Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1987 Jun;61(6):1788–1795. doi: 10.1128/jvi.61.6.1788-1795.1987

High-frequency homologous recombination in vaccinia virus DNA.

L A Ball
PMCID: PMC254181  PMID: 3573150

Abstract

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.

Full text

PDF
1788

Images in this article

1791Image
on p.1791
1792Image
on p.1792
1793Image
on p.1793
1793Image
on p.1793

Selected References

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

  1. Ball L. A., Young K. K., Anderson K., Collins P. L., Wertz G. W. Expression of the major glycoprotein G of human respiratory syncytial virus from recombinant vaccinia virus vectors. Proc Natl Acad Sci U S A. 1986 Jan;83(2):246–250. doi: 10.1073/pnas.83.2.246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buller R. M., Smith G. L., Cremer K., Notkins A. L., Moss B. Decreased virulence of recombinant vaccinia virus expression vectors is associated with a thymidine kinase-negative phenotype. 1985 Oct 31-Nov 6Nature. 317(6040):813–815. doi: 10.1038/317813a0. [DOI] [PubMed] [Google Scholar]
  3. Condit R. C., Motyczka A., Spizz G. Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus. Virology. 1983 Jul 30;128(2):429–443. doi: 10.1016/0042-6822(83)90268-4. [DOI] [PubMed] [Google Scholar]
  4. Davis D. B., Munyon W., Buchsbaum R., Chawda R. Virus type-specific thymidine kinase in cells biochemically transformed by herpes simplex virus types 1 and 2. J Virol. 1974 Jan;13(1):140–145. doi: 10.1128/jvi.13.1.140-145.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drillien R., Spehner D. Physical mapping of vaccinia virus temperature-sensitive mutations. Virology. 1983 Dec;131(2):385–393. doi: 10.1016/0042-6822(83)90506-8. [DOI] [PubMed] [Google Scholar]
  6. Ensinger M. J., Rovinsky M. Marker rescue of temperature-sensitive mutations of vaccinia virus WR: correlation of genetic and physical maps. J Virol. 1983 Nov;48(2):419–428. doi: 10.1128/jvi.48.2.419-428.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Esposito J., Condit R., Obijeski J. The preparation of orthopoxvirus DNA. J Virol Methods. 1981 Feb;2(3):175–179. doi: 10.1016/0166-0934(81)90036-7. [DOI] [PubMed] [Google Scholar]
  8. Hruby D. E., Ball L. A. Control of expression of the vaccinia virus thymidine kinase gene. J Virol. 1981 Nov;40(2):456–464. doi: 10.1128/jvi.40.2.456-464.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hruby D. E., Ball L. A. Mapping and identification of the vaccinia virus thymidine kinase gene. J Virol. 1982 Aug;43(2):403–409. doi: 10.1128/jvi.43.2.403-409.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hruby D. E., Maki R. A., Miller D. B., Ball L. A. Fine structure analysis and nucleotide sequence of the vaccinia virus thymidine kinase gene. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3411–3415. doi: 10.1073/pnas.80.11.3411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Isle H. B., Venkatesan S., Moss B. Cell-free translation of early and late mRNAs selected by hybridization to cloned DNA fragments derived from the left 14 million to 72 million daltons of the vaccinia virus genome. Virology. 1981 Jul 15;112(1):306–317. doi: 10.1016/0042-6822(81)90636-x. [DOI] [PubMed] [Google Scholar]
  12. Jones E. V., Moss B. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA. J Virol. 1984 Jan;49(1):72–77. doi: 10.1128/jvi.49.1.72-77.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mackett M., Smith G. L. Vaccinia virus expression vectors. J Gen Virol. 1986 Oct;67(Pt 10):2067–2082. doi: 10.1099/0022-1317-67-10-2067. [DOI] [PubMed] [Google Scholar]
  14. Morgan J. R., Cohen L. K., Roberts B. E. Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus. J Virol. 1984 Oct;52(1):206–214. doi: 10.1128/jvi.52.1.206-214.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nakano E., Panicali D., Paoletti E. Molecular genetics of vaccinia virus: demonstration of marker rescue. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1593–1596. doi: 10.1073/pnas.79.5.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Panicali D., Paoletti E. Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4927–4931. doi: 10.1073/pnas.79.16.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stott E. J., Ball L. A., Young K. K., Furze J., Wertz G. W. Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge. J Virol. 1986 Nov;60(2):607–613. doi: 10.1128/jvi.60.2.607-613.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Weir J. P., Bajszár G., Moss B. Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA. Proc Natl Acad Sci U S A. 1982 Feb;79(4):1210–1214. doi: 10.1073/pnas.79.4.1210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Weir J. P., Moss B. Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations. J Virol. 1983 May;46(2):530–537. doi: 10.1128/jvi.46.2.530-537.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES