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. 1987 Feb 11;15(3):971–985. doi: 10.1093/nar/15.3.971

Recombination of DNAs in Xenopus oocytes based on short homologous overlaps.

E Grzesiuk, D Carroll
PMCID: PMC340502  PMID: 3029712

Abstract

Linear molecules of pBR322 and closely related plasmid DNAs were injected into Xenopus oocyte nuclei. Such molecules were degraded unless their ends were recombined. Non-homologous ends were joined rarely, if at all, but measurable recombination was supported by homologous sequences of less than 10 base pairs (bp). The efficiency of recombination increased as the length and degree of homology improved, in the range of about 8-20 bp. The homologous sequences had to be very close to the original molecular ends (within about 20 bp); internal homologies, even when they included better matches, were never used. These observations are best accommodated by a model of recombination which envisions exonucleolytic resection to expose homologous sequences, followed by annealing of single-stranded tails, tidying up and sealing of the new joint. Some of the recombined plasmids had novel tetracycline resistance genes; their properties give some insight into the function of the tet gene product.

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Selected References

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

  1. Ayares D., Chekuri L., Song K. Y., Kucherlapati R. Sequence homology requirements for intermolecular recombination in mammalian cells. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5199–5203. doi: 10.1073/pnas.83.14.5199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Brutlag D. L., Clayton J., Friedland P., Kedes L. H. SEQ: a nucleotide sequence analysis and recombination system. Nucleic Acids Res. 1982 Jan 11;10(1):279–294. doi: 10.1093/nar/10.1.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carroll D. Genetic recombination of bacteriophage lambda DNAs in Xenopus oocytes. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6902–6906. doi: 10.1073/pnas.80.22.6902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carroll D., Wright S. H., Wolff R. K., Grzesiuk E., Maryon E. B. Efficient homologous recombination of linear DNA substrates after injection into Xenopus laevis oocytes. Mol Cell Biol. 1986 Jun;6(6):2053–2061. doi: 10.1128/mcb.6.6.2053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chakrabarti S., Joffe S., Seidman M. M. Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells. Mol Cell Biol. 1985 Sep;5(9):2265–2271. doi: 10.1128/mcb.5.9.2265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  9. Doherty M. J., Morrison P. T., Kolodner R. Genetic recombination of bacterial plasmid DNA. Physical and genetic analysis of the products of plasmid recombination in Escherichia coli. J Mol Biol. 1983 Jul 5;167(3):539–560. doi: 10.1016/s0022-2836(83)80097-7. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Folger K., Thomas K., Capecchi M. R. Analysis of homologous recombination in cultured mammalian cells. Cold Spring Harb Symp Quant Biol. 1984;49:123–138. doi: 10.1101/sqb.1984.049.01.016. [DOI] [PubMed] [Google Scholar]
  12. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Lin F. L., Sperle K., Sternberg N. Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences. Proc Natl Acad Sci U S A. 1985 Mar;82(5):1391–1395. doi: 10.1073/pnas.82.5.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maniatis T. Targeting in mammalian cells. Nature. 1985 Sep 19;317(6034):205–206. doi: 10.1038/317205a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Peden K. W. Revised sequence of the tetracycline-resistance gene of pBR322. Gene. 1983 May-Jun;22(2-3):277–280. doi: 10.1016/0378-1119(83)90112-9. [DOI] [PubMed] [Google Scholar]
  20. Perucho M., Hanahan D., Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell. 1980 Nov;22(1 Pt 1):309–317. doi: 10.1016/0092-8674(80)90178-6. [DOI] [PubMed] [Google Scholar]
  21. Roth D. B., Porter T. N., Wilson J. H. Mechanisms of nonhomologous recombination in mammalian cells. Mol Cell Biol. 1985 Oct;5(10):2599–2607. doi: 10.1128/mcb.5.10.2599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Roth D. B., Wilson J. H. Relative rates of homologous and nonhomologous recombination in transfected DNA. Proc Natl Acad Sci U S A. 1985 May;82(10):3355–3359. doi: 10.1073/pnas.82.10.3355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rubnitz J., Subramani S. Rapid assay for extrachromosomal homologous recombination in monkey cells. Mol Cell Biol. 1985 Mar;5(3):529–537. doi: 10.1128/mcb.5.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Shen P., Huang H. V. Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics. 1986 Mar;112(3):441–457. doi: 10.1093/genetics/112.3.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Smith A. J., Berg P. Homologous recombination between defective neo genes in mouse 3T6 cells. Cold Spring Harb Symp Quant Biol. 1984;49:171–181. doi: 10.1101/sqb.1984.049.01.020. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. 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]
  36. Upcroft P., Carter B., Kidson C. Analysis of recombination in mammalian cells using SV40 genome segments having homologous overlapping termini. Nucleic Acids Res. 1980 Jun 25;8(12):2725–2736. doi: 10.1093/nar/8.12.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. 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]
  40. Wilson J. H., Berget P. B., Pipas J. M. Somatic cells efficiently join unrelated DNA segments end-to-end. Mol Cell Biol. 1982 Oct;2(10):1258–1269. doi: 10.1128/mcb.2.10.1258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wyllie A. H., Laskey R. A., Finch J., Gurdon J. B. Selective DNA conservation and chromatin assembly after injection of SV40 DNA into Xenopus oocytes. Dev Biol. 1978 May;64(1):178–188. doi: 10.1016/0012-1606(78)90069-6. [DOI] [PubMed] [Google Scholar]
  42. 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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