Cloning of human bone morphogenetic protein-2 (hBMP-2) cDNA by PCR method
In this study, we attempt to amplify the complementary DNAs (cDNAs) for human bone morphogenetic proteins (hBMPs), which have a strong activity of bone induction, from a cDNA library derived from a human osteosarcoma cell line MG63, using the polymerase chain reaction (PCR) method with two specific...
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| Published in | Japanese Journal of Oral Biology Vol. 35; no. 1; pp. 64 - 74 |
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| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Japanese Association for Oral Biology
1993
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0385-0137 |
| DOI | 10.2330/joralbiosci1965.35.64 |
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| Abstract | In this study, we attempt to amplify the complementary DNAs (cDNAs) for human bone morphogenetic proteins (hBMPs), which have a strong activity of bone induction, from a cDNA library derived from a human osteosarcoma cell line MG63, using the polymerase chain reaction (PCR) method with two specific primers. Four recombinant clones, which encoded the mature region of hBMP-2, have been amplified. However, they did not contain termination codon at their 3′-terminus. PCR was performed again in order to ligate termination codon to one of the cDNAs. Sequence analysis revealed several mutations including one frameshift mutation. After elimination of the defected nucleotide moiety, a cDNA preparation encoding mature region of hBMP was obtained. It was confirmed that the amino acid sequence translated from this cDNA was identical to that of the mature region of hBMP-2. |
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| AbstractList | In this study, we attempt to amplify the complementary DNAs (cDNAs) for human bone morphogenetic proteins (hBMPs), which have a strong activity of bone induction, from a cDNA library derived from a human osteosarcoma cell line MG63, using the polymerase chain reaction (PCR) method with two specific primers. Four recombinant clones, which encoded the mature region of hBMP-2, have been amplified. However, they did not contain termination codon at their 3′-terminus. PCR was performed again in order to ligate termination codon to one of the cDNAs. Sequence analysis revealed several mutations including one frameshift mutation. After elimination of the defected nucleotide moiety, a cDNA preparation encoding mature region of hBMP was obtained. It was confirmed that the amino acid sequence translated from this cDNA was identical to that of the mature region of hBMP-2. |
| Author | Iimura, Tadahiro Tamura, Masato Oida, Shinichiro Sasaki, Satoshi Enomoto, Shoji Takeda, Kohsuke Maruoka, Yutaka Nifuji, Akira |
| Author_xml | – sequence: 1 fullname: Takeda, Kohsuke organization: First Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University – sequence: 1 fullname: Nifuji, Akira organization: Department of Molecular Pharmacology, Division of Functional Disorder Research, Medical Research Institute, Tokyo Medical and Dental University – sequence: 1 fullname: Maruoka, Yutaka organization: Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University – sequence: 1 fullname: Tamura, Masato organization: Department of Molecular Pharmacology, Division of Functional Disorder Research, Medical Research Institute, Tokyo Medical and Dental University – sequence: 1 fullname: Sasaki, Satoshi organization: Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Tokyo Medical and Dental University – sequence: 1 fullname: Oida, Shinichiro organization: Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Tokyo Medical and Dental University – sequence: 1 fullname: Iimura, Tadahiro organization: Department of Biochemistry and Molecular Biology, Faculty of Dentistry, Tokyo Medical and Dental University – sequence: 1 fullname: Enomoto, Shoji organization: Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University |
