can be prepared by a well known method. For example, the polypeptide is injected into a rabbit or the like and IG fraction is purified by ammonium sulfate precipitation. Monoclonal antibodies can be produced by preparing hybridoma from spleen cells of mice immunized with the polypeptide of the present invention and myeloma cells and culturing the hybridoma to secrete the monoclonal antibody in the culture medium, intraperitoneally injecting the antibody obtained into an animal to obtain a large quantity of the antibody.

The polypeptides, DNA, and antibodies of the present invention can be applied as follows. The 15 polypeptides and DNA of the present invention can be used for therapy and/or diagnosis of patients with low la(OH)-ase activity, such as patients with defects in la(OH)-ase or renal failure. The present inventors have identified the mutation of the DNA of the present invention in vitamin Ddependent type I rickets case, specifically, P382S (mutation from CCT to TCT), R335P (mutation from CGG to CCG), G125E (mutation from GGA to GAA), R107H (mutation from CGC to CAC). The 20 present invention is also applicable to treat these patients. The mutations in the patients can be identified by extracting DNA from peripheral leukocytes of a patient, amplifying the DNA by PCR using the primer in which each exon is set as intron, and determining the nucleotide sequence or the DNA by direct sequencing method. The DNA of the present invention can be used in gene therapy. In this case, the DNA of the invention is inserted into an appropriate vector, and the vector is introduced into 25 the body in vivo or ex vivo, using retrovirus method, liposome method, or adenovirus method. The polypeptides of present invention can be used as an immobilized enzyme to produce active vitamin D derivatives, that is, hydroxylate la position of vitamin D or its derivatives without a hydroxyl group at la position. Furthermore, the antibodies of the present invention can be used for therapy of such as vitamin D excessiveness, granulomatous diseases, and lymphoma as well as purification of the polypeptides of present invention.

The inventors also enabled screening genes encoding a polypeptide capable of converting an inactive form of various transcriptional regulatory factors into an active form using the abovedescribed screening system of ligands binding to nuclear receptors. Therefore, the present invention also relates to a method for screening a gene encoding a polypeptide that converts an inactive form of a transcriptional regulatory factor into an active form.

There are several reports on the mechanism of the conversion of a transcriptional regulatory factor into its active form. For example, NFKB, a tissue specific factor, is bound to a factor named IKB in the cytoplasm. When it is treated with TPA, IKB dissociates, and NFKB translocates into a nucleus.

Considering the effect of TPA treatment, the phosphorylation by protein kinase C is probably involved C08091 in the conversion of NFKB into an active form. In the case of HSTF, its phosphorylation level is low before the heat-shock, and is high after the heat-shock. This indicates that the phosphorylation is involved in the conversion of HSTF into its active form. Phosphorylation is also considered to be involved in the conversion of AP1 into its active form.

GAL4 is an inactive form when GAL80 binds thereto before the induction by galactose. After the induction by galactose, the complex dissociates and GAL4 becomes an active form. hsp90 binds to a glucocorticoid receptor before the hormone induction. After the induction, the complex dissociate to form an active form of glucocorticoid receptor (Jikken Igaku (Experimental Medicine) Vol. 7, No.4 (1989)).

The "polypeptides that convert an inactive form of a transcriptional regulatory factor into an active form" used herein includes polypeptides functioning in activation of transcriptional regulatory factors by dissociation of inhibitory factors, or by its qualitative alteration, such as phosphorylation.

The "inactive form of a transcriptional regulatory factor" include, for example, a complex of nonphosphorylated NFOB and IOB, non-phosphorylated HSTF, non-phosphorylated AP1, as described 15 above, but is not limited thereto.

In this screening method, a gene encoding an inactive form of a transcriptional regulatory S:factor, instead of a nuclear receptor gene, is introduced into a vector to construct the "expression unit 1" described above, and a vector into which the binding sequence of the transcriptional regulatory factor and a reporter gene downstream thereof is constructed as the "expression unit The 20 expression units are introduced into cells, and a test gene is introduced into the cells. If the test gene introduced has activity to convert an inactive form of the transcriptional regulatory factor into an active form, the inactive transcriptional regulatory factor, which is the product of the expression unit 1, is converted into the active form, and then active transcriptional regulatory factor binds to its binding sequence in the expression unit 2 to induce expression of the reporter gene. In contrast, when the .i 25 test gene introduced does not have activity to convert an inactive transcriptional regulatory factor into an active form, the reporter gene in the expression unit 2 will not be induced. Therefore, one can judge whether or not a test gene has activity to convert an inactive transcriptional regulatory factor into its active form using the present screening method by detecting the reporter activity.

When a gene library is used as a test gene, one can isolate a gene encoding a polypeptide with the activity to convert an inactive form of transcriptional regulatory factor into an active form from the library.

Brief Description of the Drawings Figure 1 schematically shows the expression cloning system mediated by VDR.

Figure 2 is a graph showing the serum concentration of la,25(OH) 2

D

3 in 3- and 7-week-old VDR+/- and VDR-/- mice.

Figure 3 is a micrograph of cells stained with X-gal. presents COS-1 cells transformed with a expression cDNA library; negative control; positive control; and stained cells with cDNA that was extracted from the positive cells in and amplified by PCR.

C08091 Figure 4 shows the putative amino acid sequence of CYP1AD. The first methionine is assigned as position 1. Asterisk indicates the terminal codon. Putative mitochondria targeting signal is surrounded by square. Underline indicates sterol binding domain. Dotted underline indicates hembinding domain.

Figure 5 shows homology of 'CYP1AD' to rat 25(OH)-ase (CYP27) and mouse 24(OH)-ase (CYP24). Amino acid sequence homologies in sterol binding domain and hem-binding domain are also indicated.

Figure 6 shows a photograph of 10% SDS-PAGE pattern of CYP1AD protein translated in vitro.

Figure 7 shows the result of CAT assay for detecting in vivo activity of CYP1AD. The bottom panel shows a representative CAT assay, and the top panel shows the relative CAT activity as average and SEM from three independent experiments.

Figure 8 shows the normal phase HPLC analysis of 25(OH)D3 metabolites.

Figure 9 shows the reverse phase HPLC analysis of 25(OH)D 3 metabolites.

Figure 10 shows the northern blot analysis for analyzing tissue distribution of CYP1AD 15 transcripts.

Figure 11 shows the northern blot analysis of 3- and 7-week-old, VDR+/- and VDR-/mice, with(+) or without(-) overdosage of 1a,25(OH)2D3 (50 ng/mouse).

