CZ20004017A3 - The human BMP-7 promoter and method for detecting bone-affecting substances using this promoter - Google Patents

Abstract

The present invention provides a method for detecting low molecular weight compounds that positively or negatively regulate the expression of human BMP-7 by using the activity of a reporter gene and a 5' upstream region of a gene containing a BMP-7 promoter, and further using animal cells into which a recombinant expression vector linked to a suitable reporter gene has been introduced. The low molecular weight compounds and their derivatives obtained by the method of the present invention have morphogenetic activity and inhibitory activity for bone and cartilage, which is caused by the expression of human BMP-7, and these compounds are useful as prophylactic or therapeutic agents for bone or cartilage diseases. Furthermore, the low molecular weight compounds and their derivatives are useful as therapeutic agents for kidney diseases.

Description

Technical area

The present invention relates to a DNA region of the 5'-preceding DNA of human bone morphogenetic protein (BMP, hereinafter referred to as 7), which contains a promoter. Furthermore, the present invention relates to a method for screening for low molecular weight compounds that positively or negatively regulate the expression of human BMP-7 using animal or yeast cells into which a recombinant expression vector containing the 5'-preceding DNA region containing the human BMP-4 promoter integrated into a suitable reporter gene is inserted, and using the reporter activity as an indicator.

State of the art

Currently, bone morphogenetic activity has been described for bone morphogenetic factor, BMP, which belongs to the TGF (transforming growth factor) β superfamily (Science 150: 893-897, 1965; Science 242: 1528-1534, 1988). Known BMPs are BMP-1 to BMP-14. BMP-2 to BMP-14 are known to have bone morphogenetic activity. BMP-2 to BMP-14 are considered effective for the therapeutic and preventive treatment of various bone dysfunctions and bone diseases, however, they occur in very small amounts under natural circumstances. Therefore, large amounts of BMP-2 to BMP-14 used for this treatment require the production of recombinant protein. The production of recombinant protein is usually very expensive compared to the production of low molecular weight compounds. On the other hand, there are many limitations in their use as drugs, which are determined by their physical properties and the route of administration, which are given by their protein nature. For these reasons, small molecule organic compounds having the same activity as the said BMP proteins are very promising drugs. The substance obtained by using the method of the present invention is active in inducing the expression of human BMP-7, a bone morphogenetic protein, and also has the same activity as human BMP-7, the use of which is very useful. On the contrary, in the context of bone and cartilage hyperplasia, inhibition of the expression of human BMP-7 can prevent osteohyperplasia. The present invention allows the detection of inhibition of the expression of human BMP-7 and provides a method for the investigation of substances that prevent such hyperplasia. In addition, it is known that human BMP-7 has the ability to enhance the differentiation of kidney cells (Proc. Natl. Acad. Sci. USA, vol. 93, pp. 9021-9026, 1996). Therefore, the experimental system of the present invention can be used to investigate agents suitable for the treatment of kidney diseases.

For such a research method, only an example using the mouse BMP-2 promoter has been described (WO 97/15308); there is no example using the human BMP-7 promoter. In addition, since the materials for the research method of the present invention are all obtained from human sources, the substances obtained in such research should have clinical utility.

The essence of the invention

The present invention provides a 5'-preceding DNA region containing the human BMP-7 promoter. By using the 5'-preceding region of a gene containing the human BMP-7 promoter and animal cells into which a recombinant expression vector has been inserted that has been integrated with a suitable reporter gene, low molecular weight compounds that positively or negatively regulate • ·

expression of human BMP-7, using reporter activity.

The low molecular weight compounds and their derivatives have morphogenetic activity and inhibitory activity for bone and cartilage caused by the expression of human BMP-7 and are useful as preventive or therapeutic agents for bone or cartilage diseases, as drugs for bone metastases, or as therapeutic or prophylactic agents for excessive osteogenesis. Furthermore, these low molecular weight compounds and their derivatives are useful as preventive or therapeutic agents for kidney diseases.

Description of images in the attached drawings

Fig. 1- is the exon-intron structure of the 10.8 kb region 5' upstream of the human BMP-7 gene and a restriction enzyme site map. The exon region is indicated in black and the empty square indicates the intron region.

Fig. 2 is a recombinant expression vector (pMSS115) containing the 5'-preceding region of the human BMP-7 gene. The promoter region (4.4 kb) (bases 3813 to 8222 of SEQ ID NO: 1 in the Sequence Listing, from the XbaI site to the 3rd XbaI site from the 5'-end, as shown in Fig. 1) was inserted into the NheI restriction site of the pGL3basic vector.

Fig. 3 shows the results of measuring the activity of the human BMP-7 promoter (transiently expressed).

The present invention relates to a DNA whose nucleotide sequence is represented by bases 1 to 10877 of SEQ ID NO: 1 in

A list of sequences encoding the promoter region of human bone morphogenetic protein 7, or a fragment thereof.