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| References | 11) Oida, S., Nifuji, A., Tamura, M., Maruoka, Y., Iimura, T. and Sasaki, S.: PCR amplification and cloning of a mouse bone morphogenetic protein (BMP) cDNA. Jpn. J. Oral Biol. 34: 612-615, 1992. 16) Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977. 17) Keohavong, P. and Thilly, W. G.: Fidelity of DNA polymerase in DNA amplification. Proc. Natl. Acad. Sci. USA 86: 9253-9257, 1989. 4) Gray, A. M. and Mason, A. J.: Requirement for Activin A and Transforming Growth Factor-β1 Pro-Regions in Homodimer Assembly. Science 247: 1328-1330, 1990. 6) Rosen, V. and Thies, R. S.: The BMP proteins in bone formation and repair. Trends Genet. 8: 97-102, 1992. 1) Urist, M. R.: Bone: Formation by Autoinduction. Science 150: 893-899, 1965. 7) Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G.T., Mullis, K. B. and Erlich, H. A.: Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. Science 239: 487-491, 1988. 21) Cox, K., Holtrop, M., D'Alessandro, J. S., Wang, E. A., Wozney, J. M. and Rosen, V.: Histological and ultrastructural comparison of the in vivo activities of rhBMP2 and rhBMP5. J. Bone. Mineral Res. 6: S155, 1991. 3) Celeste, A. J., Taylor, R., Yamaji, N., Wang, J., Ross, J. and Wozney, J.: Molecular cloning of BMP-8: A protein present in bovine bone which is highly related to the BMP-5/6/7 subfamily of osteoinductive molecules. J. Cell. Biochem. Supplement 16F: W502, 1992. 9) Watson, J.D., Gilman, M., Witkowski, J. and Zoller, M.: Recombinant DNA, 2nd edn., pp.79-98, W. H. Freeman and Company, New York, 1992. 2) Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M. and Wang, E. A.: Novel Regulators of Bone Formation: Molecular Clones and Activities. Science 242: 1528-1534, 1988. 8) Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G.T., Erlich, H. A. and Arnheim, N.: Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 230: 1350-1354, 1985. 23) Toriumi, D. M., Kotler, H. S., Luxenberg, D.P., Holtrop, M. E. and Wang, E.A.: Mandibular Reconstruction with a Recombinant Bone-Inducing Factor. Arch Otolaryngol. Head Neck Surg. 117: 1101-1112, 1991. 15) 村松正實, 他: ラボマニュアル遺伝子 (村松正實編), 増補版, pp.51-55, 丸善, 東京, 1990. 10) 二藤彰, 大井田新一郎, 田村正人, 下川仁弥太, 石崎明, 山口聰, 内田哲馬, 丸岡豊, 百瀬文雄, 佐々木哲: 骨形成因子関連遺伝子群のPCR法によるcDNAクローニングとその発現. 日骨代謝誌9: 269, 1991. 5) 大井田新一郎: 骨誘導因子 (BMPとOIF). THE BONE 5: 37-43, 1991. 18) Tindall, K. R. and Kunkel, T. A.: Fidelity of DNA Synthesis by the Thermus aquaticus DNA Polymerase. Biochemistry 27: 6008-6013, 1988. 24) Sampath, T. K., Coughlin, J. E., Whetstone, R. M., Banach, D., Corbett, C., Ridge, R. J., özkaynak, E., Oppermann, H. and Rueger, D. C.: Bovine Osteogenic Protein Is Composed. of Dimers of OP-1 and BMP-2A, Two Members of Transforming Growth Factor-β Superfamily. J. Biol. Chem. 265: 13198-13205, 1990. 19) Eckert, K. A. and Kunkel, T. A.: High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucl. Acids Res. 18: 3739-3744, 1990. 14) 村松正實, 他: ラボマニュアル遺伝子工学 (村松正實編), 増補版, pp.113-116, 丸善, 東京, 1990. 22) Sampath, T. K., Özkaynak, E., Jones, W. K., Sasak, H., Tucker, R., Tucker, M., Kusmik, W., Lightholder, J., Pang, R., Corbett C., Oppermann, H. and Rueger, D. C.: Recombinant human osteogenic protein (hOP-1) induces new bone formation with a specific activity comparable to that of natural bovine osteogenic protein. J. Bone Mineral Res. 6: S155, 1991. 13) Bhat, G. J., Lodes, M. J., Myler, P. J. and Stuart, K. D.: A simple method for cloning blunt ended DNA fragments. Nucl. Acids Res. 19: 398, 1990. 12) Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J.: Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonulease. Biochemistry 18: 5294-5299, 1979. 20) Wang, E. A., Rosen, V., D'Alessandro, J.S., Bauduy, M., Cordes, P., Harada, T., Israel, D. I., Hewick, R. M., Kerns, K. M., Lapan, P., Luxenberg, D. P., McQuaid, D., Moutsatsos, I. K., Nove, J. and Wozney, J. M. Recombinant human bone morphogenetic protein induces bone formation. Proc. Natl. Acad. Sci. USA 87: 2220-2224, 1990. |