Figure 12 shows the relative amount of the hydroxylase gene in 3- or 7-week-old, VDR+/+, VDR+/- and VDR-/- mice, with(+) or without(-) overdosage of 1a,25(OH) 2

D

3 (50 ng/mouse).

20 Best Mode for Implementing the Invention The present invention is demonstrated with reference to examples below, but is not to be construed being limited thereto.

Example 1 Isolation of cDNA encoding an enzyme that hydroxylates 10 position of vitamin D 25 The inventors developed an expression cloning system mediated by a nuclear receptor for cloning a full-length cDNA encoding la (OH)-ase. The system is based on the mechanism that 3 a precursor of 1a,25(OH) 2 D3, can activate the transactivating function of VDR only in the presence of la (OH)-ase (Figure In other words, the ligand-dependent transactivating function of VDR (AF-2) is induced by la,25(OH)2D3, but not by 25(OH)D 3 25(OH)D 3 is converted into 1a,25(OH) 2

D

3 only in cells expressing la (OH)-ase. Therefore, the cells can be detected by X-gal staining A. Frederick et al., Current Protocols in Molecular Biology (Wiley, New York, 1995)) as the result of the expression of the lacZ reporter gene in the presence of 25(OH)D 3 In the kidney of 7-week-old VDR-deficient mice mice), the serum concentration of 1a,25(OH) 2

D

3 was extremely high (Figure which suggested the high la (OH)-ase activity.

Therefore, the kidney of 7-week-old VDR-/- mice was used to prepare an expression library. poly(A) RNA was purified Takeyama et al., Biochem. Biophys. Res. Commun. 222, 395 (1996); H. Mano et al., J. Biol. Chem. 269, 1591 (1994)), and total cDNA was prepared from poly(A)* RNA Gubler and B. J. Hoffman, Gene 25, 263 (1983); M. Kobori and H. Nojima, Nucleic Acid Res. 21, 2782 (1993)). The total cDNA was inserted into the Hindlll position of pcDNA3 (Invitrogen), a expression C08091 vector that is derived from SV40, functions in mammals, and autonomously replicates in COS-1 cells.

The reporter plasmid, 17M2-G-lacZ, was constructed by inserting yeast GAL4 (UAS) x2 and P-globulin promoter into the multicloning site of Basic expression vector (Clontech). The function of AF-2 induced by a ligand was detected using VDR-ligand-binding domain fused with GAL4-DNA binding domain (VDR-DEF) [GAL4-VDR(DEF)] Ebihara et al., Mol. Cell. Biol. 16, 3393 (1996); T. Imai et al., Biochem. Biophys. Res. Commun. 233, 765 (1997)). Cos-1 cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum were transiently transformed with of GAL4-VDR (DEF) expression vector, 1pg of 17M2-G-lacZ, 0.2pg each of ADX expression vector and ADR expression vector Sakaki, S. Kominami, K. Hayashi, M. AkiyoshiShibata, Y.

Yabusaki, J. Biol. Chem. 271, 26209 (1996); F. J. Dilworth et al., J. Biol. Chem. 270, 16766 (1995)), and 0.1pg of the expression cDNA library, using Lipofectin (GIBCO BRL). 108M 25(OH)D3 was added to the culture medium 12 hours after the transformation. Cells were fixed with 0.05% glutaraldehyde 48 hours after the transformation and were then incubated with X-gal at 37 0 C for 4 hours to identify I-galactosidase positive cells expressing la(OH)-ase by X-gal staining (Figure 3(c)) A. Fredrick et al., Current Protocols in Molecular Biology (Wiley, New York, 1995)). In the negative control, the expression cDNA library was not used (Figure In the positive controls, the e expression library was not used, and la,25(OH) 2

D

3 was used instead of 25(OH)D 3 (Figure The stained cells were selectively collected by micromanipulation using a micropipette with 40pm diameter under an inverted microscope S. Tong et al., J. Bone Miner. Res. 9, 577 (1994)), 20 then transferred into PCR buffer solution. The PCR products were electrophoresed on 1% agarose gel, and fragments of about 2.0 to 2.5kb, which is the expected cDNA size of the full-length la (OH)ase, are purified and subcloned into pcDNA3. Sequence analysis of cDNA isolated from randomly selected 64 clones showed that 13 clones encode completely identical ORF. COS-1 cells into which the single cDNA clone was introduced were positive in X-gal staining (Figure 25 The full-length cDNA was obtained by the colony hybridization screening of the same library using the cDNA as a probe. The amino acid sequence deduced from ORF is a novel polypeptide with 507 amino acids (Figure 4).

The polypeptide, hereinafter called "CYP1AD," has a mitochondria-targeting signal and has significant homologies with P450 family members W. Nebert, DNA Cell. Biol. 10, 1 (1991)).

Especially, the homology with rat vitamin D 3 25-hydroxylase is 41.7% and that with mouse 25(OH)D 3 24-hydroxylase is 31.6% (Figure Masumoto, Y. Ohyama, K. Okuda, J. Biol. Chem. 263, 14256 (1988); E. Usui, M. Noshiro, Y. Ohyama, K. Okuda, FEBS Lett. 262, 367 (1990); Y. Ohyama and K.

Okuda, J. Biol. Chem. 266, 8690 (1991); S. Itoh et al., Biochem. Biophys. Acta. 1264, 26 (1995)). The homologies for sterol domain, especially conserved domain, in these enzymes are 93% and respectively, and those for hem binding domain are 70% and 80%, respectively.

The 10% SDS-PAGE analysis of CYP1AD protein, which was translated in vitro in the presence of [35S] methionine using Reticulocyte Lysate System (Promega) Sasaki et al., Biochemistry 34, 370 (1995)) revealed that the molecular weight of the polypeptide is approximately 55kDa (Figure 6), which is identical to the molecular weight of partially purified la (OH)-ase Wakino et al., C08091 Gerontology 42, 67 (1996); Eva Axen, FEBS Lett. 375, 277 (1995); M. Burgos-Trinidad, R. Ismail, R.

A. Ettinger, J. M. Prahl, H. F. DeLuca, J. Biol. Chem. 267, 3498 (1992); M. Warner et al., J. Biol.

Chem. 257,12995 (1982)).