SEQ ID NO: 1 of the Sequence Listing shows the sequence of the 5' upstream region of the human BMP-7 gene.

The present invention relates to a method for preparing DNA according to SEQ ID NO: 1, which comprises the following steps:

(1) digestion of human placental genomic DNA with the restriction enzyme HindIII;

(2) isolation by agarose gel electrophoresis;

(3) cloning of the isolated DNA fragment digested with HindIII into the lambda phage vector ΔDASH II treated with the same enzyme;

(4) packaging said vector into phage;

(5) creation of a genomic DNA library by phage infection of Escherichia coli;

(6) PCR investigation; and (7) subcloning into a plasmid vector.

The plasmid vector used is not specifically specified and one of the commercially available vectors may be used. A preferred example is the pUC18 vector.

The present invention relates to a recombinant expression vector characterized by the integration of all or part of the DNA set forth in SEQ ID NO: 1 into a reporter gene. More specifically, the recombinant expression vector is prepared by placing an appropriate region from the 5 ^' upstream region of the human BMP-7 gene as set forth in SEQ ID NO: 1 in front of the reporter gene. The reporter gene, such as the luciferase or β-galactosidase gene, indicates the expression status of its original product. The original vector for the recombinant expression vector is not specifically specified, and a commercial plasmid vector may be used. A preferred vector for use in the present invention is pGL3-basic. Using pGL3-basic yields pMSS115 (9.2 kb), which is a recombinant expression vector containing the human BMP-7 promoter and

9» ·· • « • · * « · · • 9 · ♦ » · • · 9 9 luciferase reporter gene. The present invention refers to this vector as a recombinant expression vector. It is necessary to introduce the recombinant expression vector into mammalian cells, preferably human osteoblast cells, such as SaOS-2 cells, with liposomes. Animal cells stably transfected with the recombinant expression vectors are selected using resistance markers.

The present invention relates to a method for researching substances affecting, in which a recombinant expression vector characterized by the integration of the complete or partial DNA shown in SEQ ID NO: 1 of the Sequence Listing into a reporter gene is used. It further relates to a method for researching substances affecting bone, in which said substance affecting bone is an osteogenesis-inducing substance or an osteogenesis-inhibiting substance. Low molecular weight compounds that induce or inhibit the expression of human BMP-4 can be obtained by isolating a promoter that regulates gene expression, linking it to a suitable reporter gene and introducing the gene structure into suitable mammalian cells, thereby obtaining a test system. Substances that regulate the expression of human BMP-7 in a test system act on the promoter and increase or decrease the expression of the reporter gene. Thus, a simple and easy measurement of the activity of the reporter gene allows the investigation of said substances.

Animal cells transfected with the vector can be used to screen a library of chemical compounds by high-throughput screening (Nature, vol. 384, Suppl., pp. 14-16, 1996) and to screen active substances from natural substances. A substance increasing or decreasing activity is selected by treating the cells with this substance for a certain period of time and then measuring the activity of a reporter gene. The substance thus obtained can regulate expression either by acting directly on the transcription factor or indirectly on the human BMP-7 promoter by regulating the signal transduction system. Therefore, these compounds are effective as therapeutic agents for bone and cartilage diseases, tumor metastasis to the bone or osteohyperplasia.

Furthermore, these compounds are useful as therapeutic agents for kidney diseases.

The substance obtained by the method of the present invention has morphogenetic activity on cartilage or bone and is effective as a therapeutic or prophylactic agent in the field of orthopedic surgery (in diseases such as fractures, osteoarthritis such as articular osteoarthritis and osteoarthritis of the hip joint, arthrosteitis, cartilage damage such as meniscus damage, bone regeneration and cartilage deficiency caused by injury or tumor dissection, bone reconstruction such as spinal fusion and enlargement of the spinal canal, and congenital bone and cartilage diseases such as dysosteogenesis and achondroplasia), or in the field of dentistry (in bone reconstruction such as cleft palate, mandibular reconstruction and residual arch construction) and in the field of osteoporosis treatment. Furthermore, the substances of the present invention can be used in bone grafts in plastic surgery. These substances are also effective in therapy in the field of veterinary medicine. On the other hand, the present invention can provide substances that inhibit bone or cartilage morphogenesis. In this case, such substances are used as agents for the prevention or therapy of bone or cartilage hyperplasia.

Ί • · · · ··· «** · · · · · ·

Furthermore, the present invention can provide substances with the ability to enhance the differentiation of kidney cells, and these substances can be used for the treatment and prevention of kidney diseases.

Examples of embodiments of the invention

The present invention will now be further illustrated by the following examples. These examples are in all respects illustrative only and are not intended to limit the scope of the present invention in any way.

