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. 1985 Jan;5(1):70–74. doi: 10.1128/mcb.5.1.70

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

K R Folger, K Thomas, M R Capecchi
PMCID: PMC366679  PMID: 3982420

Abstract

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baas P. D., Jansz H. S. Asymmetric information transfer during phi X174 DNA replication. J Mol Biol. 1972 Feb 14;63(3):557–568. doi: 10.1016/0022-2836(72)90447-0. [DOI] [PubMed] [Google Scholar]
  2. Bauer J., Krämmer G., Knippers R. Asymmetric repair of bacteriophage T7 heteroduplex DNA. Mol Gen Genet. 1981;181(4):541–547. doi: 10.1007/BF00428750. [DOI] [PubMed] [Google Scholar]
  3. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Capecchi M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. doi: 10.1016/0092-8674(80)90358-x. [DOI] [PubMed] [Google Scholar]
  5. Enea V., Vovis G. F., Zinder N. D. Genetic studies with heteroduplex DNA of bacteriophage fl. Asymmetric segregation, base correction and implications for the mechanism of genetic recombination. J Mol Biol. 1975 Aug 15;96(3):495–509. doi: 10.1016/0022-2836(75)90175-8. [DOI] [PubMed] [Google Scholar]
  6. Folger K. R., Thomas K., Capecchi M. R. Nonreciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei. Mol Cell Biol. 1985 Jan;5(1):59–69. doi: 10.1128/mcb.5.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Folger K. R., Wong E. A., Wahl G., Capecchi M. R. Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol. 1982 Nov;2(11):1372–1387. doi: 10.1128/mcb.2.11.1372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hudziak R. M., Laski F. A., RajBhandary U. L., Sharp P. A., Capecchi M. R. Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes. Cell. 1982 Nov;31(1):137–146. doi: 10.1016/0092-8674(82)90413-5. [DOI] [PubMed] [Google Scholar]
  9. Lai C. J., Nathans D. A map of temperature-sensitive mutants of simian virus 40. Virology. 1975 Jul;66(1):70–81. doi: 10.1016/0042-6822(75)90179-8. [DOI] [PubMed] [Google Scholar]
  10. Lu A. L., Clark S., Modrich P. Methyl-directed repair of DNA base-pair mismatches in vitro. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4639–4643. doi: 10.1073/pnas.80.15.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Luciw P. A., Bishop J. M., Varmus H. E., Capecchi M. R. Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA. Cell. 1983 Jul;33(3):705–716. doi: 10.1016/0092-8674(83)90013-2. [DOI] [PubMed] [Google Scholar]
  12. Miller L. K., Cooke B. E., Fried M. Fate of mismatched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3073–3077. doi: 10.1073/pnas.73.9.3073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nevers P., Spatz H. C. Escherichia coli mutants uvr D and uvr E deficient in gene conversion of lambda-heteroduplexes. Mol Gen Genet. 1975 Aug 27;139(3):233–243. doi: 10.1007/BF00268974. [DOI] [PubMed] [Google Scholar]
  14. Pukkila P. J., Peterson J., Herman G., Modrich P., Meselson M. Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair in Escherichia coli. Genetics. 1983 Aug;104(4):571–582. doi: 10.1093/genetics/104.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Razzaque A., Mizusawa H., Seidman M. M. Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3010–3014. doi: 10.1073/pnas.80.10.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spatz H. C., Trautner T. A. One way to do experiments on gene conversion? Transfection with heteroduplex SPP1 DNA. Mol Gen Genet. 1970;109(1):84–106. doi: 10.1007/BF00334048. [DOI] [PubMed] [Google Scholar]
  17. Wagner R., Jr, Meselson M. Repair tracts in mismatched DNA heteroduplexes. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4135–4139. doi: 10.1073/pnas.73.11.4135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wake C. T., Gudewicz T., Porter T., White A., Wilson J. H. How damaged is the biologically active subpopulation of transfected DNA? Mol Cell Biol. 1984 Mar;4(3):387–398. doi: 10.1128/mcb.4.3.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. White R. L., Fox M. S. Genetic consequences of transfection with heterduplex bacteriophage lambda DNA. Mol Gen Genet. 1975 Nov 24;141(2):163–171. doi: 10.1007/BF00267681. [DOI] [PubMed] [Google Scholar]
  20. Wildenberg J., Meselson M. Mismatch repair in heteroduplex DNA. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2202–2206. doi: 10.1073/pnas.72.6.2202. [DOI] [PMC free article] [PubMed] [Google Scholar]

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