| References_xml | – reference: 14) 村松正實, 他: ラボマニュアル遺伝子工学 (村松正實編), 増補版, pp.113-116, 丸善, 東京, 1990. – reference: 12) Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. and Rutter, W. J.: Isolation of Biologically Active Ribonucleic Acid from Sources Enriched in Ribonulease. Biochemistry 18: 5294-5299, 1979. – reference: 24) Sampath, T. K., Coughlin, J. E., Whetstone, R. M., Banach, D., Corbett, C., Ridge, R. J., özkaynak, E., Oppermann, H. and Rueger, D. C.: Bovine Osteogenic Protein Is Composed. of Dimers of OP-1 and BMP-2A, Two Members of Transforming Growth Factor-β Superfamily. J. Biol. Chem. 265: 13198-13205, 1990. – reference: 7) Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G.T., Mullis, K. B. and Erlich, H. A.: Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase. Science 239: 487-491, 1988. – reference: 4) Gray, A. M. and Mason, A. J.: Requirement for Activin A and Transforming Growth Factor-β1 Pro-Regions in Homodimer Assembly. Science 247: 1328-1330, 1990. – reference: 15) 村松正實, 他: ラボマニュアル遺伝子 (村松正實編), 増補版, pp.51-55, 丸善, 東京, 1990. – reference: 9) Watson, J.D., Gilman, M., Witkowski, J. and Zoller, M.: Recombinant DNA, 2nd edn., pp.79-98, W. H. Freeman and Company, New York, 1992. – reference: 23) Toriumi, D. M., Kotler, H. S., Luxenberg, D.P., Holtrop, M. E. and Wang, E.A.: Mandibular Reconstruction with a Recombinant Bone-Inducing Factor. Arch Otolaryngol. Head Neck Surg. 117: 1101-1112, 1991. – reference: 19) Eckert, K. A. and Kunkel, T. A.: High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucl. Acids Res. 18: 3739-3744, 1990. – reference: 17) Keohavong, P. and Thilly, W. G.: Fidelity of DNA polymerase in DNA amplification. Proc. Natl. Acad. Sci. USA 86: 9253-9257, 1989. – reference: 22) Sampath, T. K., Özkaynak, E., Jones, W. K., Sasak, H., Tucker, R., Tucker, M., Kusmik, W., Lightholder, J., Pang, R., Corbett C., Oppermann, H. and Rueger, D. C.: Recombinant human osteogenic protein (hOP-1) induces new bone formation with a specific activity comparable to that of natural bovine osteogenic protein. J. Bone Mineral Res. 6: S155, 1991. – reference: 16) Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977. – reference: 13) Bhat, G. J., Lodes, M. J., Myler, P. J. and Stuart, K. D.: A simple method for cloning blunt ended DNA fragments. Nucl. Acids Res. 19: 398, 1990. – reference: 3) Celeste, A. J., Taylor, R., Yamaji, N., Wang, J., Ross, J. and Wozney, J.: Molecular cloning of BMP-8: A protein present in bovine bone which is highly related to the BMP-5/6/7 subfamily of osteoinductive molecules. J. Cell. Biochem. Supplement 16F: W502, 1992. – reference: 1) Urist, M. R.: Bone: Formation by Autoinduction. Science 150: 893-899, 1965. – reference: 2) Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M. and Wang, E. A.: Novel Regulators of Bone Formation: Molecular Clones and Activities. Science 242: 1528-1534, 1988. – reference: 21) Cox, K., Holtrop, M., D'Alessandro, J. S., Wang, E. A., Wozney, J. M. and Rosen, V.: Histological and ultrastructural comparison of the in vivo activities of rhBMP2 and rhBMP5. J. Bone. Mineral Res. 6: S155, 1991. – reference: 10) 二藤彰, 大井田新一郎, 田村正人, 下川仁弥太, 石崎明, 山口聰, 内田哲馬, 丸岡豊, 百瀬文雄, 佐々木哲: 骨形成因子関連遺伝子群のPCR法によるcDNAクローニングとその発現. 日骨代謝誌9: 269, 1991. – reference: 18) Tindall, K. R. and Kunkel, T. A.: Fidelity of DNA Synthesis by the Thermus aquaticus DNA Polymerase. Biochemistry 27: 6008-6013, 1988. – reference: 8) Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G.T., Erlich, H. A. and Arnheim, N.: Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 230: 1350-1354, 1985. – reference: 11) Oida, S., Nifuji, A., Tamura, M., Maruoka, Y., Iimura, T. and Sasaki, S.: PCR amplification and cloning of a mouse bone morphogenetic protein (BMP) cDNA. Jpn. J. Oral Biol. 34: 612-615, 1992. – reference: 20) Wang, E. A., Rosen, V., D'Alessandro, J.S., Bauduy, M., Cordes, P., Harada, T., Israel, D. I., Hewick, R. M., Kerns, K. M., Lapan, P., Luxenberg, D. P., McQuaid, D., Moutsatsos, I. K., Nove, J. and Wozney, J. M. Recombinant human bone morphogenetic protein induces bone formation. Proc. Natl. Acad. Sci. USA 87: 2220-2224, 1990. – reference: 5) 大井田新一郎: 骨誘導因子 (BMPとOIF). THE BONE 5: 37-43, 1991. – reference: 6) Rosen, V. and Thies, R. S.: The BMP proteins in bone formation and repair. Trends Genet. 8: 97-102, 1992. |
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| SubjectTerms | cDNA (complementary DNA) cloning hBMP (human Bone Morphogenetic Protein) mature region PCR (Polymerase Chain Reaction) |
| Title | Cloning of human bone morphogenetic protein-2 (hBMP-2) cDNA by PCR method |
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