Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1989 Feb;63(2):705–713. doi: 10.1128/jvi.63.2.705-713.1989

Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase.

C L Thompson 1, U Hooda-Dhingra 1, R C Condit 1
PMCID: PMC247742  PMID: 2911120

Abstract

We have mapped the temperature-sensitive (ts) lesions of three mutants, ts51, ts53, and ts65, and two other mutants, ts7 and ts20, to regions on the vaccinia virus genome that encode the 147- and 22-kilodalton subunits of the viral DNA-dependent RNA polymerase, respectively. Plasmid and bacteriophage clones from the HindIII J region and the region spanning the HindIII J-H junction were used in marker rescue experiments to map the mutations. Sequence analysis of the region encoding the 22-kilodalton subunit in the wild-type, ts7, and ts20 viruses revealed a single base change in the mutants compared with that in the wild-type virus. The identification of these RNA polymerase mutants provides us with tools to understand transcription and its regulation in vaccinia virus.

Full text

PDF
711

Images in this article

707Image
on p.707
708Image
on p.708
711Image
on p.711

Selected References

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

  1. Bajszár G., Wittek R., Weir J. P., Moss B. Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants. J Virol. 1983 Jan;45(1):62–72. doi: 10.1128/jvi.45.1.62-72.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baroudy B. M., Moss B. Purification and characterization of a DNA-dependent RNA polymerase from vaccinia virions. J Biol Chem. 1980 May 10;255(9):4372–4380. [PubMed] [Google Scholar]
  3. Bertholet C., Stocco P., Van Meir E., Wittek R. Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. EMBO J. 1986 Aug;5(8):1951–1957. doi: 10.1002/j.1460-2075.1986.tb04449.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broyles S. S., Moss B. Homology between RNA polymerases of poxviruses, prokaryotes, and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. Proc Natl Acad Sci U S A. 1986 May;83(10):3141–3145. doi: 10.1073/pnas.83.10.3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  6. Cochran M. A., Puckett C., Moss B. In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. J Virol. 1985 Apr;54(1):30–37. doi: 10.1128/jvi.54.1.30-37.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Condit R. C., Motyczka A. Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus. Virology. 1981 Aug;113(1):224–241. doi: 10.1016/0042-6822(81)90150-1. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Coupar B. E., Boyle D. B., Both G. W. Effect of in vitro mutations in a vaccinia virus early promoter region monitored by herpes simplex virus thymidine kinase expression in recombinant vaccinia virus. J Gen Virol. 1987 Sep;68(Pt 9):2299–2309. doi: 10.1099/0022-1317-68-9-2299. [DOI] [PubMed] [Google Scholar]
  10. Ensinger M. J. Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase. J Virol. 1987 Jun;61(6):1842–1850. doi: 10.1128/jvi.61.6.1842-1850.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ensinger M. J., Weir J. P., Moss B. Fine structure marker rescue of temperature-sensitive mutations of vaccinia virus within a central conserved region of the genome. J Virol. 1985 Dec;56(3):1027–1029. doi: 10.1128/jvi.56.3.1027-1029.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  13. Hooda-Dhingra U., Thompson C. L., Condit R. C. Detailed phenotypic characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase. J Virol. 1989 Feb;63(2):714–729. doi: 10.1128/jvi.63.2.714-729.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jones E. V., Puckett C., Moss B. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome. J Virol. 1987 Jun;61(6):1765–1771. doi: 10.1128/jvi.61.6.1765-1771.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mahr A., Roberts B. E. Arrangement of late RNAs transcribed from a 7.1-kilobase EcoRI vaccinia virus DNA fragment. J Virol. 1984 Feb;49(2):510–520. doi: 10.1128/jvi.49.2.510-520.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mahr A., Roberts B. E. Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment. J Virol. 1984 Feb;49(2):497–509. doi: 10.1128/jvi.49.2.497-509.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miner J. N., Weinrich S. L., Hruby D. E. Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster. J Virol. 1988 Jan;62(1):297–304. doi: 10.1128/jvi.62.1.297-304.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morrison D. K., Moyer R. W. Detection of a subunit of cellular Pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions. Cell. 1986 Feb 28;44(4):587–596. doi: 10.1016/0092-8674(86)90268-0. [DOI] [PubMed] [Google Scholar]
  19. Nevins J. R., Joklik W. K. Isolation and properties of the vaccinia virus DNA-dependent RNA polymerase. J Biol Chem. 1977 Oct 10;252(19):6930–6938. [PubMed] [Google Scholar]
  20. Niles E. G., Condit R. C., Caro P., Davidson K., Matusick L., Seto J. Nucleotide sequence and genetic map of the 16-kb vaccinia virus HindIII D fragment. Virology. 1986 Aug;153(1):96–112. doi: 10.1016/0042-6822(86)90011-5. [DOI] [PubMed] [Google Scholar]
  21. Plucienniczak A., Schroeder E., Zettlmeissl G., Streeck R. E. Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome. Nucleic Acids Res. 1985 Feb 11;13(3):985–998. doi: 10.1093/nar/13.3.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rohrmann G., Yuen L., Moss B. Transcription of vaccinia virus early genes by enzymes isolated from vaccinia virions terminates downstream of a regulatory sequence. Cell. 1986 Sep 26;46(7):1029–1035. doi: 10.1016/0092-8674(86)90702-6. [DOI] [PubMed] [Google Scholar]
  23. Rosel J. L., Earl P. L., Weir J. P., Moss B. Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. J Virol. 1986 Nov;60(2):436–449. doi: 10.1128/jvi.60.2.436-449.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Seto J., Celenza L. M., Condit R. C., Niles E. G. Genetic map of the vaccinia virus HindIII D Fragment. Virology. 1987 Sep;160(1):110–119. doi: 10.1016/0042-6822(87)90051-1. [DOI] [PubMed] [Google Scholar]
  26. Spencer E., Shuman S., Hurwitz J. Purification and properties of vaccinia virus DNA-dependent RNA polymerase. J Biol Chem. 1980 Jun 10;255(11):5388–5395. [PubMed] [Google Scholar]
  27. Thompson C. L., Condit R. C. Marker rescue mapping of vaccinia virus temperature-sensitive mutants using overlapping cosmid clones representing the entire virus genome. Virology. 1986 Apr 15;150(1):10–20. doi: 10.1016/0042-6822(86)90261-8. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Weir J. P., Moss B. Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase. Virology. 1987 May;158(1):206–210. doi: 10.1016/0042-6822(87)90254-6. [DOI] [PubMed] [Google Scholar]
  30. Weir J. P., Moss B. Determination of the transcriptional regulatory region of a vaccinia virus late gene. J Virol. 1987 Jan;61(1):75–80. doi: 10.1128/jvi.61.1.75-80.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Yuen L., Moss B. Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6417–6421. doi: 10.1073/pnas.84.18.6417. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES