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. 1991 Dec 11;19(23):6619–6625. doi: 10.1093/nar/19.23.6619

Amplification of polyomavirus DNA sequences stably integrated in rat cells.

L St-Onge 1, M Bastin 1
PMCID: PMC329231  PMID: 1661409

Abstract

To investigate the mechanism by which the polyomavirus large T antigen (T-Ag) promotes amplification of integrated viral sequences, we constructed a rat cell line, Hy2-ts5, carrying two different inserts of polyomavirus DNA. The first insert, designated the middle T (pmt) locus, was devised to analyze homologous recombination between two defective copies of pmt lying 3.3 kb apart on the same chromosome. Reconstitution of a functional pmt by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. The second locus contained the polyomavirus large T (plt) gene carrying a temperature-sensitive mutation and producing a nonfunctional large T-Ag at 39 degrees C. A shift to the permissive temperature for as little as 24 h induced the production of a functional large T-Ag which, in turn, promoted homologous recombination in the pmt locus at a rate close to 1.0 per cell generation. The particularity of this system is that it allowed recombination products to be analyzed as early as a single cell doubling following the initial recombinational event. Amplification occurred by successive duplications of a discrete sequence in the viral insert. Unequal sister chromatid exchange was ruled out as the recombination mechanism promoted by large T-Ag. Instead, we proposed a model of nonconservative recombination involving mispairing between homologous 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. Asselin C., Vass-Marengo J., Bastin M. Mutation in the polyomavirus genome that activates the properties of large T associated with neoplastic transformation. J Virol. 1986 Jan;57(1):165–172. doi: 10.1128/jvi.57.1.165-172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett E. R., Naujokas M., Hassell J. A. Requirements for species-specific papovavirus DNA replication. J Virol. 1989 Dec;63(12):5371–5385. doi: 10.1128/jvi.63.12.5371-5385.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Botchan M., Topp W., Sambrook J. Studies on simian virus 40 excision from cellular chromosomes. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 2):709–719. doi: 10.1101/sqb.1979.043.01.079. [DOI] [PubMed] [Google Scholar]
  4. Bouchard L., Mathieu F., Bastin M. Polyoma large T can activate middle T expression by a hit-and-run mechanism. Oncogene. 1988 Apr;2(4):379–386. [PubMed] [Google Scholar]
  5. Colantuoni V., Dailey L., Basilico C. Amplification of integrated viral DNA sequences in polyoma virus-transformed cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3850–3854. doi: 10.1073/pnas.77.7.3850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Colantuoni V., Dailey L., Valle G. D., Basilico C. Requirements for excision and amplification of integrated viral DNA molecules in polyoma virus-transformed cells. J Virol. 1982 Aug;43(2):617–628. doi: 10.1128/jvi.43.2.617-628.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. FRIED M. ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF POLYOMA VIRUS. Virology. 1965 Apr;25:669–671. doi: 10.1016/0042-6822(65)90098-x. [DOI] [PubMed] [Google Scholar]
  8. Huberman J. A. New views of the biochemistry of eucaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase alpha. Cell. 1981 Mar;23(3):647–648. doi: 10.1016/0092-8674(81)90426-8. [DOI] [PubMed] [Google Scholar]
  9. Levinson G., Gutman G. A. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol Biol Evol. 1987 May;4(3):203–221. doi: 10.1093/oxfordjournals.molbev.a040442. [DOI] [PubMed] [Google Scholar]
  10. Liskay R. M., Stachelek J. L. Evidence for intrachromosomal gene conversion in cultured mouse cells. Cell. 1983 Nov;35(1):157–165. doi: 10.1016/0092-8674(83)90218-0. [DOI] [PubMed] [Google Scholar]
  11. Luria S. E., Delbrück M. Mutations of Bacteria from Virus Sensitivity to Virus Resistance. Genetics. 1943 Nov;28(6):491–511. doi: 10.1093/genetics/28.6.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Miller J., Bullock P., Botchan M. Simian virus 40 T antigen is required for viral excision from chromosomes. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7534–7538. doi: 10.1073/pnas.81.23.7534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mouchès C., Pasteur N., Bergé J. B., Hyrien O., Raymond M., de Saint Vincent B. R., de Silvestri M., Georghiou G. P. Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito. Science. 1986 Aug 15;233(4765):778–780. doi: 10.1126/science.3755546. [DOI] [PubMed] [Google Scholar]
  14. Otto E., Young J. E., Maroni G. Structure and expression of a tandem duplication of the Drosophila metallothionein gene. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6025–6029. doi: 10.1073/pnas.83.16.6025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pellegrini S., Dailey L., Basilico C. Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication. Cell. 1984 Apr;36(4):943–949. doi: 10.1016/0092-8674(84)90044-8. [DOI] [PubMed] [Google Scholar]
  16. Piché A., Bourgaux P. Resolution of a polyomavirus-mouse hybrid replicon: viral function required for recombination. J Virol. 1987 Mar;61(3):845–850. doi: 10.1128/jvi.61.3.845-850.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rubnitz J., Subramani S. Extrachromosomal and chromosomal gene conversion in mammalian cells. Mol Cell Biol. 1986 May;6(5):1608–1614. doi: 10.1128/mcb.6.5.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schaffhausen B., Benjamin T. L. Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro. J Virol. 1981 Oct;40(1):184–196. doi: 10.1128/jvi.40.1.184-196.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Schimke R. T. Gene amplification in cultured animal cells. Cell. 1984 Jul;37(3):705–713. doi: 10.1016/0092-8674(84)90406-9. [DOI] [PubMed] [Google Scholar]
  20. Schimke R. T. Gene amplification in cultured cells. J Biol Chem. 1988 May 5;263(13):5989–5992. [PubMed] [Google Scholar]
  21. Shah D. M., Horsch R. B., Klee H. J., Kishore G. M., Winter J. A., Tumer N. E., Hironaka C. M., Sanders P. R., Gasser C. S., Aykent S., Siegel N. R., Rogers S. G., Fraley R. T. Engineering herbicide tolerance in transgenic plants. Science. 1986 Jul 25;233(4762):478–481. doi: 10.1126/science.233.4762.478. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. St-Onge L., Bouchard L., Laurent S., Bastin M. Intrachromosomal recombination mediated by papovavirus large T antigens. J Virol. 1990 Jun;64(6):2958–2966. doi: 10.1128/jvi.64.6.2958-2966.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stark G. R. DNA amplification in drug resistant cells and in tumours. Cancer Surv. 1986;5(1):1–23. [PubMed] [Google Scholar]
  26. Stark G. R., Wahl G. M. Gene amplification. Annu Rev Biochem. 1984;53:447–491. doi: 10.1146/annurev.bi.53.070184.002311. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Zhu Z. Y., Veldman G. M., Cowie A., Carr A., Schaffhausen B., Kamen R. Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins. J Virol. 1984 Jul;51(1):170–180. doi: 10.1128/jvi.51.1.170-180.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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