Example 1: Isolation of the 5'-preceding region of the human BMP-7 gene

Human placental genomic DNA (CloneTech) was digested with various restriction enzymes (BamHI, BglII, EcoRI, HindIII, PstI, SacI, SalI, Smal, SPhl and XbaI), separated by agarose gel electrophoresis, transferred to a nylon membrane and processed by Southern hybridization under standard conditions using BMP-7 cDNA (EMBO J., 9: 2085-2093, 1990) as a probe. It was found that digestion with the restriction enzyme EcoRI together with other restriction enzymes resulted in the acquisition of a DNA fragment of approximately 11 kb in size, which contained the longest human BMP-7 gene. Then, the human placental genomic DNA was digested with the restriction enzyme HindIII and separated by agarose gel electrophoresis to extract a DNA fragment of approximately 11 kb in size from the agarose gel. The obtained DNA fragment was cloned into the lambda phage vector ÁDASH II (Stratagene Ltd.) digested with the restriction enzyme HindIII.

The vector was processed in vitro with Gigapack III XL extract (Stratagene Ltd.), was used for infection of Escherichia coli

XLl-Blue MRA (Stratagene Ltd.), for the creation of a genomic library

DNA. The library was divided into several pools. Each pool was amplified by screening (Nucleic Acids Research 21: 2627• ·

99

2631, 1993) using PCR; more specifically, PCR using PCR primers (SEQ ID NO: 2 and SEQ ID NO: 3 of the Sequence Listing) corresponding to the translation region for selecting the desired set, to obtain the 5'-preceding region of the human BMP-7 gene (10.8 kb). Then, the 5'-preceding 10.8 kb fragment was subcloned into the pUC18 vector (Amersham Pharmacia Biotech.). The vector was designated E. coli pKOT 314. E. coli pKOT 314 was deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry 1-3, Higashi 1chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan, 30.3.1998, under accession number FERM P-16737 and was transferred to the International Depository Authority under the Budapest Treaty 17.2.1-998 (accession number FERM BP-6651).

Example 2: Determination of the DNA sequence of the 5'-preceding region of the human BMP-7 gene

The sequence of the obtained 5'-preceding region of the human BMP7 gene was determined using an ALF DNA sequencer (Amersham Pharmacia Biotech), according to the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977). The sequence thus analyzed is given in SEQ ID NO: 1 of the Sequence Listing. Bases 5557 to 10780 of SEQ ID NO: 1 have been previously described (EMBO J., 9: 2085-2093, 1990). However, there are many differences from the sequence of the present invention.

Example 3: Construction of a recombinant expression vector containing the human BMP-7 promoter and a luciferase reporter gene

As shown in Fig. 1, the human BMP-7 promoter occurs upstream of exon 1. The 4.4 kb region containing the promoter located from the second XbaI to the third XbaI from the 5'-end, as described in Fig. 1, was inserted into the NheI restriction site of the luciferase reporter vector pGL3-basic (Pro Mega Ltd.) to be assembled upstream of the reporter gene, resulting in the recombinant expression vector pMSS115 (9.2 kb). This vector is shown in Fig. 2.

Example 4: Measurement of human BMP-7 promoter activity (insertion of recombinant expression vector into human cells and transient expression)

For transient expression of human BMP-4 recombinant expression vector, the recombinant expression vector (pMSS1-15) was mixed with the pRL-SV40 vector (Pro Mega Co.) containing the firefly luciferase gene, which was used as an internal control for measuring the efficiency of gene insertion, at the same amount used. Then, the cationic liposome Lipofectamine (Lifetech Oriental Co.) was mixed with the DNA solution, and this mixture was added to human osteosarcoma cells HOS, MG63, and SaOS-2 for transfection. The firefly luciferase activity and the firefly luciferase activity were measured by Pikka Gene Dual Kit (Toyo Ink. Co.). The results are shown in Fig. 3. The promoter activity was expressed as the ratio of the firefly luciferase activity to the firefly luciferase activity. From the results, it is clear that the DNA SEQ ID NO: 1 of the Sequence Listing has promoter activity

Example 5: Insertion of a recombinant expression vector into human cells and stable expression

For stable expression of human BMP-7 recombinant expression vector, the above vectors were mixed in a ratio of

10:1 with the pPUR vector (CloneTech Ltd.) containing the puromycin resistance gene and further mixed with cationic liposome Lipofectamine (Lifetech Oriental Co.) for transfection of human osteosarcoma HOS cells. Cells into which the selected gene was inserted were selected on culture medium containing puromycin (Sigma, Ltd.).

Example 6: Searching for active compounds with low molecular weight

Selected cells were seeded in a 96-well plate, treated with various chemical compounds from the libraries for 1-3 days, dissolved in cytolytic reagent (Pro Mega Ltd.) - and enzymatic activity was measured using a luciferase assay kit (Pro Mega Ltd.). In this way, various substances inducing or inhibiting the expression of human BMP-7 could be tested.

