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Influence of cellular sequences on instability of plasmid integration sites in human cells

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Somatic Cell and Molecular Genetics

Abstract

To learn more about mechanisms of genome instability in human cells, I investigated DNA sequences that promote high rates of recombination by analyzing rare unstable plasmid integration sites in simian virus 40-transformed human fibroblasts. Previous studies had hypothesized that rearrangement or loss of integrated sequences could be attributed to adjacent cellular DNA. Consistent with this interpretation, a cloned fragment containing both the integrated plasmid and 2.0 kb of adjacent cell DNA from one such unstable integration site in the cell line LM205 demonstrated a much higher incidence of rearrangements when integrated into other chromosome locations than did the original plasmid. To further test this hypothesis, portions of cellular DNA from this region were integrated in duplicate in other locations to determine their ability to promote restriction-fragment-length polymorphism, an indicator of high rates of homologous recombination. Although two types of instability were observed, neither could be attributed solely to the cell sequences being tested in the plasmid. The first type of instability was a transient deletion or amplification of the plasmid DNA soon after integration, which appeared to be a general phenomenon often associated with any type of newly integrated sequence. A second type of instability continued indefinitely for many cell generations, as did that observed in cell line LM205. Because this was rare (one of 78 clones tested), it could not be attributed solely to cell sequences contained within the plasmid. However, the rearrangements in this cell clone occurred exclusively within the cell DNA adjacent to the integration site, again suggesting a role forcis-acting cell sequences in this process. The inability to identify specific cell sequences responsible for instability may therefore indicate that a complex combination of sequences is involved, possibly within both the plasmid and cell DNA.

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Literature cited

  1. Smith, G.R., and Stahl, F.W. (1985).BioEssays 2244–249.

    Google Scholar 

  2. Nicolas, A., Treco, D., Schultes, N.P., and Szostak, J.W. (1989).Nature 33835–39.

    PubMed  Google Scholar 

  3. White, J.H., DiMartino, J.F., Anderson, R.W., Lusnak, K., Hilbert, D., and Fogel, S. (1988).Mol. Cell. Biol. 81253–1258.

    PubMed  Google Scholar 

  4. Voelkel-Meiman, K., Keil, R.L., and Roeder, G.S. (1987).Cell 481071–1079.

    PubMed  Google Scholar 

  5. Sun, H., Treco, D., Schultes, N.P., and Szostak, J.W. (1989).Nature 33887–90.

    PubMed  Google Scholar 

  6. Jeffreys, A.J., Royle, N.J., Wilson, V., and Wong, Z. (1988).Nature 332278–281.

    PubMed  Google Scholar 

  7. Kobori, J.A., Strauss, E., Minard, K., and Hood, L. (1986).Science 234173–179.

    PubMed  Google Scholar 

  8. Steinmetz, M., Stephan, D., and Lindahl, K.F. (1986).Cell 44895–904.

    PubMed  Google Scholar 

  9. Adair, G.M., Siciliano, M.J., Brotherman, K.A., and Nairn, R.S. (1989).Somat. Cell Mol. Genet. 15271–277.

    PubMed  Google Scholar 

  10. Carroll, S.M., Gaudray, P., DeRose, M.L., Emery, J.F., Meinkoth, J.L., Nakkim, E., Subler, M., von Hoff, D.D., and Wahl, G.M. (1987).Mol. Cell. Biol. 71740–1750.

