Advertisement

Detection and Characterization of Mutations in Mammalian Cells with the pSP189 Shuttle Vector System

  • Michael M. Seidman

Abstract

In the early 1980s the attraction of recombinant DNA technology was beginning to be felt in fields that had previously been refractory to molecular analysis. One such field was mammalian cell mutagenesis. Cloning technology offered the opportunity to recover mutant genes for which there were effective selection protocols (such as HPRT). Sequence determination of the entire gene could then display the nature of the mutations. Despite the feasibility of such protocols, the time and effort required discouraged most investigators. An alternative approach, based on shuttle vectors, appeared more attractive. These were plasmids whose design was based on advances in two fields. The extensive analysis of DNA tumor viruses such as SV40 had defined the genetic information necessary for viral replication in monkey and human cells. The biology and molecular biology of bacterial plasmids was also well developed. Thus, constructs with SV40 virus replication functions (the T-antigen gene and an origin of replication) linked to components of bacterial plasmids (the plasmid origin and a drug resistance marker) had been shown to replicate in monkey cells, and could be recovered and introduced into bacteria (Peden et al., 1980; Lusky and Botchan, 1981; these references also discuss the problem and resolution of the replication poison sequence found on pBR322). These experiments were primarily demonstrations of principle; there was no actual use of the shuttle technology. It seemed logical, however, to those interested in mammalian mutagenesis, to add a third component, a bacterial marker gene. The resultant vector, perhaps treated with a DNA damaging agent, would then be introduced into mammalian cells, allowed to replicate, recovered, and reintroduced into bacteria. Bacterial colonies with mutations in the marker gene would be recognized by standard microbiological selection or screening procedures, and the nature of the mutations identified by direct sequence analysis. This logic was quite compelling, and a number of groups set out to develop the technology.

Keywords

Shuttle Vector Bacterial Plasmid Plasmid Origin Drug Resistance Marker Klenow Polymerase 
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.

References

  1. Bredberg, A., Kraemer, K. H., and Seidman, M. M. (1986). Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells. Proc. Natl. Acad. Sci. USA 83:8273–8277.PubMedCrossRefGoogle Scholar
  2. Calos, M. P., Lebkowski, J. S., and Botchan, M. R. (1983). High mutation frequency in DNA transfected into mammalian cells. Proc. Natl. Acad. Sci. USA 80:3015–3019.PubMedCrossRefGoogle Scholar
  3. Drinkwater, N., and Klinedinst, D. K. (1986). Chemically induced mutagenesis in a shuttle vector with a low background mutant frequency. Proc. Natl. Acad. Sci. USA 83:3402–3406.PubMedCrossRefGoogle Scholar
  4. Hsia, C. H., Lebkowski, J. S., Leong, P. M., Calos, M., and Miller, J. H. (1989). Comparison of ultraviolet irradiation induced mutagenesis of the lad gene in E. coli and in human cells. J. Mol. Biol. 205:103–113.PubMedCrossRefGoogle Scholar
  5. Lebkowski, J. S., DuBridge, R. B., Anteil, E. A., Greisen, K. S., and Calos, M. P. (1984). Transfected DNA is mutated in monkey mouse, and human cell lines. Mol Cell. Biol. 4:1951–1960.PubMedGoogle Scholar
  6. Lebkowski, J. S., Clancy, S., Miller, J. H., and Calos, M. P. (1985). The lac I shuttle: Rapid analysis of mutational specificity of ultraviolet light in human cells. Proc. Natl. Acad. Sci. USA 82:8606–8610.PubMedCrossRefGoogle Scholar
  7. Lusky, M., and Botchan, M. (1981). Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature 293:79–81.PubMedCrossRefGoogle Scholar
  8. Menck, C. F. M., Sarasin, A., and James, M. R. (1987). SV40 based E. coli shuttle vectors infectious for monkey cells. Gene 53:21–29.PubMedCrossRefGoogle Scholar
  9. Parris, C. N., and Kraemer, K. H. (1992). Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation. Exp. Cell Res. 201:462–469.PubMedCrossRefGoogle Scholar
  10. Parris, C. N., and Seidman, M. M. (1992). A signature sequence distinguishes sibling and independent mutations in a shuttle vector plasmid. Gene 117:1–5.PubMedCrossRefGoogle Scholar
  11. Parris, C. N., Levy, D. D., Jessee, J., and Seidman, M. M. (1994). Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells. J. Mol. Biol. 236:491–502.PubMedCrossRefGoogle Scholar
  12. Peden, K. W. C., Pipas, J. M., Pearson-White, S., and Nathans, D. (1980). Isolation of mutants of an animal virus in bacteria. Science 209:1392–1396.PubMedCrossRefGoogle Scholar
  13. Razzaque, A. S., Mizusawa, H., and Seidman, M. M. (1983). Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc. Natl Acad. Sci. USA 80:3010–3014.PubMedCrossRefGoogle Scholar
  14. Sarker, S., Dasgupta, U. B., and Summers, W. C. (1984). Error prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of E. coli. Mol. Cell. Biol 4:2227–2230.Google Scholar
  15. Seetharam, S., and Seidman, M. M. (1992). Modulation of ultraviolet mutational hotspots by cellular stress. J. Mol. Biol. 228:1031–1036.PubMedCrossRefGoogle Scholar
  16. Seetharam, S., Kraemer, K. H., Waters, H. L., and Seidman, M. M. (1991). Ultraviolet mutational spectrum in a shuttle vector plasmid propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts. Mutat. Res. 254:97–105.PubMedCrossRefGoogle Scholar
  17. Seidman, M. M. (1989). The development of transient SV40 based shuttle vectors for mutagenesis studies:problems and solutions. Mutat. Res. 220:55–60.PubMedCrossRefGoogle Scholar
  18. Seidman, M. M., Dixon, K., Razzaque, A., Zagursky, R. J., and Berman, M. L. (1985). A shuttle vector plasmid for studying carcinogen induced point mutations in mammalian cells. Gene 38:233–237.PubMedCrossRefGoogle Scholar
  19. Seidman, M. M., Bredberg, A., Seetharam, S., and Kraemer, K. H. (1987). Multiple point mutations in a shuttle vector propagated in human cells. Evidence for an error prone polymerase activity. Proc. Natl. Acad. Sci. USA 84:4944–4948.PubMedCrossRefGoogle Scholar
  20. Wei, D., Maher, V. M., and McCormick, J. J. (1995). Site specific rates of excision repair of benzo[a] pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: Correlation with the mutation spectra. Proc. Natl. Acad. Sci. USA 92:2204–2208.PubMedCrossRefGoogle Scholar
  21. Zernik-Kobak, M., Pirsel, M., Doniger, J., DiPaolo, J. A., Levine, A. S., and Dixon, K. (1990). Polyomavirus based shuttle vectors for studying mechanisms of mutagenesis in rodent cells. Mutat. Res. 242:57–65.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Michael M. Seidman
    • 1
  1. 1.OncorPharmGaithersburgUSA

Personalised recommendations