Sequence Listing <110> Hoechst Marion Roussel, Ltd.

<120> Human BMP-7 promoter and method for researching bone-affecting substances using this promoter <130> JH98K005 PCT sequence in English <14 0>

<141>

<150> 10-120174 <151> 1998-04-30 <160> 3 <170> Patentln Ver. 2.1 <210> 1 <211> 10877 <212> DNA <213> Human <220>

<221> various characteristics <222> (1)....(10877) <223> 5'-preceding gene sequence of human BMP-7 including exon 1 regions <400><l aagcttggac atcagatagc tgtgtcattc atgctaactc ttattagttg ttgcatcata 60 tgtaagatga gctcaaatct gttaatcttc ccatacgatc tttcacatg agttgggact 120 aacctgttat atttagagta atggtttacc aatggggagg gaataaagat aaaactcgaa 180 gtaaaagaaa gaatacaatt ttttaaatga ggagaaacac agtgaaggag aggagagaag 240 gagaaaaca acagagatgg agagaagcac gacgtggggt ggtgaggagt tcccaggact 300 ctgggagcga ggagcccagt ctaatccctg attcactgta agactttggg cccagcagt 360 tctctttggg tgtcaatttc tctatcttca agatgaagga attgaaccac gcttctgcag 420

• *· fl · *· ♦ ·« ·« 17 · · · · * · ··· ·· «·· ·· »» ·♦ • · ♦ · » · · · • · · · . • · · · • « · · · gacttttgtt gtaactgact ggaaactccg tacacaatgg cttaaactga acagtcctgg 480 gtggggctgg cttcagggat agtgggctcc aggggctcac tgatgctgtc agtctcctgc 540 cttttcttgt tttactgtct tgtggcttct ctcacagggc aggctctccc tgaagggagg 600 gaagatgaca cccagcagca gcccctgacc tagcaacccc agtgggagga gagaccccttt 660 taccggtagt tctagcaaaa gttctaaaag ttgaatctca ctggcctgga ttaggtcaca 720 tgactgtcac tgaacccatc gcatgggcat ctctggttga ccaggctggg tcatgtgccc 780 agtctgaatt ttagagaaga ggatagttca acctgaaata caggactga tggtgaagaa 840 ggggcatttc tactgaggaa aacagaggaa atcttggcca ggcacagtgg cttatgcctg 900 taatcccagc actttgagag gccaagatgg atggattgtt tgaggcccgg agttcaagac 960 cagtctggcc agcgtggtgt aaccctgtct tctactaaaa attack tagccaggca 1020 tggtggtgca cacctgtaat tccagctact cggggggct gaggaggaggag aattgcttga 1080 aaccgggagg cagaggttac agtgagctga gattgtgcca ttgcactcca gcctgggtga 1140 cagagggaga ctctgtctaa aaaaaaaaag aaaagaaaag aaaacagagg aaatatttgc 1200 agaagaagga aaaaggcag gggtctcaaa ttaggtgagc ttccaaagtg ttttctgtgg 1260 aaccctggga ccagagaaaa tggtagtgaa aagaaacagg cctggggact ctgggccccct 1320 gctgggatca cctgataaca ggcagggtgt ccagtccgat ttgaacttca gataaacaac 1380 gagtaaattt cagtatgagt atataataata catatactcc aatataaaagg gatatagtca 1440 tactaaaatt cattcattgt ttatctgaaa ttcaaattga actggacacc tattttttttt 1500 tttgcaaaat gtggtaaccc tagtccctcc cctgctctat gtaaccaaag aagctctgtt 1560 tctgttttct gtgtcgagca tgtgtgaaaa actccttgac caaaagttttt agcgctgcac 1620 aaaagagaag gcctgtatcc ttttaactga catgattgcc atgccctctc cagctcccca 1680 gtgctgtatc cagcagggct tctggctgca ggcaacagaa accagcttgg gcaggctggt 1740 gcggaaaagg agaccactag aggatcttgg gggctcacag aacccctggg aaggccagag 1800 aaccaggctt cggggctctc tagttgggac acggccccag atcactctgc agaatagtct 1860 gggcctgcac tgatagcttc tggggtgggt gcctctgact ggctgaaact tggtcacctg 1920 tcagggctct ggctgcaaag caggctagac agtgcctctc tggatattca ccagatgggg 1980 catcctcaac cataagagag ggctcagatc tagctgggag gagggagatg ctgaaaagca 2040 ggagcaatga acactcactc ctgctcttgc agatggaatt ctggttttttg atgtttctcg 2100 ggagaggggggg agtttatcag atcagtcacc agggccaaac agcagagtttt tgctgggaag 2160' ggactctggg gaaggttagg gtcagattttt gcctaaaggg gcagacagtt ccagccttttt 2220 caaccactgg ctcgtagcc ctcgaatcat gaattcaatg gagctgtctt gaaatgcacc 2280 atccttggtg gtcctttggt ttgggtgatg ggttggggga ggctgggcac afccctccaga 2340 cacagcagag ccctttatga ggccaggggc aaaccagatt catcgaatct gactggtaga 2400 tgtgtcttct ttggtctaat aaccaaaacg agttgaatta gttgccaaca gataaacatt 2460 gagagatgtc acattcaaac tcaagtttct agcttctttg gaaaatcaga actttctggt 2520 gatgttggaa tcaaatttct ggatggcatc acccatgggc atgtggtttc cagttcaccc 2580 cagtctgcac cctccttatt tatccaggcc agtgtctcct gccacctgtc accatgtctg 2640 tgctcatgat tgccttccat taaattaaga ggaagagaaa tattccttga gcttcatgtt 2700