Example 2 Detection of in vivo activity of CYPIAD To confirm that CYP1AD has ability to activate the transactivating function of VDR by converting 25(OH)D 3 into active vitamin D in vivo, COS-1 cells were co-transformed with 0.5pg of GAL4-VDR(DEF) expression vector, lpg of 17M2-G-CAT Kato et al., Science 270, 1491 (1995)), each of ADX expression vector and ADR expression vector Sakaki, S. Kominami, K.

Hayashi, M. Akiyoshi-Shibata, Y. Yabusaki, J. Biol. Chem. 271, 26209 (1996); F. J. Dilworth et al., J.

Biol. Chem. 270, 16766 (1995)), and lpg of CYP1AD expression vector, in the presence of 25(OH)D3 or la,25(OH) 2

D

3 A representative CAT assay is shown at the bottom panel of Figure 7. The relative CAT activities are shown at the top panel of Figure 7, as the average and SEM of three independent experiments. 25(OH)D 3 activated the CAT reporter gene when CYP1AD was expressed, while only 15 lo,25(OH) 2

D

3 activated the reporter gene without using CYP1AD expression vector. However, 3 did not significantly activate the reporter gene in the absence of ADX or ADR. These results strongly suggest that CYP1AD is la(OH)-ase, which converts 25(OH)D 3 into 1a,25(OH) 2

D

3 Example 3 Chemical analysis of CYP1AD products 20 To chemically determine the enzyme product of CYP1AD, normal phase HPLC and reversed phase HPLC were performed B. Mawer et al., J. Clin. Endocrinol. Metab. 79, 554 (1994); H. Fujii et al., EMBO in press (1997)). The cells (5x10 6 transformed with ADR expression vector, ADX expression vector and CYP1AD expression vector (Figure or the cells (5x10 6 not transformed S. (Figure were incubated in the presence of 3 H]25(OH)D 3 (105 dpm; 6.66 terabecquerel/mmol, 25 Amersham International) at 37°C for 6 hours. The culture media were extracted with chloroform, and the extract was analyzed by normal phase HPLC using TSK-gel silica 150 column (4.6x250mm, Tosoh), with hexane/isopropanol/methanol (88:6:6) for mobile phase, at the flow rate of 1.OmL/min.

The eluate was collected and its radioactivity was measured using a liquid scintillation counter B.

Mawer et al., J. Clin. Endocrinol. Metab. 79, 554 (1994); H. Fujii et al., EMBO J. in press, (1997)).

The standard samples of vitamin D derivatives, namely, la,(OH)D 3 25(OH)D 3 24,25(OH) 2

D

3 2

D

3 and la,24,25(OH) 3

D

3 were applied to chromatography to determine their retention time by UV absorbance at 264nm (Figure Likewise, reverse phase HPLC was performed with a column filled with Cosmasil 5C18-AR (4.6x150 mm Nacalai Tesque) at flow rate of 1.OmL/min to confirm the existence of 3 H]1a,25(OH) 2

D

3 The chromatograms of standard samples for vitamin D derivatives, and the reaction product in the presence or absence of CYP1AD, are shown in Figure and respectively.

The retention times of enzyme products in normal phase HPLC and reverse phase HPLC were completely identical to that of sample, la,25(OH)2D3 standard. The results indicate that the cDNA of CYP1AD encodes mouse la (OH)-ase, which hydroxylates 25(OH)D 3 to 1a,25(OH)2D3.

C08091 Example 4 Analysis of tissue distribution of CYPIAD transcripts The tissue distribution of CYP1AD transcripts in 7-week-old normal and VDR-/- mice was examined. Poly(A)' RNA was extracted from brain, lung, heart, liver, spleen, kidney, small intestine, skeletal muscle, skin, and bone, and analyzed by northern blot technique using cDNA of CYP1AD and P-actin as probes Takeyama et al., Biochem. Biophys. Res. Commun. 222, 395 (1996); H. Mano et al., J. Biol. Chem. 269, 1591 (1994)). As the result, the transcript of CYP1AD was detected as a single band in the kidney. The size of the transcript (2.4 kbp) is identical to that of cloned cDNA (Figure 10). Except for kidney, in, la(OH)-ase activity has been reported in other tissues than kidney W. Norman, J. Roth, L. Orchi, Endocr. Rev. 3, 331 (1982); H. F. DeLuca, Adv. Exp. Med. Biol. 196, 361 (1986); M. R. Walters, Endocr. Rev 13, 719 (1992); G. A. Howard, R. T. Turner, D. J. Sherrard, D.

J. Baylink, J. Biol. Chem. 256, 7738 (1981); T. K. Gray, G. E. Lester, R. S. Lorenc, Science 204, 1311 (1979)). However, the transcript of 10(OH)-ase was not detected in tissues other than kidney in this experiment.

15 The northern blot analysis of the expression of the CYP1AD gene and the 24(OH)-ase (CYP24) gene was performed in 3- and 7-week-old and VDR-/- mice, with or without administration of excess la,25(OH) 2

D

3 (50ng/mouse). A representative northern blot analysis is shown in Figure 11. The relative amount of the hydroxylase gene standardized with the P-actin gene transcripts was measured in at least 5 mice for each group (Figure 12). Interestingly, the marked 20 induction of the gene was seen in VDR-/- mice (2.5 and 50 times in 3- and 7-week-old mice, respectively)(Figure 11, 12). In VDR+/+ mice and VDR+/- mice, the administration of la,25(OH)2D3 significantly inhibited expression of the la (OH)-ase gene, whereas the inhibition did not occurred in 3- and 7-week-old VDR-/- mice. Therefore, the overexpression of la (OH)-ase appears to cause raise in the serum level of la,25(OH)2D 3 in 7-week-old VDR-/- mice compared with the normal level i 25 (Figure Considering these results, it can be considered that ligand-bound VDR is involved in the negative regulation of the la (OH)-ase gene expression by 1a,25(OH) 2

D

3 In VDR-/- mice, the expression of the 24(OH)-ase gene was decreased to the undetectable level, and the reaction against 2

D

3 was not seen (Figure 11, 12). The 24(OH)-ase gene converts 25(OH)D 3 to 24,25(OH) 2

D

3 which is an inactive form of vitamin D, and its gene expression is positively regulated by la,25(OH) 2

D

3 These results confirmed that the ligand-bound VDR is involved in the gene expression induced by la,25(OH) 2

D

3 through vitamin D responsive element in the promoter of the 24(OH)-ase gene Zierold, H. M. Darwish, H. F. DeLuca, J. Biol. Chem. 270, 1675 (1995); Y.

