Advertisement

Somatic Cell and Molecular Genetics

, Volume 18, Issue 2, pp 179–188 | Cite as

Analysis of sequence specificity of 5-bromodeoxyuridine-induced reversion in cells containing multiple copies of a mutantgpt gene

  • Joel B. Rotstein
  • Mark T. Kresnak
  • Georgy M. Samadashwily
  • Richard L. Davidson
Article

Abstract

For studies on molecular mechanisms of mutagenesis, it would be advantageous to transfer mutant genes with specific alterations into mammalian cells and use the transformed cells in reversion analyses. In the present paper, we describe an efficient method for analyzing reversion events occurring in cells that possess multiple copies of a mutational target gene. This method involves amplification of the chromosomally integrated target genes with the polymerase chain reaction (PCR) and restriction endonuclease digestion of the amplified product. Single reversion events that either create or destroy restriction endonuclease recognition sequences that encompass the site of the original mutation can be identified in a background of 10–20 copies of the gene that retain the mutant sequence. Using this method, we have analyzed revertants induced by 5-bromodeoxyuridine (BrdU) in a Chinese hamster ovary cell line that possesses multiple copies of a mutant bacterial gpt gene containing a specific alteration. The results of this study not only demonstrate the effectiveness of this method for analyzing reversion of a single gene copy in transfectants possessing multiple copies of a mutant target gene, but also demonstrate that the sequence specificity for BrdU-induced mutations is the same in Chinese hamster cells as previously observed with mouse cells.

Keywords

Chinese Hamster Ovary Cell Multiple Copy Sequence Specificity Chinese Hamster Ovary Restriction Endonuclease Digestion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature cited

  1. 1.
    Ashman, C.R., and Davidson, R.L. (1984).Mol. Cell. Biol. 42266–2272.PubMedGoogle Scholar
  2. 2.
    Ashman, C.R., Jagadeeswaran, P., and Davidson, R.L. (1986).Proc. Natl. Acad. Sci. U.S.A. 833356–3360.PubMedGoogle Scholar
  3. 3.
    Hauser, J., Seidman, M.M., Sidur, K., and Dixon, K. (1986).Mol. Cell. Biol. 6277–285.PubMedGoogle Scholar
  4. 4.
    Lebkowski, J.S., Miller, J.H., and Calos, M.P. (1986).Mol. Cell. Biol. 61838–1842.PubMedGoogle Scholar
  5. 5.
    DuBridge, R.B., Jang, P., Hsia, H.C., Leong, P.M., Miller, J.H., and Calos, M.P. (1987).Mol. Cell. Biol. 7379–387.PubMedGoogle Scholar
  6. 6.
    Ashman, C.R., and Davidson, R.L. (1987).Proc. Natl. Acad. Sci. U.S.A. 843354–3358.PubMedGoogle Scholar
  7. 7.
    Ashman, C.R., and Davidson, R.L. (1987).Somat. Cell Mol. Genet. 13563–568.PubMedGoogle Scholar
  8. 8.
    Davidson, R.L., and Ashman, C.R. (1987).Somat. Cell Mol. Genet. 13415–417.PubMedGoogle Scholar
  9. 9.
    Davidson, R.L., Broeker, P., and Ashman, C.R. (1988).Proc. Natl. Acad. Sci. U.S.A. 854406–4410.PubMedGoogle Scholar
  10. 10.
    Gelbert, L.M., and Davidson, R.L. (1988).Proc. Natl. Acad. Sci. U.S.A. 859143–9147.PubMedGoogle Scholar
  11. 11.
    Gelbert, L.M., Wilson, M.M., and Davidson, R.L. (1990).Somat. Cell Mol. Genet. 16173–184.PubMedGoogle Scholar
  12. 12.
    Kresnak, M.T., and Davidson, R.L. (1991).Somat. Cell Mol. Genet. 17399–410.PubMedGoogle Scholar
  13. 13.
    Kresnak, M.T., and Davidson, R.L. (1992).Proc. Natl. Acad. Sci. U.S.A. (in press).Google Scholar
  14. 14.
    Davidson, R.L., and Gerald, P.S. (1976).Somat. Cell Genet. 2165–176.PubMedGoogle Scholar
  15. 15.
    Hirt, B. (1967).J. Mol. Biol. 26365–369.PubMedGoogle Scholar
  16. 16.
    Hanahan, D. (1983).J. Mol. Biol. 166557–580.PubMedGoogle Scholar
  17. 17.
    Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).Google Scholar
  18. 18.
    Engelke, D.R., Hoener, P.A., and Collins, F.S. (1988).Proc. Natl. Acad. Sci. U.S.A. 85544–548.PubMedGoogle Scholar
  19. 19.
    Innis, M.A., Myambo, K.B., Gelfand, D.H., and Brow, M.A.D. (1988).Proc. Natl. Acad. Sci. U.S.A. 859436–9440.PubMedGoogle Scholar
  20. 20.
    Mihovilovic, M., and Lee, J.E. (1989).Biotechniques 714–16.PubMedGoogle Scholar
  21. 21.
    Winship, P.R. (1989).Nucleic Acids Res. 171266.PubMedGoogle Scholar
  22. 22.
    Skopek, T.R., and Hutchinson, F. (1982).J. Mol. Biol. 15919–33.PubMedGoogle Scholar
  23. 23.
    Lasken, R.S., and Goodman, M.F. (1985).Proc. Natl. Acad. Sci. U.S.A. 821301–1305.PubMedGoogle Scholar
  24. 24.
    Muller, M., Martial, J., and Verly, W.G. (1988).J. Biochem. 253637–643.Google Scholar

Copyright information

© Plenum Publishing Corporation 1992

Authors and Affiliations

  • Joel B. Rotstein
    • 1
  • Mark T. Kresnak
    • 1
  • Georgy M. Samadashwily
    • 1
  • Richard L. Davidson
    • 1
  1. 1.Department of GeneticsUniversity of Illinois College of Medicine at ChicagoChicago

Personalised recommendations