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gcatctctgt atttatgtta cctggttggt gcccgttggc ttttgagtgt atgacttctg 2760 caataaagga ggaattgaag aagggctcag aggtatgttc ctcaacact gatagggcat 2320 ctactgtgtg caaggcctcc atgggtactg ggaggtccct acaggactaa gacaaaccct 2880 gtgttttggt gtacctcaat gtttaaggac ttaagtcctg gaacaagata gccggcgtca 2940 gaattctgat tttaccattc actcaccatt ggtgtggctt tgggcaggtt ccctcaagta 3000 tttgagcctc aatttcctca tctgtaaaat gggggataata ataataaaaa tcatacaagg 3060 tagttgggag aattaatgag ttaattttaa aagacacctg gaatcataga tattgaagtt 3120 ttaatttgaa gccccaattt gtcagctata acttcagaaa tctagagcct cgatcgtcaa 3180 catacagcct gaagacgaca gcctccgagc cccccaggag ctagttagaa gtgcagaatc 3240 tcagctcact cagactgact gtgggaatct atgttctaac agacccccag gggattcaca 3300 cgcacagttg ggttggagaa gtgttgctttt aggttaaagc aaggaaggaa ggggctgctc 3360 ccaggtcact gaacgtgcac tgatgcttaa aggcctggc tgtgtagaca gatgtggctt 3420 tggatcctgg ctccattaca gattagattg gctgtctgac tctggacaag gtgctctcgg 3480 gatgttggtc ctcctctgca atctgaggag cagaactagc ctctccatgc agtggggagt 3540 tgcgggtgat gtgggcttgg gccgtgcagg cctagcaggt gcccagcaga agcaagtgct 3600 cccaacaggt gacaggtcgg ccgctccccct gtcacgtttt ggaaggagga aagggctacc 3660 tctagtgctg aaccgaggat agttagtgct caaaaccata ccagatttcc tccaatcaag 3720 gaagaataa cagccctgat aataataaac aaaaccactg cttccccctta gctggcttcc 3780 agatcctaga aatcctgcat ggtatcagct tctctagagt gtgtgtgtcg tctgtattttt 3840 ccatttggaa ctgtggccac tgccatgtgt acttaagact gatggagaac gtttaatctc 3900 ttattctgct gcttttaaag ttgtgcaaag aaaatctcag agtgggcaag cgtgaggagt 3960 ccaagcctcc cgtgcaatga gagatctgcg tgggaaacaa aatttcacca caggtgtgct 4020 ct-ataaattt cctctgcagg gtccacctct gcacacaacg attttcaagc ctgtgcagga 4080 ggcagctctt ggccctctat tccttgttgg ggttggaggt ccaggtcatc tggcctttgc 4140 caggtggtgt ggacagagag agagcagccc actggcttcc ttggctgtgc tccccaggga 4200 gcttccaggc cagctgagcc tcctctaggg cagatggatg aagatgaggt ccccacctcg 4260 ctgaacaatg tgtgtgtcag cagaattctt ttcttcttttt tactgcctta gagagacatg 4320 ctgacttggt caaaatcact tcagcaagat gtgactgatg atgtaggaaa tgaaaactcc 4380 tggcccagca ctgggaggcc agcaggcgag gggcgcggac actgggaccg ggccacccga 444θ cacaataaca ccccttacaa ttccaggccg tctttcatcc aggcaaaggc ggccccaggt 4500 gtggagcaga ctggggcaga tcagattacg gtcctggagc ccagtgctgg gtgccctggc 4560 caggggaacc acaactgggg gtgtgtgggt tggggctag ctgctggggga aatgagatag 4620 gacccaggga agtccctggg ccccagctgg cctgcagggg gcctaggcac aatgtgagga 4680 ttggaatgca gtgatgacct tttcatctct catgttccct tccacttcac tctctttttt 4740 gctctggtac ttttgctctt gctctcagag tcaggataca tctgcttttc tctgcttggc 4800 aaaaagcaga ccatcatgaa aagttttgtt gtaatttgaa actcaggagg ctttcctgtt 4860 accat ccttttcatc cagatggtaa cacaataaca tttatcaagg gttttctctg 4920 tgttccaggc actgtgcaaa gccctttcca tgaattaagt tctgcaacct cacagcgatc 4980

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9 9

ccagaagaca ggcgctatca gtccccccat ··· tttacagatg ·· ♦·» » ggggaactga ggtgtagácja 5040 ggttaagtcc cttgcccatg gtgcacagct ggagagaca gagctggagt gtgaatgcgg 5100 ctgggcaggc tccagtgccc aggctacctc ctccacacaa gacttgccct cggcaatctc 5160 aaagccttttt ctggtggtgg gctcagctcc caaactggca tcggatgcac tcccagccag 5220 atttcttg cttgccggtt ttcattcatt cattcattca ttcattcatt cattcattcg 5280 gcattcactg agggcctggt gcagtcttgg ggatgcctct cgggggaa acagggaaaa 5340 gaaagacccc cccaccaagc atggatcaca gaaaagataa ggctaaatgg gggtttgtgg 5400 gacttcagag gaaaccttat ctcttgaggt cttggatatg aagagcatgt tgtctcttcc 5460 tcttgcatga gaaaagatgg cgtctcagag gaagggttgc tggggtgagg gatctgggag 5520 atgccttagc ttggcgcctg cacagtcagc cctcagtcaa ccggtctcttt taggtttttgg 5580 ctgtgcttat tactattcat tcaacaggta ctaattgagc acctgctgtg tgccaggctc 5640 agaataggct caggtgagat gcacaaagaa gggtaaacta gaatccttgc ttagacactg 5700 acggatcagt tgtttcatat gtaaattgta gcaccaagac ctgctgcccc tgcccccagc 5760 ctcacctgct tgtgaagatc cctccaaaag atttgagagt agataaaaaag cagagactac 5820 tactgaagaa cagggctgct ttggctcctt attatttcag actttggaag aaaatgacct 5880 cctttttctc tactggcact ggaggtggca tagctgtccc tagcaagcca gcgctggagg 5940 gcgtgtgcag ggctggggac cgagcctggt ttctgttccc tgctctgcag gctcaagcac 6000 ttgctgttcc tccacctggg atgcctttcc ctggaaaagc ctgtctctttt cttgtctttc 6060 aggactcagg tcagtggcat ctcctccaaa aactcccctt cccaccctcc atcacctcac 6120 cctgtttatc tgcgcccccg cccccactgc ctgtcactta ttgcaggctg aagtgaccca 6180 ggctctccag ttgtacactc tcagatggac cctggacgac tgtggcactc ctgcaatttc 6240 cccagtctcc ctggggtagg attcctgctt gccaggatgc ccacctttcc ttctccctcc 6300 tgcatgtcct cctctgcctg getting started tgttcccaga gagagtgata gacaagatct 6360 gcctctcctt cagtccctga atcttattta aggctcttgc tttgcttccc tggcctggag 6420 gcggctcctt gatggagtct gccatgtggg ttcgctcatg gccatgtctt cctgcccagc 6480 letggtgcttg gccctgggac tggccacata atatctgggc caggtgcaaa attagtacgg 6540 ggcagggggt actttgttca taggtgattc agaaccacat atggtgacct cagatagga 6600 aaccaagtgt ggggcccttta agagctgggg ggccctgtac gactgtccag gttgcaggcc 6660 ccacagctcg cctcctgata tcctgtgctc catgcttgtc tgttgaagga aggagtgaat 6720 ggatgaagag caggtggtgg gggtggtttg agggccttgc tggtggtgg gtagaggccc 6780 ctccctggca tggggctcaa gacctgttcc atcccacagc ctggagcctg tgtgtaaatg 6840 gccaggacct gcaggctggc attttctgc tccttgcctg cctctggcct cccctttctc 6900 cacccatgtg gcccctcagg ttgccatcta gtccaaaagt ccccaaggga gacccagagg 6960 gccacttggc caaactactt ctgctccaga aaactgtaga agaccataat tctcttcccc 7020 agctctcctg ctccaggaag gacagcccca aagtgaggct tagccagagc ccctcccaga 7080 caagcgcccc cgcttcccca acctcagccc ttcccagttc atcccaaagg ccctctgggg 7140 accactctc tcacccagcc ccaggagggggg aaggagacag gatgaacttt tacèccgctg 7200 ccctcactgc cactctgggt gcagtaattc ccttgagatc ccacaccggc agagggaccg 7260