Ohyama et al., J. Biol. Chem. 269, 10545 (1994)). Therefore, the ligand-bound VDR adversely regulates the expression of la(OH)-ase and 24(OH)-ase genes by 1a,25(OH)2D3.

Example Isolation of human gene encoding an enzyme that hydroxylates the la position of vitamin D A normal human kidney cDNA library was prepared by extracting poly(A) RNA from normal human kidney tissue using the Sacll(500bp)-Eco-RI(1200bp) fragment of mouse la (OH)-ase as a probe and inserting the RNA into A-ZAPII. A human gene encoding the enzyme that hydroxylates la C08091 position of vitamin D was obtained by screening the library prepared above by plaque hybridization method. The nucleotide sequence of the isolated gene is shown in SEQ ID NO: 4, and the putative amino acid sequence is shown in SEQ ID NO: 2.

Industrial Applicability The present invention provides a method for screening genes encoding polypeptides capable of converting a ligand precursor into a ligand, and a method for determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand. The method of the present invention, unlike the existing expression cloning method, advantageously utilizes the nature of nuclear receptors that regulate transcription by being bound by a ligand. Since a desired gene can be detected by the reporter activity, the method of the invention enables simply and efficiently detecting and isolating a gene even if it encodes a polypeptide that is expressed at a low level. The present invention also provides a polypeptide that converts a ligand precursor into a ligand, namely, a polypeptide that converts an inactive form of vitamin D3 into its active form and a gene encoding it, which are obtained by the screening method as described above. The polypeptide and gene of the 15 present invention can be used for treating and/or preventing defects in la(OH)-ase or renal failure.

The polypeptide of the present invention can also be used to produce active vitamin D derivatives, namely, hydroxylate la position of vitamin D or its derivatives without a hydroxy group at la position.

The antibodies against the polypeptide of the present invention can be used to purify the polypeptide of the present invention, and to treat vitamin D excessiveness, granulomatous diseases, lymphoma, 20 and the like.

In addition, the present invention provides a method for screening ligands that bind to nuclear receptors, and a method for determining whether or not a test compound is a ligand of the nuclear receptor. The method also takes advantage of the nature of nuclear receptors and uses the reporter activity for the detection. These methods are thus simple and efficient as well as the method 25 described above. For example, the method is useful in searching ligands for orphan receptors, for which ligands are unknown.

Furthermore, the present invention provides a method for screening genes encoding polypeptides capable of converting an inactive form of transcriptional regulatory factor into an active form, based on the screening method described above. This method enables easily isolating genes that encode polypeptides capable of converting an inactive form of various transcriptional regulatory factors into the active form by detecting the reporter activity.

C08091 EDITORIAL NOTE APPLICATION NUMBER 29262/02 The following Sequence Listing pages 1 to 7 are part of the description. The claims pages follow on pages "17" to "19".

1/7 Sequence Listing <110> CHUGAI SEIYAKU KABUSHIKI KAISHA.

