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. 1987 Oct;7(10):3561–3565. doi: 10.1128/mcb.7.10.3561

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

M M Seidman 1
PMCID: PMC368009  PMID: 3683393

Abstract

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

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Selected References

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

  1. Alwine J. C. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins. Mol Cell Biol. 1985 May;5(5):1034–1042. doi: 10.1128/mcb.5.5.1034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson R. A., Eliason S. L. Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing. Mol Cell Biol. 1986 Sep;6(9):3246–3252. doi: 10.1128/mcb.6.9.3246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ayares D., Spencer J., Schwartz F., Morse B., Kucherlapati R. Homologous recombination between autonomously replicating plasmids in mammalian cells. Genetics. 1985 Oct;111(2):375–388. doi: 10.1093/genetics/111.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brenner D. A., Smigocki A. C., Camerini-Otero R. D. Double-strand gap repair results in homologous recombination in mouse L cells. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1762–1766. doi: 10.1073/pnas.83.6.1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chakrabarti S., Seidman M. M. Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative. Mol Cell Biol. 1986 Jul;6(7):2520–2526. doi: 10.1128/mcb.6.7.2520. [DOI] [PMC free article] [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. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  9. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  10. Jayaram M. Association of reciprocal exchange with gene conversion between the repeated segments of 2-micron circle. J Mol Biol. 1986 Oct 5;191(3):341–354. doi: 10.1016/0022-2836(86)90131-2. [DOI] [PubMed] [Google Scholar]
  11. Kucherlapati R. S., Eves E. M., Song K. Y., Morse B. S., Smithies O. Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA. Proc Natl Acad Sci U S A. 1984 May;81(10):3153–3157. doi: 10.1073/pnas.81.10.3153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lee T. N., Nathans D. Evolutionary variants of simian virus 40: replication and encapsidation of variant DNA. Virology. 1979 Jan 30;92(2):291–298. doi: 10.1016/0042-6822(79)90134-x. [DOI] [PubMed] [Google Scholar]
  13. Lin F. L., Sperle K., Sternberg N. Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process. Mol Cell Biol. 1984 Jun;4(6):1020–1034. doi: 10.1128/mcb.4.6.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Orr-Weaver T. L., Szostak J. W. Yeast recombination: the association between double-strand gap repair and crossing-over. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4417–4421. doi: 10.1073/pnas.80.14.4417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peden K. W., Pipas J. M., Pearson-White S., Nathans D. Isolation of mutants of an animal virus in bacteria. Science. 1980 Sep 19;209(4463):1392–1396. doi: 10.1126/science.6251547. [DOI] [PubMed] [Google Scholar]
  16. Potter H., Dressler D. On the mechanism of genetic recombination: the maturation of recombination intermediates. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4168–4172. doi: 10.1073/pnas.74.10.4168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Roth D. B., Wilson J. H. Nonhomologous recombination in mammalian cells: role for short sequence homologies in the joining reaction. Mol Cell Biol. 1986 Dec;6(12):4295–4304. doi: 10.1128/mcb.6.12.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Seidman M. M., Dixon K., Razzaque A., Zagursky R. J., Berman M. L. A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells. Gene. 1985;38(1-3):233–237. doi: 10.1016/0378-1119(85)90222-7. [DOI] [PubMed] [Google Scholar]
  19. Shapira G., Stachelek J. L., Letsou A., Soodak L. K., Liskay R. M. Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4827–4831. doi: 10.1073/pnas.80.15.4827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Smithies O., Gregg R. G., Boggs S. S., Koralewski M. A., Kucherlapati R. S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature. 1985 Sep 19;317(6034):230–234. doi: 10.1038/317230a0. [DOI] [PubMed] [Google Scholar]
  21. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  22. Subramani S., Berg P. Homologous and nonhomologous recombination in monkey cells. Mol Cell Biol. 1983 Jun;3(6):1040–1052. doi: 10.1128/mcb.3.6.1040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Szostak J. W., Orr-Weaver T. L., Rothstein R. J., Stahl F. W. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. doi: 10.1016/0092-8674(83)90331-8. [DOI] [PubMed] [Google Scholar]
  24. Thomas K. R., Folger K. R., Capecchi M. R. High frequency targeting of genes to specific sites in the mammalian genome. Cell. 1986 Feb 14;44(3):419–428. doi: 10.1016/0092-8674(86)90463-0. [DOI] [PubMed] [Google Scholar]
  25. Upcroft P., Carter B., Kidson C. Mammalian cell function mediating recombination of genetic elements. Nucleic Acids Res. 1980 Dec 11;8(23):5835–5844. doi: 10.1093/nar/8.23.5835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Volkert F. C., Young C. S. The genetic analysis of recombination using adenovirus overlapping terminal DNA fragments. Virology. 1983 Feb;125(1):175–193. doi: 10.1016/0042-6822(83)90072-7. [DOI] [PubMed] [Google Scholar]
  27. Wake C. T., Vernaleone F., Wilson J. H. Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. Mol Cell Biol. 1985 Aug;5(8):2080–2089. doi: 10.1128/mcb.5.8.2080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wake C. T., Wilson J. H. Defined oligomeric SV40 DNA: a sensitive probe of general recombination in somatic cells. Cell. 1980 Aug;21(1):141–148. doi: 10.1016/0092-8674(80)90121-x. [DOI] [PubMed] [Google Scholar]
  29. de Saint Vincent B. R., Wahl G. M. Homologous recombination in mammalian cells mediates formation of a functional gene from two overlapping gene fragments. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2002–2006. doi: 10.1073/pnas.80.7.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

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