i5 · » ♦ *· »·» » ♦ »9 · 9 9 9 *· gtgggttctg agtggtctgg ggactccctg tgacagcg.tg catggctcgg tattgattga 7320 gggatgaatg gaggagaga gacaggagag gaggccgatg gggaggtctc aggcacagac 7380 ccttggaggg gaagaggatg tgaagaccag cggctggctc cccaggcact gccacgagga 7440 gggctgatgg gaagccctag tggtggggct gggctgtctg gtctcaggct gaggggtggc 7500 tggaagata caggccccgg aagaggagga ggtgggaaga acccccccag ctcacacgca 7560 gttcacttat tcactcaaca aatcgtgact gcgcacgtac agtggctacc aggcgctggg 7620 ttcaaggcac tgcgggtacc agaggtgcgg agaagatcgc tgatccgggc cccagtgctc 7680 tgggtgtcta gcgggggtaa gaaggcaata aagaaggcac ggagtaactc aaacagcaat 7740 tccagacagc aagagaaact acaggaaga aaacaaacgt gcgaggggcg aggcgaggaa 7800 acaacctcag cttggcaggt cttggaggtc tctgggagga gaaagcagcg tctgatgggg 7860 gcgggaggtg gtgagtgggg agaggtccag gcggagggaa tggcgagcgc agagacaggc 7920 tggcaacggc ttcagggagg cgcggagggg tcagcgtggc tggcttaaaa ggatacatgg 7980 gactgagggg caagaccggc tcaagggtca ccgcttccag gaagccttct atttccgcgc 8040 caacctcggc gctcccccaa cttttcccac cgcggtccgc agcccacccg tcctgctcgg 8100 gccgccttcc tggtccggac cgcgagtgcc gagagggcag ggccggctcc aattcctcca 8160 gccgcatccc cgcgacgtcc cgccaggctc taggcacccc gtgggcactc agtaaacatt 8220 tgtcgagcgc tctagaggga atgaatgaac ccactgggca cagctggggg gagggcgggg 8280 ccgagggcag gtgggaggcc gccggcgcgg gaggggcccc tcgaagcccg tcctcctcct 8340 cctcctcct cgcccaggcc ccagcgcgta ccactctggc gctcccgagg cggcctcttg 8400 tgcgatccag ggcgcacaag gctgggagag cgccccgggg ccèctgctat ccgcgccgga 8460 gttggagaag ggtgggttgc cgccgcccga gggcgagagc gccagaggag cgggaagaag 8520 gagcgctcgc ccgcccgcct gcctcctcgc tgcctccccg gcgttggctc tctggactcc 8580 taggcttgct ggctgctcct cccacccgca cccgcctcct cactcgcctt ttcgttcgcc 8640 ggggctgctt tccaagccct gcggtgcgcc cgggcgagtg cggggcgagg ggcccggggc 8700 cagcaccgag cagggggcgg gggtccgggc agagcgcggc cggccggggga ggggccatgt 8760 ctggcgcggg cgcagcgggg cccgtctgca gcaagtgacc gacggccggg acggccgcct 8820 gccccctctg ccacctgggg cggtgcgggc ccggagcccg gagcccgggt agcgcgtaga 8880 gccggcgcga tgcacgtgcg ctcactgcga gctgcggcgc cgcacagctt cgtggcgctc 8940 tgggcacccc tgttcctgct gcgctccgcc ctggccgact tcagcctgga caacgaggtg 9000' cactcgagct tcatccaccg gcgcctccgc agccaggagc ggcgggagat gcagcgcgag 9060 atcctctcca tttttgggctt gccccaccgc ccgcgcccgc acctccaggg caagcacaac 9120 tcggcaccca tgttcatgct ggacctgtac aacgccatgg cggtggagga gggcggcgggg 9180 cccggcggcc agggcttctc ctacccctac aaggccgtct tcagtaccca gggcccccct 9240 ctggccagcc tgcaagatag ccatttcctc accgacgccg acatggtcat gagcttcgtc 9300 aacctcggtg agtaagggca ggcgagggta cgcgtctcct ttcggggggca ctttgagact 9360 gggagggagg gagccgcttc ttctatgcag cccgcccagc tttccgctcc tggctgaaat 9420 cgcagtgcct gcccgagggt ctcccaccca cagccctatg actcccaagc tgtgtgcgcc 9480 cccaggtcgg gcgcgctggg ttcggtgagc ctgtaggggt tactgggaag gagggatcct 9540