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Giu Leu Leu Leu Ala 315 Thr Leu Tyr Glu Leu 330 Ser Giu Ilie Thr Ala 345 Thr Ala Leu Ser Gin 365 Leu krg Leu Tyr Pro 380 Asp Ile krg Val Gly 395 Leu Cys His Tyr Ala 410 Asn Ser Phe Asn Pro 425 Pro Phe Ala Ser Leu 445 Arg Arg Leu Ala Glu 460 Thr His Phe Glu Val 475 Met Thr Arg Thr Val 490 Val Asp Arg 505 <210> 2 <21 1> 508 <212> RPT <213> Homo sapiens 5 <400> 2 Met Thr Gin Thr Leu 1 5 Tri Ala Pro Glu Leu 20 Ala Arg Arg Ser Leu Leu Ala Glu Leu Phe GIn Val GIn Gly Ala Gly Thr Val Arg Thr Leu Leu Arg Gin Glu 100 Tmp Thr Glu His Arg 115 Ala Glu Gly Glu Glu 130 Leu Leu Arg Pro Gin 145 Val Val Cys Asp Leu 165 Thr Gly Pro Pro Ala 180 Gly Ser Gly 350 Leu Val Asn Thr Ala 430 Pro Leu Leu Leu Lys Gly Ala Cys Ala 70 Val Gly Arg Trp Ala 150 Val Leu Val Arg 335 Thr Pro Val Tyr Ser 415 Arg Phe Glu Pro Val 495 Tyr Ala Asp Lys 55 His Tyr Pro Cys GIn 135 Ala Arg Val Asp 320 His Arg Leu Pro Val 400 Arg Tmp Gly Leu Glu 480 Pro 2/7 Thr Val Pro Asp Gly Ser Leu Lys 370 Gly Asn 385 Ilie Pro Asp Pro Leu Gly Phe Gly 450 GIn Met 465 Pro Gly Giu Arg Ser Val Cys 355 Ala Ser GIn Thr Glu 435 Lys Ala Ala Ser Val Tyr Pro Ser Val 75 Pro Arg Ala Ser Ala 155 Arg Ala Asn GIn 340 Ala Val Arg Asp Gin 420 Gly Arg Leu Leu I le 500 Thr Leu Ser 325 Thr Ala Leu His Pro His Ilie Lys Glu 375 Val Pro Asp 390 Thr Leu Val 405 Phe Pro Asp Pro Thr Pro Ser Cys Ilie 455 Ser Gin Ilie 470 Pro Ilie Lys 485 Asn Leu Gin Tmi His Gly 360 Val Arg Ser Pro His 440 Gly Leu Pro Phe Ala Ser Ser Leu 25 Ilie Pro 40 Gly Gly Phe Gly Val Ala Arg Pro 105 Arg GIn 120 Arg Leu Ala Arg Arg Leu Arg Asp 185 Arg 10 Gly Gly Leu Pro Ala 90 Glu Arg Arg Tyr Arg 170 Val Phe His Arg Arg Giu Tyr Ser Thr Pro Arg Leu His Tmp Leu Ala Ala Leu Val Cys Ser Phe 110 Cys Gly Leu 125 Leu Leu Ala 140 Gly Thr Leu Gin Arg Gly Gly Glu Phe 190 Val Arg His Ser Ser Phe Glu Leu Ser Phe Glu Giu Ser Pro Leu Thr Pro Leu Asn Asn 160 Arg Gly 175 Tyr Lys C08092 Phe Gly Leu Glu Gly lie Ala Ala Val Leu Leu Gly Ser A A A. Cys Leu 210 Val Gly 225 Trp Leu Trp Asp Ala Glu Ser Gly 290 Gin Ser 305 Thr Val Pro Glu Pro Gly Leu Leu 370 Pro Gly 385 lie lie Arg Asp Trp Leu Gly Phe 450 Leu Gin 465 Glu Pro Pro Glu 195 Glu Ala Ser Val Arg His Gin Met 260 Ala Ala 275 Ala His lie Leu Ser Asn Val Gin 340 Ser Ser 355 Lys Ala Asn Ser Pro Lys Pro Ala 420 Gly Glu 435 Gly Lys Met Ala Gly Ala Arg Ser 500 Gin Val Phe Val 230 Leu Val 245 Phe Ala Met Arg Leu Thr Gly Asn 310 Thr Leu 325 Thr Ala Ala Tyr Val Val Arg Val 390 Asn Thr 405 Gin Phe Gly Pro Arg Ser Leu Ala 470 Ala Pro 485 lie Asn Pro Pro 215 Ser Thr Pro Gly Phe Ala Asn Gly 280 His Phe 295 Val Thr Ser Trp Leu His Pro Ser 360 Lys Glu 375 Pro Asp Leu Val Pro Glu Thr Pro 440 Cys Met 455 Gin lie Va! Arg Leu Gin Asp Leu Pro Gin 265 Gly Leu Glu Ala Ser 345 Ala Val Lys Thr Pro 425 His Gly Leu Pro Phe 505 Thr Leu Trp 250 Arg Gin Phe Leu Leu 330 Glu Thr Leu Asp Leu 410 Asn Pro Arg Thr Lys 490 Leu Glu Thr Phe 220 Thr Met Ala 235 Gly Arg Leu His Val Glu Pro Glu Lys 285 Arg Glu Glu 300 Leu Leu Ala 315 Tyr Glu Leu lie Thr Ala Val Leu Ser 365 Arg Leu Tyr 380 lie His Val 395 Cys His Tyr Ser Phe Arg Phe Ala Ser 445 Arg Leu Ala 460 His Phe Glu 475 Thr Arg Thr Asp Arg 200 205 Arg Leu Gly lie Arg Ala Met Pro His 240 Cys Arg Asp 255 Arg Arg Glu 270 Asp Leu Glu Leu Pro Ala Gly Val Asp 320 Ser Arg His 335 Ala Leu Ser 350 Gin Leu Pro Pro Val Val Gly Asp Tyr 400 Ala Thr Ser 415 Pro Ala Arg 430 Leu Pro Phe Glu Leu Glu Val Gin Pro 480 Val Leu Val 495 <210> 3 <211> 2386 <212> DNA <213> Mus musculus <221> CDS <222> (30)...(1550) <400> 3 ctctcgaagc agactcccca aacacagac atg acc cag gca gtc aag ctc gcc 53 Met Thr Gin Ala Val Lys Leu Ala 1 tcc aga gtt ttt cac cga atc cac ctg cct ctg cag ctg gat gcc tcg 101 Ser Arg Val Phe His Arg lie His Leu Pro Leu Gin Leu Asp Ala Ser 15 ctg ggc tcc aga ggc agt gag tcg gtt ctc cgg agc ttg tct gac atc 149 Leu Gly Ser Arg Gly Ser Glu Ser Val Leu Arg Ser Leu Ser Asp lie C08092 30 35 cot ggg ccc tot aca ctc agc ttc ctg got gaa ctc ttc tgc aaa ggg 197 Pro Giy Pro Ser Thr Leu Ser Phe Leu Ala Giu Leu Phe Cys Lys Gly 50 ggg ctg too agg ctg cat gaa ctg cag gtg cat ggc got gog cgg tao 245 Gly Leu Ser Arg Leu His Giu Leu Gin Val His Gly Ala Ala Arg Tyr 65 ggg oca ata tgg tot ggo ago ttt ggg aoa ott cgo aca gtt tao gtt 293 Gly Pro lie Trp Ser Gly Ser Phe Giy Thr Leu Arg Thr Val Tyr Val 80 gc gao cot aca ctt gtg gag cag otc ctg oga caa gaa agt cac tgt 341 Ala Asp Pro Thr Leu Vai Glu GIn Leu Leu Arg Gin Glu Ser His Cys 95 100 oca gag cgc tgt agt ttc toa toa tgg goa gag cac ogt cgc cgc cac 389 Pro Giu Arg Cys Ser Phe Ser Ser Trp Ala Glu His Arg Arg Arg His 105 110 115 120 cag ogt gct tgo gga ttg ota acg gog gat ggt gaa gaa tgg cag agg 437 Gin Arg Ala Cys Gly Leu Leu Thr Ala Asp Gly Giu Giu Trp GIn Arg 125 130 135 tc cga agt ott ctg goc cog otc otc otc ogg oca caa gca gc gog 485 Leu Arg Ser Leu Leu Ala Pro Leu Leu Leu Arg Pro GIn Ala Ala Ala 140 