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. 1986 Jul;83(14):5199–5203. doi: 10.1073/pnas.83.14.5199

Sequence homology requirements for intermolecular recombination in mammalian cells.

D Ayares, L Chekuri, K Y Song, R Kucherlapati
PMCID: PMC323918  PMID: 3523485

Abstract

We have examined the homology requirements for intermolecular recombination between plasmids introduced into human, monkey, and bacterial cells. Variable-size-deletion derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo were constructed. Each of these plasmids was mixed with another pSV2neo plasmid containing a different, nonoverlapping deletion. Recombination was measured in mammalian cells and bacteria by the frequency of reconstruction of an intact neo gene. We observed that 25 base pairs of homologous sequence is sufficient to yield recombinant products, implying that synapsis and homologous pairing can occur with this level of homology. Examination of the products revealed that nonreciprocal recombination played a role in the generation of normal neo genes. In addition coconversion of linked markers was observed. Exonucleolytic action seems to play a role in gene conversion.

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Selected References

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

  1. Andrews B. J., Proteau G. A., Beatty L. G., Sadowski P. D. The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences. Cell. 1985 Apr;40(4):795–803. doi: 10.1016/0092-8674(85)90339-3. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Phenotypes of adenovirus-5 host-range mutants for early-mRNA synthesis. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):429–436. doi: 10.1101/sqb.1980.044.01.046. [DOI] [PubMed] [Google Scholar]
  4. Brenner D. A., Smigocki A. C., Camerini-Otero R. D. Effect of insertions, deletions, and double-strand breaks on homologous recombination in mouse L cells. Mol Cell Biol. 1985 Apr;5(4):684–691. doi: 10.1128/mcb.5.4.684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cox M. M., Lehman I. R. Directionality and polarity in recA protein-promoted branch migration. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6018–6022. doi: 10.1073/pnas.78.10.6018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cox M. M. The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4223–4227. doi: 10.1073/pnas.80.14.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DasGupta C., Radding C. M. Polar branch migration promoted by recA protein: effect of mismatched base pairs. Proc Natl Acad Sci U S A. 1982 Feb;79(3):762–766. doi: 10.1073/pnas.79.3.762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Gonda D. K., Radding C. M. By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology. Cell. 1983 Sep;34(2):647–654. doi: 10.1016/0092-8674(83)90397-5. [DOI] [PubMed] [Google Scholar]
  11. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  12. Johnson R. C., Simon M. I. Hin-mediated site-specific recombination requires two 26 bp recombination sites and a 60 bp recombinational enhancer. Cell. 1985 Jul;41(3):781–791. doi: 10.1016/s0092-8674(85)80059-3. [DOI] [PubMed] [Google Scholar]
  13. Kucherlapati R. S., Ayares D., Hanneken A., Noonan K., Rauth S., Spencer J. M., Wallace L., Moore P. D. Homologous recombination in monkey cells and human cell-free extracts. Cold Spring Harb Symp Quant Biol. 1984;49:191–197. doi: 10.1101/sqb.1984.049.01.022. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Landy A., Ross W. Viral integration and excision: structure of the lambda att sites. Science. 1977 Sep 16;197(4309):1147–1160. doi: 10.1126/science.331474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meselson M. S., Radding C. M. A general model for genetic recombination. Proc Natl Acad Sci U S A. 1975 Jan;72(1):358–361. doi: 10.1073/pnas.72.1.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller C. K., Temin H. M. High-efficiency ligation and recombination of DNA fragments by vertebrate cells. Science. 1983 May 6;220(4597):606–609. doi: 10.1126/science.6301012. [DOI] [PubMed] [Google Scholar]
  20. Rubnitz J., Subramani S. The minimum amount of homology required for homologous recombination in mammalian cells. Mol Cell Biol. 1984 Nov;4(11):2253–2258. doi: 10.1128/mcb.4.11.2253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Singer B. S., Gold L., Gauss P., Doherty D. H. Determination of the amount of homology required for recombination in bacteriophage T4. Cell. 1982 Nov;31(1):25–33. doi: 10.1016/0092-8674(82)90401-9. [DOI] [PubMed] [Google Scholar]
  22. Small J., Scangos G. Recombination during gene transfer into mouse cells can restore the function of deleted genes. Science. 1983 Jan 14;219(4581):174–176. doi: 10.1126/science.6294829. [DOI] [PubMed] [Google Scholar]
  23. Song K. Y., Chekuri L., Rauth S., Ehrlich S., Kucherlapati R. Effect of double-strand breaks on homologous recombination in mammalian cells and extracts. Mol Cell Biol. 1985 Dec;5(12):3331–3336. doi: 10.1128/mcb.5.12.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  25. Sussman D. J., Milman G. Short-term, high-efficiency expression of transfected DNA. Mol Cell Biol. 1984 Aug;4(8):1641–1643. doi: 10.1128/mcb.4.8.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Thomas C. A., Jr Recombination of DNA molecules. Prog Nucleic Acid Res Mol Biol. 1966;5:315–337. doi: 10.1016/s0079-6603(08)60237-8. [DOI] [PubMed] [Google Scholar]
  28. Wake C. T., Wilson J. H. Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2876–2880. doi: 10.1073/pnas.76.6.2876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wasmuth J. J., Vock Hall L. Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids. Cell. 1984 Mar;36(3):697–707. doi: 10.1016/0092-8674(84)90350-7. [DOI] [PubMed] [Google Scholar]
  30. Watt V. M., Ingles C. J., Urdea M. S., Rutter W. J. Homology requirements for recombination in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4768–4772. doi: 10.1073/pnas.82.14.4768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zieg J., Simon M. Analysis of the nucleotide sequence of an invertible controlling element. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4196–4200. doi: 10.1073/pnas.77.7.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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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