• 16 • 99 9 ♦ 9· 999 9 9 9 9 9 · 9 9 9 9 99 999 • ** • · · * « • · · · * · · · · • » · » ♦ ·· ·· »9 • 9 • 9 » 9 ccgaagtccc ctccatgtta cgccgccggc cgcatctctg gggctggagg caagggccgt 9600 tcaaagcgcg gggctcggtc atgtgagctg tcccgggccg gcgcggctcg cgtaacctgg 9660 atgtaaaggg cccttccccgg cgaggctgcc ttgccgccct tcctgggccc ctctcagccc 9720 tgcctggccc tggcatcgcg gccgtcgcac ccccttacccc tccctgtcaa gccctacctg 9780 tcccctcgtg gtgcgcccgc cttagcgtac cgcgcgctcc gagcgcttgg ggcccctctc 9840 cgggccgccg gatgcccccat tctctttgg ctggagctgg ggaaagcg gtgccattgc 9900 taatt tgttttcttt ctttgtttat t tttttttttttttt ttttctttttc 9960 ttttctttttc ttttttttttt ttttttgaga cggagttcac tcttgtcgcc cagtctggag 10020 tgcaatggcg cgatctctgc tcaccgcaac ctctgcctcc cgggttcaag cgattctcgt 10080 gcctcagcct cccgagtagc tgggattaca gcatgcgcca ccatgcctgg ctaattttgt 10140 atttttagta gagacagggt ttctccatgt taggcaggct ggtctcgaac tcccgatctc 10200 aggtgatcct cccgcctcag cctcccaaag tggtgctggg attacaggcg tgagccactg 10260 tgccctgccg ctagtcttct attttaagta tttagtggta ggtcccgggc cggcagaatc 10320 tattttcagc atttaccacg tgtggcgcgc aaaccacagg ttttggcgat tgggttgcgc 10380 gggatctcag actgacacgc gggggcggct gggggtcccg gtttccgact ggagccgcga 104 40 cgaccccggc gacgcgagcc tggggctgca gcgagggccg gggagctccc cctccatatg 10500 tgcgcgcaca ttctccagac ttgctcaaac taacccccgg cggcgccagc gcgctgcggg 10560 actgatgatc aaatatttgg tttccgagat aacacacccc gatagcgctg tttcctgagc 10620 cgctttcatt ctacttgtgt aacttgctgc gaaaacccga accaagtcaa gacagcaaac 10680 tcaccccacg ggcgctgtgt caacatggaa atatatgatac tgaagcccca cgctgggcac 10740 ctggggcgtg gactggggc gcgggggaag cgcagatccg ccttcatgct tcccccctcc 10800 tgataaggtc cctggagttc ccgggaggcc attgtctgta cttaataata actaaatcca 10860 actagtgaac caagctt 10877

<210> 2 <211> 30 <212> DNA <213> Human <220>

<221> various characteristics <222> (1)....(30) <223> Primer (in the direction of the coding sequence) for cloning the 5'-preceding human BMP-7 gene sequence corresponding to the exon 1 region <400> 2 gggcgcagcg gggcccgtct gcagcaagtg <210> 3 <211> 30 <212> DNA <213> Human <220>

<221> various characteristics <222> complement((1) .... (30) ) <223> Reverse primer for cloning 5' upstream human BMP-7 gene sequence corresponding to exon 1 region <400> 3 agaggatctc gcgctgcatc tcccgccgct *· *· · ·· ♦· · » · • ··· · · 4 4 4 • 4 4 4 4 4 4 4 4 4 4 9

4 4 4 4 4 4 4 4

444 44 494 444 44

Claims (7)

Patent claims (1) digestion of human placental genomic DNA with the restriction enzyme HindIII; DNA having a nucleotide sequence consisting of bases No. 1 to 10877 of SEQ ID NO: 1 of the Sequence Listing, which encodes the human bone morphogenetic protein 7 promoter region, or a fragment thereof. (2) agarose gel electrophoresis; 2. A method of preparing the DNA of SEQ ID NO: 1 of the Sequence Listing comprising the steps of: 3. A recombinant expression vector comprising integrated, in the reporter gene, all or part of the DNA shown in SEQ ID NO: 1 of the Sequence Listing. (3) cloning the isolated HindIII digested DNA fragment into the λDASH II lambda phage vector treated with the same enzyme; 4. A method for researching bone-modifying substances using the recombinant expression vector of claim 3. (4) packaging said vector into phage; 5. The method for researching bone-modifying substances according to claim 4, wherein the bone-modifying substance is an osteogenesis-inducing substance. • · • · φ φ * φφφφφφ .φφ .φφφφ 10 · · · · 17 ··· · ♦ φφφ * (5) creating a genomic DNA library by infection of Escherichia coli with phage; 6. The method for researching bone-modifying substances according to claim 4, wherein the bone-modifying substance is an osteogenesis inhibiting substance. (6) PCR screening; and (7) subcloning into the plasmid vector. A method for researching substances effective in the treatment of kidney disease, characterized in that it utilizes the recombinant expression vector of claim 3.