145 150 ggc tat got gga act otg gao aac gtg gto cgt gac ctt gtg oga oga 533 Gly Tyr Ala Gly Thr Leu Asp Asn Val Val Arg Asp Leu Val Arg Arg 155 160 165 ota agg cgc cag cgg gga cgt ggo tot ggg ota ccc ggc cta gtt ctg 581 Leu Arg Arg GIn Arg Gly Arg Gly Ser Gly Leu Pro Gly Leu Val Leu 170 175 180 gao gtg gca gga gag ttt tao aaa ttt ggc cta gaa agt ata ggc gog 629 Asp Val Ala Gly Glu Phe Tyr Lys Phe Gly Leu Giu Ser lie Gly Ala 185 190 195 200 gtg otg otg gga tog cgc ctg ggc tgc cta gag got gaa gto cot cot 677 Val Leu Leu Gly Ser Arg Leu Gly Cys Leu Glu Ala Glu Val Pro Pro 205 210 215 gao aca gaa aco ftc ata oat gca gtg ggc tca gtg t gtg tot aca 725 Asp Thr Giu Thr Phe lie His Ala Val Gly Ser Vai Phe Val Ser Thr 220 225 230 ctc *g acc atg gog atg ccc aac tgg ttg cac cac ott ata cot gga 773 Leu Leu Thr Met Ala Met Pro Asn Trp Leu His His Leu lie Pro Gly 235 240 245 ccc tgg goc cgc otc tgc oga gao tgg gat cag atg t goc Ut goc 821 Pro Trp Ala Arg Leu Cys Arg Asp Trp Asp Gin Met Phe Ala Phe Ala 250 255 260 cag agg cac gtg gag ctg oga gaa ggt gaa got gog atg agg aac cag 869 Gin Arg His Val Glu Leu Arg Glu Gly Giu Ala Ala Met Arg Asn Gin 265 270 275 280 gga aag cot gag gag gat atg cog tot ggg cat cac tta aco cac ttc 917 Gly Lys Pro Gu Glu Asp Met Pro Ser Gly His His Leu Thr His Phe 285 290 295 ott Ut cgg gaa aag gtg tot gto cag too ata gtg ggg aat gtg aca 965 Leu Phe Arg Glu Lys Val Ser Val Gin Ser lie Val Gly Asn Val Thr 300 305 310 gag ota ota ctg got gga gtg gao acg gta too aat acg otc too tgg 1013 Glu Leu Leu Leu Ala Gly Val Asp Thr Val Ser Asn Thr Leu Ser Tri 315 320 325 aca otc tat gag ott too cgg cac ccc gat gtc cag act gca otc cac 1061 Thr Leu Tyr Glu Leu Ser Arg His Pro Asp Val Gin Thr Ala Leu His C08092 330 tct gag atc aca gct Set Glu Ilie Thr Ala 345 act gct ctg tcc cag Thr Ala Leu Ser Gin 365 tig aga ttg tac cct Leu Arg Leu Tyr Pro 380 gac ato cgt gta. gga Asp Iie Arg Vai Giy 395 cta tgt cac tat gcc Leu Cys His Tyr Ala 410 aac tct Uft aat cca Asn Set Phe Asn Pro 425 cca Ut gca tct cUt Pro Phe Aia Ser Leu 445 aga cgc ttg gca gag Arg Arg Leu Aia Giu 460 acc cat Utt gaa gtg Thr His Phe Glu Vai 475 atg acc: cgg act gtc Met Thr Arg Thr Vai 490 gta gat aga Vai Asp Arg 505 aggatggggt ctftgftata caaactccag gaagcaggtc gaaccaccat ctitctcct gccttcagat tttaacacat cagcctgggg agggattcgc ttatcacggc acaagctaag ccatgtgtgt gccttctgag tttgagac agagtcttgc cctcaccttt cccaagtat tttatatctc ctgccagagt gaacctggac catgtggcag ttaatctftc ctctagggaa ccgctacctt ggttctcag ggcctgccct tctcca <210> 4 <21 1> 2362 <212> DNA <213> Homo sapiens <221 ODS <222> (1524) <400> 4 335 ggg acc cgt Giy Thr Arg 350 otg ccc ctg Leu Pro Leu gtg gta Oct Val Val Pro aac tat gta Asn Tyr Val 400 act tca agg Thr Set Arg 415 gct cgc tgg Aia Arg Trp 430 ccc tic ggc Pro Phe Giy ctt gag cta Leu Giu Leu cta cot gag Leu Pro Giu 480 ctg gtc cct Leu Val Pro 495 taaccattcg cacaagaggc ctg acctatg cctgctcagt ccftaaagtg ccctgatcct tgattgcatc aagagtaatg tatgtattcc gggttacaga ctatcccftg gatcgtccac gtaaatctgc ccactctcaa 340 ggc tcc tgt gcc cac Giy Set Cys Aia His 355 tta aag gct gtg atc Leu Lys Ala Val Ilie 370 ggg aat tcc cgt gtc Gly Asn Set Arg Val 385 aft ccc caa gat acg Ilie Pro Gin Asp Thr 405 gao ccc aca cag Uft Asp Pro Thr Gin Phe 420 ctg ggg gag ggt ccg Leu Gly Giu Gly Pro 435 Uft ggc aaa cgg ago Phe Gly Lys Arg Set 450 caa atg got ttg too Gin Met Ala Leu Ser 465 cca ggt got ctt cot Pro Gly Ala Leu Pro gag agg agc atc aat Giu Arg Set Ilie Asn 500 gaagacagcc aacatogtct cco cat ggc Pro His Gly 360 aaa gaa gtg Lys Giu Val 375 cca gao aga Pro Asp Arg 390 cta gtc tcc Leu Val Set oca gao ccc Pro Asp Pro aco coo oac Thr Pro His 440 tgc atc: ggg Cys Ilie Gly 455 cag ato: Ug Gin Ile Leu 470 ato aag ccc Ilie Lys Pro 485 ota cag tt Leu Gin Phe ctotoaagao tctgaccgag aggcatcgoa aggatccaat caactccagg gtctaagcat ctctacctga gctcfttftC aacttccttg tgtatcagto ttctcagact Utttctotc ttaagcct tccatgttta 1109 1157 1205 1253 1301 1349 1397 1445 1493 1541 1590 1650 1710 1770 1830 1890 1950 2010 2070 2130 2190 2250 2310 2370 2386 acactctcct tgtactiggc gcctctcctg ccaacgcagg gtagtgttcg tggtctgcac actagtctac atgctgtcct cttgagotac gttatttcag tcaccaggct cottgoctaa gtggatccac tggaggcctg ctgactcagc atcatcctc ggftaactao ftgatgctct ctggctgcat tgggctta ggaaattcac cacttccagc caccatacat ctgcccacco Uftacagcgt tttatca C08092 atg acc cag acc otc Met Thr Gin Thr Leu 1 5 tgg gcg ccc gag ttg Trp Ala Pro Glu Leu gca cgc cgg agc ttg Ala Arg Arg Ser Leu ctg gcc gaa ctt ttc Leu Ala Glu Leu. Phe cag gtg cag ggc gcc Gin Val Gin Gly Ala ggg aca gtg cgc acc Gly Thr Val Arg Thr ctg ctg cga cag gag Leu Leu Arg Gin Glu 100 tgg acg gag cac cgc Trp Thr Giu His Arg 115 gcg gaa ggc gaa gaa Ala Giu Gly Giu Glu 130 ctc otc cgg cot caa Leu Leu Arg Pro Gin 145 gta gto tgo gac ott Val Val Cys Asp Leu 165 acg ggg cog ccc gcc Thr Gly Pro Pro Ala 180 ftc gga otg gaa ggc Phe Gly Leu Glu Gly 195 tgc ctg gag got caa Cys Leu Giu Ala Gin 