Abstract
A mutagenic DNA‐adduct, O6‐methylguanine, was introduced into codon 12 of the synthetic c‐Ha‐ras gene by cassette mutagenesis. Transfection of this modified ras gene into normal NIH3T3 cells by the calcium phosphate procedure resulted in significant induction of focus formation. The ras gene inserted into the transformed cells was found to have a G to A transition at the position of the modified base. These results indicate that an O6‐methylguanine residue in DNA may lead to a mutation and be one cause of activation of the ras gene.
Keywords: c‐Ha‐ras, O6‐Methylguanine, Transformation, Mutation, Cassette mutagenesis
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REFERENCES
- 1. ) Barbacid , M.ras genes , Ann. Rev. Biochem. , 56 , 779 – 827 ( 1987. ). [DOI] [PubMed] [Google Scholar]
- 2. ) Nishimura , S. and Sekiya , T.Human cancer and cellular oncogenes . Biochem. J. , 243 , 313 – 327 ( 1987. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. ) Singer , B. and Kusmierek , J. T.Chemical mutagenesis . Ann. Rev. Biochem. , 52 , 655 – 693 ( 1982. ). [DOI] [PubMed] [Google Scholar]
- 4. ) Snow , E. T. , Foote , R. S. and Mitra , S.Base‐pairing properties of O6‐methylguanine in template DNA during in vitro DNA replication . J. Biol. Chem. , 259 , 8095 – 8100 ( 1984. ). [PubMed] [Google Scholar]
- 5. ) Singer , B. , Chavez , F. , Goodman , M. F. , Essigmann , J. M. and Dosanjh , M. K.Effect of 3′ flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6‐methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used , Proc. Natl. Acad. Sci. USA , 86 , 8271 – 8274 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. ) Zarbl , H. , Sukumar , S. , Arthur , A. V. , Martin‐Zanca , D. and Barbacid , M.Direct mutagenesis of Ha‐ras‐1 oncogenes by N‐nitroso‐N‐methylurea during initiation of mammary carcinogenesis in rats . Nature , 315 , 382 – 385 ( 1985. ). [DOI] [PubMed] [Google Scholar]
- 7. ) Mitra , G. , Pauly , G. T. , Kumar , R. , Pei , G. K. , Hughes , S. H. , Moschel , R. C. and Barbacid , M.Molecular analysis of (O6‐substituted guanine‐induced mutagenesis of ras oncogenes . Proc. Natl. Acad. Sci. USA , 86 , 8650 – 8654 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. ) Lindahl , T. , Sedgwick , B. , Sekiguchi , M. and Nakabeppu , Y.Regulation and expression of the adaptive response to alkylating agents . Ann. Rev. Biochem. , 57 , 133 – 157 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 9. ) Tano , K. , Shiota , S. , Collier , J. , Foote , R. S. and Mitra , S.Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6‐alkylguanine . Proc. Natl. Acad. Sci USA , 87 , 686 – 690 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. ) Major , G. N. , Gardner , E. J. , Carne , A. F. and Lawley , P. D.Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6‐methylguanine . Nucleic Acids Res. , 18 , 1351 – 1359 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. ) Hayakawa , H. , Koike , G. and Sekiguchi , M.Expression and cloning of complementary DNA for a human enzyme that repairs O6‐methylguanine in DNA . J. Mol. Biol. , 213 , 739 – 747 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 12. ) Capon , D. J. , Chen , E. Y. , Levinson , A. D. , Seeburg , P. H. and Goeddel , D. V.Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue . Nature , 302 , 33 – 37 ( 1983. ). [DOI] [PubMed] [Google Scholar]
- 13. ) Miura , K. , Inoue , Y. , Nakamori , H. , Iwai , S. , Ohtsuka , E. , Ikehara , M. , Noguchi , S. and Nishimura , S.Synthesis and expression of a synthetic gene for the activated human c‐Ha‐ras protein . Jpn. J. Cancer Res. , 77 , 45 – 51 ( 1986. ). [PubMed] [Google Scholar]
- 14. ) Miura , K. , Kamiya , H. , Tominaga , M. , Inoue , Y. , Ikehara , M. , Noguchi , S. , Nishimura , S. and Ohtsuka , E.Overexpression of cellular and activated Ha‐ras proteins by mutating a synthetic gene . Chem. Pharm. Bull. , 35 , 4878 – 4882 ( 1987. ). [DOI] [PubMed] [Google Scholar]
- 15. ) Kamiya , H. , Miura , K. , Ohtomo , N. , Koda , T. , Kakinuma , M. , Nishimura , S. and Ohtsuka , E.Transformation of NIH3T3 cells with synthetic c‐Ha‐ras genes . Jpn. J. Cancer Res , 80 , 200 – 203 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. ) Perucho , M. , Goldfarb , M. , Shimizu , K. , Lama , C. , Fogh , J. and Wigler , M.Human‐tumor‐derived cell lines contain common and different transforming genes . Cell , 27 , 467 – 476 ( 1981. ). [DOI] [PubMed] [Google Scholar]
- 17. ) Saiki , R. K. , Gelfand , D. H. , Stoffel , S , Sharf , 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. ). [DOI] [PubMed] [Google Scholar]
- 18. ) Haliassos , A. , Chomel , J. C. , Grandjouan , S. , Kruh , J. , Kaplan , J. C. and Kitzis , A.Detection of minority point mutations by modified PCR technique; a new approach for a sensitive diagnosis of tumor‐progression markers . Nucleic Acids Res. , 17 , 8093 – 8099 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. ) de Vos , A. M. , Tong , L. , Milburn , M. V. , Matias , P. M. , Jancarik , J. , Noguchi , S. , Nishimura , S. , Miura , K. , Ohtsuka , E. and Kim , S‐H.Three‐dimensional structure of an oncogene protein: catalytic domain of human c‐Ha‐ras p21 . Science , 239 , 888 – 893 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 20. ) Tong , L. , Milburn , M. V. , de Vos , A. M. and Kim , S‐H.Structure of ras protein . Science , 245 , 244 ( 1989. ). [DOI] [PubMed] [Google Scholar]
- 21. ) Milburn , M. V. , Tong , L. , de Vos , A. M. , Brüunger , A. , Yamaizumi , Z. , Nishimura , S. and Kim , S‐H.Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins . Science , 247 , 939 – 945 ( 1990. ). [DOI] [PubMed] [Google Scholar]
- 22. ) Brünger , A. T. , Milburn , M. V. , Tong , L. , de Vos , A. H. , Jancarik , J. , Yamaizumi , Z. , Nishimura , S. , Ohtsuka , E. and Kim , S‐H.Crystal structure of an active form of RAS protein, a complex of a GTP analog and the HRAS p21 catalytic domain . Proc. Natl. Acad. Sci. USA , 87 , 4849 – 4853 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. ) Chang , E. H. , Furth , M. E. , Scolnick , E. M. and Lowy , D. R.Tumorigenic transformation of mammalian cells induced by a normal human gene homologous to the oncogene of Harvey murine sarcoma virus . Nature , 297 , 479 – 483 ( 1982. ). [DOI] [PubMed] [Google Scholar]