    PubMed  Google Scholar 

  11. Hyrien, O., Debatisse, M., Buttin, G., and de Saint Vincent, B.R. (1987).EMBO J. 62401–2408.

    PubMed  Google Scholar 

  12. Letsou, A., and Liskay, R.M. (1986). InGene Transfer (ed.) Kucherlapati, R. (Plenum Press, New York), pp. 383–409.

    Google Scholar 

  13. Bender, M.A., and Brockman, W.M. (1981).J. Virol. 38872–879.

    PubMed  Google Scholar 

  14. Hiscott, J.B., Murphy, D., and Defendi, V. (1981).Proc. Natl. Acad. Sci. U.S.A. 781736–1740.

    PubMed  Google Scholar 

  15. Hwang, S.P., and Kucherlapati, R.S. (1983).Somat. Cell Genet. 9457–468.

    PubMed  Google Scholar 

  16. Mounts, P., and Kelly, T.J., Jr. (1984).J. Mol. Biol. 177431–460.

    PubMed  Google Scholar 

  17. Sager, R., Anisowics, A., and Howell, N. (1981).Cell 2341–50.

    PubMed  Google Scholar 

  18. Butner, K.A., and Lo, C.W. (1986).J. Mol. Biol. 187547–556.

    PubMed  Google Scholar 

  19. Glanville, N. (1985).Mol. Cell. Biol. 51456–1464.

    PubMed  Google Scholar 

  20. Heartlein, M.W., Knoll, J.H.M., and Latt, S.A. (1988).Mol. Cell. Biol. 83611–3618.

    PubMed  Google Scholar 

  21. Meinkoth, J., Killary, A.M., Fournier, R.E.K., and Wahl, G.M. (1987).Mol. Cell. Biol. 71415–1424.

    PubMed  Google Scholar 

  22. Perucho, M., Hanahan, D., and Wigler, M. (1980).Cell 22309–317.

    PubMed  Google Scholar 

  23. Wahl, G.M., de Saint Vincent, Vincent, B.R., and DeRose, M.L. (1984).Nature 307516–520.

    PubMed  Google Scholar 

  24. Murnane, J.P., and Yezzi, M.J. (1988).Somat. Cell Mol. Genet. 14273–286.

    PubMed  Google Scholar 

  25. Murnane, J.P., and Young, B.R. (1989).Gene 84201–205.

    PubMed  Google Scholar 

  26. Debatisse, M., Saito, I., Buttin, G., and Stark, G.R. (1988).Mol. Cell. Biol. 817–24.

    PubMed  Google Scholar 

  27. Murnane, J.P. (1986).Mol. Cell. Biol. 6549–558.

    PubMed  Google Scholar 

  28. Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. (1978).Cell 14725–731.

    PubMed  Google Scholar 

  29. Capizzi, R.L., and Jameson, J.W. (1973).Mutat. Res. 17147–148.

    PubMed  Google Scholar 

  30. Southern, E.M. (1975).J. Mol. Biol. 98503–517.

    Google Scholar 

  31. Struhl, K. (1985).BioTechniques 3452–453.

    Google Scholar 

  32. Morgan, W.F., Fero, M.L., Land, M.C., and Winegar, R.A. (1988).Mol. Cell. Biol. 84204–4211.

    PubMed  Google Scholar 

  33. Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P., and Garapin, A.C. (1981).J. Mol. Biol. 1501–14.

    PubMed  Google Scholar 

  34. Savage, J.R. (1975).J. Med. Genet. 12103–122.

    Google Scholar 

  35. Perry, P., and Wolff, S. (1974).Nature 251156–158.

    PubMed  Google Scholar 

  36. Morgan, W.F., and Cleaver, J.E. (1982).Mutat. Res. 104361–366.

    PubMed  Google Scholar 

  37. Blackwell, T.K., Moore, M.W., Yancopoulos, G.D., Suh, H., Lutzker, S., Selsing, E., and Alt, F.W. (1986).Nature 324585–589.

    PubMed  Google Scholar 

  38. Brinster, R.L., Chen, H.Y., Warren, R., Sarthy, A., and Palmiter, R. (1982).Nature 29639–42.

    PubMed  Google Scholar 

  39. Gross, D.S., and Garrard, W.T. (1988).Annu. Rev. Biochem 57159–197.

    PubMed  Google Scholar 

  40. Enver, T., Brewer, A.C., and Patient, R.K. (1985).Nature 318680–683.

    PubMed  Google Scholar 

  41. Cannizzaro, L.A., Durst, M., Mendez, M.J., Hecht, B.K., and Hecht, F. (1988).Can. Genet. Cytogenet. 3393–98.

    Google Scholar 

  42. Sager, R. (1988).Cancer Surv. 7325–333.

    PubMed  Google Scholar 

  43. Sager, R., Gadi, I.K., Stephens, L., and Grabowy, C.T. (1985).Proc. Natl. Acad. Sci. U.S.A. 827015–7019.

    PubMed  Google Scholar 

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  1. Laboratory of Radiobiology and Environmental Health, University of California, 94143, San Francisco, California

    John P. Murnane

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Murnane, J.P. Influence of cellular sequences on instability of plasmid integration sites in human cells. Somat Cell Mol Genet 16, 195–209 (1990). https://doi.org/10.1007/BF01233356

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  • DOI: https://doi.org/10.1007/BF01233356

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