210 gtg ggo tog gtg t Val Gly Ser Val Phe 225 tgg ctg ogc cac ctt Tr Leu Arg His Leu 245 tgg gac cag atg ttt Trp Asp Gin Met Phe 260 gca gag gca gcc atg Ala Glu Ala Ala Met 275 tct ggg gcg cac ctg Ser Gly Ala His Leu 290 tgo aag Cys Lys 55 999 Gly ggg ctg tcg Gly Leu Ser gcg cac tic ggg ocg gtg Ala His Phe Giy Pro Vai 70 75 gtg tao gtg got goc cct Val Tyr Val Ala Ala Pro 90 gga ccc ogg ccc gag cgc Gly Pro Arg Pro Giu Arg 105 cgo tgc cgo cag cgg got Arg Cys Arg Gin Arg Ala 6/7 aag tao gc too aga gtg Lys Tyr Ala Ser Arg Val 10 ggo gco too cta ggc tao Gly Ala Ser Leu Giy Tyr 25 gca gao ato cca ggo ccc Ala Asp lie Pro Gly Pro 40 tgg caa Trp Gin 135 otc cgo agt Leu Arg Ser gcg gc gc cgc tao gco Ala Ala Ala Arg Tyr Ala 150 155 gtg cgg ogt ctg agg cgc Val Arg Arg Leu Arg Arg 170 ctg gt cgg gao gtg gcg Leu Val Arg Asp Val Ala 185 ato gc gog gil ctg otc lie Ala Ala Val Leu Leu 200 ftc cat ogc "gt cgc 48 Phe His Arg Val Arg oga gag tao cac tca 96 Arg Glu Tyr His Ser tct aog ccc ago Ut 144 Ser Thr Pro Ser Phe agg cia cac gag ctg 192 Arg Leu His Glu Leu tgg ota gc ago Ut 240 Trp Leu Ala Ser Phe gca otc gto gag gag 288 Ala Leu Val Giu Glu tgc ago tic tog ccc 336 Cys Ser Phe Ser Pro 110 tgo gga ctg otc act 384 Cys Gly Leu Leu Thr 125 otc ctg goc cog otc 432 Leu Leu Ala Pro Leu 140 gga aco ctg aao aac 480 Gly Thr Leu Asn Asn 160 cag cgg gga cgt ggc 528 Gin Arg Gly Arg Gly 175 ggg gaa Ut tao aag 576 Gly Giu Phe Tyr Lys 190 ggc tcg cgc ftg ggo 624 Gly Ser Arg Leu Gly 205 aco ttc ato cgc got 672 Thr Phe lie Arg Ala 220 atg gog atg ccc cac 720 Met Ala Met Pro His 240 cgc otc tgc cga gao 768 Arg Leu Cys Arg Asp 255 gtg gag cgg oga gag 816 Val Glu Arg Arg Glu 270 gag aag gao ctg gag 864 Glu Lys Asp Leu Glu 285 gaa gag ftg oct gc 912 Glu Glu Leu Pro Ala 300 gtg oca Vai Pro 215 ccc Pro gac acg gag Asp Thr Glu gtg too aog cig ftg aco Val Ser Thr Leu Leu Thr 230 235 gtg cot ggg ccc tgg ggc Val Pro Gly Pro Trp Gly 250 gca ttt got cag agg cao Ala Phe Ala Gin Arg His 265 agg aac gga gga cag ccc Arg Asn Gly Gly Gin Pro 280 aco cac ftc Thr His Phe 295 ctg tic ogg Leu Phe Arg C08092 oag too Gin Ser 305 aog gtg Thr Val 71 atc otg gga aat gtg aoa gag ttg cta ttg gcg gga "gtg gac 960 Ilie Leu Gly Asn Val Thr Glu Leu Leu Leu Ala Gly Val Asp 310 315 320 toc aac aog ctc tot tgg got otg tat gag oto too ogg oao 1008 Ser Asn Thr Leu Ser Trp Ala Leu Tyr Giu Leu Ser Arg His 325 000 gaa gto oag aoa Pro Giu Val Gin Thr 340 goa oto oao Ala Leu His toa gag Ser Giu 345 ato aoa got Ilie Thr Ala goo Ala 350 oag Gin 335 otg ago Leu Ser otg 000 Leu Pro oct ggo too Pro Gly Ser ctg otg Leu Leu 370 oct gga Pro Gly 385 agt goo tao 000 toa Ser Ala Tyr Pro Ser 360 gog gtg gto aag gaa Ala Val Val Lys Giu 375 tot ogt gto ooa gao Ser Arg Val Pro Asp 390 aaa aat aog ctg gto Lys Asn Thr Leu Val gco aot gil otg too Ala Thr Val Leu Ser 365 gtg ota aga otg tao Val Leu Arg Leu Tyr 380 aaa gao aft oat gtg Lys Asp Ilie His Val 395 act otg tgt oao tat Thr Leu Cys His Tyr aat Asn oot gtg gta Pro Val Val ggt gao tat Gly Asp Tyr 400 goc act toa Ala Thr Ser 415 ooa got ogo Pro Ala Arg att ato 000 Ilie Ilie Pro 405 agg gao oct goo oag Arg Asp Pro Ala Gin 420 t ooa gag Phe Pro Glu 410 ooa aat Pro Asn 425 tot Ut cgt Ser Phe Arg tgg otg Trp Leu ggo t Giy Phe 450 ttg oaa Leu Gin 465 gag ooa Glu Pro ggg gag ggt 000 aoo 000 Gly Giu Gly Pro Thr Pro 435 440 ggo aag ogo ago tgt atg Gly Lys Arg Ser Cys Met 455 atg got ttg goo oag ato Met Ala Leu Ala Gin Ilie 470 ggt gcg goo ooa gtt aga Gly Ala Ala Pro Va! Arg oao ooa Utt goa tot His Pro Phe Ala Ser 445 ggg aga ogo otg goa Gly Arg Arg Leu Ala 460 ota aoa oat Ut gag Leu Thr His Phe Glu 475 000 aag aoo ogg act Pro Lys Thr Arg Thr 000 tt Pro Phe cot gaa Pro Giu gaaagagact aagacoaagg agtgaagtgt gocaggtgag cccaaggatg tgootott goataagcto aagototftg tototgaota gataagoago aaggtgaato actattaggt cctgcccagg aaaccaaata 485 agg ago ato Arg Ser Ilie 500 gtoatcatoa tataoatot gaggoggoto aaaaccatgg aaatoagat tttgggott agtagctgtg agaggaaggg ggtgtcaooa ttacttagta tgcoctagoo gggtttgooo octctggct toatt aao ota cag Asn Leu Gin ooottoat ooootaatgo tgaooaatgt tototctgot ttaaotaata oatagtgt oatotggtot tgaagtctta taoaoattt ggctotgtot tggtftacgg catoaottag Uttatattga aaaaaaaaaa 490 Utt Utg Phe Leu 505 catoataggg ctatotgacc gtgaagtatg tgcttggooo atgotggatg attgatgotg gnaootggt fttgftt tagattgaat aocctoo tttcttataa ttcaggca aaatttttaa aaaaaaaaaa gao aga Asp Arg ataagattt aaaotggata oaottggoot ttgatoat goctgaagga ctggctrrgo ggtcctgt atgtcccotg otgaaocatg ttctttgtct ototcctttg gagaoatct atattcacaa aaaaaaaa gag oil gaa Giu Leu Glu gtg oag oot Val Gin Pro 480 gto otg gta Val Leu Val 495 tagtoocatg ttgtaggoao gaaooaooat gaotcaggaa gtatgcatco aagaftcaao atttgtoaaa otttgcatgt ooagggcotg tggcagaagg tgcocctagg otototggoo tgggootgto attttagaat 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1534 1594 1654 1714 1774 1834 1894 1954 2014 2074 2134 2194 2254 2314 2362 C08092

Claims (18)

1. 2. A method for determining whether or not a test gene encodes a polypeptide that converts a ligand precursor into a ligand, the method comprising introducing a test gene into a cell comprising a vector carrying a gene encoding a nuclear receptor and a vector carrying the binding sequence of the nuclear 1s receptor and a reporter gene located downstream of said binding sequence, contacting a ligand precursor to the cell into which the test gene is introduced, and detecting the reporter activity.
2. 3. A method for screening a gene encoding a polypeptide that converts an inactive 20 form of vitamin D3 into an active form, the method comprising 0 0 introducing a test gene into a cell comprising a vector carrying a gene encoding a nuclear vitamin D receptor and a vector carrying the binding sequence of the vitamin D receptor and a reporter gene located downstream of said binding sequence, contacting an inactive form of vitamin D3 to the cell into which the test 25 gene is introduced, detecting the reporter activity, and isolating the test gene from the cell that shows the reporter activity.
3. 4. A method for determining whether or not a test gene encodes a polypeptide that "converts an inactive form of vitamin D 3 into an active form, the method comprising introducing a test gene into a cell comprising a vector carrying a gene 0* 00[ encoding a nuclear vitamin D receptor and a vector carrying the binding sequence of the :%goo vitamin D receptor and a reporter gene located downstream of said binding sequence, contacting an inactive form of vitamin D3 with the cell into which the test gene is introduced, and (R'.tBP] I 7&9 lspec.doe:GCC COMS ID No: SBMI-00850129 Received by IP Australia: Time 16:12 Date 2004-07-30 JUL. 2004 16:11 SPRUSON AND FERGUSON 61292615486 NO. 5559-P. 18 detecting the reporter activity. An isolated gene encoding a polypeptide that converts a ligand precursor into a ligand comprising the sequence ofSEQ ID NO:3 or 4.
4. 6. An isolated gene encoding a polypeptide that converts an inactive form of vitamin D 3 into an active formnn by hydroxylating position la comprising the sequence of SEQ ID NO:3 or 4.
5. 7. An isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 1 or its derivative comprising said sequence in which one or more amino acids are substituted, deleted, or added, and having activity to convert an inactive form of vitamin D 3 into an active form.
6. 8. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or its derivative comprising said sequence in which one or more amino acids are substituted, deleted, or added, and having activity to convert an inactive form of vitamin D 3 into an active form.
7. 9. An isolated polypeptide encoded by a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 3, wherein the polypeptide has activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position Ia. An isolated polypeptide encoded by a DNA that hybridizes with the nucleotide sequence of SEQ ID NO: 4, wherein the polypeptide has activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position I a. S"
8. 11. An isolated DNA encoding the polypeptide of any one of claims 7 to
9. 12. An isolated DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 3 and encoding a polypeptide having activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position 1 ta. 25 13. An isolated DNA hybridizing with a DNA having the nucleotide sequence of SEQ ID NO: 4 and encoding a polypeptide having activity to convert an inactive form of vitamin D 3 into an active form by hydroxylating position I a,
10. 14. A vector comprising the DNA of any one of claims 11 to 13.
11. 15. A transformant expressively retaining the DNA of any one of claims 11 to 13.
12. 16. A method for producing the polypeptide of any one of claims 7 to 10, the method comprising culturing the transformant of claim
13. 17. An isolated antibody that specifically binds to the polypeptide of any one of S a 0 claims 7 to
14. 18. A method for screening a gene encoding a polypeptide that converts an inactive form of transcriptional regulatory factor into an active form, the method comprising (RA:IBPFI 17891 spec.doc.GCC COMS ID No: SBMI-00850129 Received by IP Australia: Time 16:12 Date 2004-07-30 JUL. 2004 16:11 SPRUSON AND FERGUSON 61292615486 NO. 5559 P. 6- 19 introducing a test gene into cells into which a vector comprising a gene encoding an inactive form of transcriptional regulatory factor and a vector comprising the binding sequence of said inactive transcriptional regulatory factor and a reporter gene located downstream thereof are introduced, detecting the reporter activity, and isolating the test gene from the cells showing the reporter activity.
15. 19. The method of claim 18, wherein the inactive transcriptional regulatory factor is a complex of non-phosphorylated NFiB and IKB, non-phosphorylated HSTF, or non-phosphorylated API.
16. 20. A method for screening a gene encoding a polypeptide that converts a ligand precursor into a ligand substantially as hereiubefore described with reference to any one of the examples.
17. 21. A method for screening a gene encoding a polypeptide that converts an inactive form of vitamin D 3 into an active form substantially as hereinbefore described with is reference to any one of the examples.
18. 22. A method for screening a gene encoding a polypeptide that converts an inactive form of transcriptional regulatory factor into an active form substantially as hereinbefore described with reference to any one of the examples. Dated 30 July 2004 Chugal Seiyaku Kabushiki Kaisha *0 *0 0 Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S91spdoCC (R:\UBFFJ 17891 spcc.doc.CC COMS ID No: SBMI-00850129 Received by IP Australia: Time 16:12 Date 2004-07-30