Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1987 Oct;61(10):3152–3162. doi: 10.1128/jvi.61.10.3152-3162.1987

Molecular genetic analysis of a vaccinia virus gene with an essential role in DNA replication.

E Evans, P Traktman
PMCID: PMC255892  PMID: 3041037

Abstract

We have identified a gene encoded by vaccinia virus which is essential for DNA replication. The gene, located in the HindIII D fragment of the viral genome, is transcribed early after infection into two transcripts of 3.0 and 3.7 kilobases which share a 3' terminus. The lesions of three temperature-sensitive DNA replication mutants with defects in this gene have been localized by marker rescue with progressively smaller DNA fragments. We have determined by hybrid selection that the gene encodes an 82-kilodalton protein. An antibody has been prepared against this polypeptide and used to quantitate expression of the protein after infection with wild-type virus or with a viral mutant whose lesion maps within this gene. The temporal pattern of expression in the mutant is unaffected, but the product encoded by the mutant is significantly more thermolabile than the wild-type protein.

Full text

PDF

Images in this article

3156Image
on p.3156
3157Image
on p.3157
3158Image
on p.3158
3159Image
on p.3159
3160Image
on p.3160

Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer W. R., Ressner E. C., Kates J., Patzke J. V. A DNA nicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties. Proc Natl Acad Sci U S A. 1977 May;74(5):1841–1845. doi: 10.1073/pnas.74.5.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Ultraviolet mapping of the adenovirus 2 early promoters. Cell. 1977 Sep;12(1):45–55. doi: 10.1016/0092-8674(77)90184-2. [DOI] [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Campbell J. L. Eukaryotic DNA replication. Annu Rev Biochem. 1986;55:733–771. doi: 10.1146/annurev.bi.55.070186.003505. [DOI] [PubMed] [Google Scholar]
  6. Challberg M. D. A method for identifying the viral genes required for herpesvirus DNA replication. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9094–9098. doi: 10.1073/pnas.83.23.9094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Challberg M. D., Englund P. T. Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus. J Biol Chem. 1979 Aug 25;254(16):7812–7819. [PubMed] [Google Scholar]
  8. Chernos V. I., Belanov E. F., Vasilieva N. N. Temperature-sensitive mutants of vaccinia virus. I. Isolation and preliminary characterization. Acta Virol. 1978 Mar;22(2):81–90. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Drillien R., Spehner D., Kirn A. Complementation and genetic linkage between vaccinia virus temperature-sensitive mutants. Virology. 1982 Jun;119(2):372–381. doi: 10.1016/0042-6822(82)90096-4. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Earl P. L., Jones E. V., Moss B. Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3659–3663. doi: 10.1073/pnas.83.11.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ensinger M. J. Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR. J Virol. 1982 Sep;43(3):778–790. doi: 10.1128/jvi.43.3.778-790.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Foglesong P. D., Bauer W. R. Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. J Virol. 1984 Jan;49(1):1–8. doi: 10.1128/jvi.49.1.1-8.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Golini F., Kates J. R. Transcriptional and translational analysis of a strongly expressed early region of the vaccinia virus genome. J Virol. 1984 Feb;49(2):459–470. doi: 10.1128/jvi.49.2.459-470.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. JOKLIK W. K. The preparation and characteristics of highly purified radioactively labelled poxvirus. Biochim Biophys Acta. 1962 Aug 20;61:290–301. doi: 10.1016/0926-6550(62)90091-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Miller J. S., Paterson B. M., Ricciardi R. P., Cohen L., Roberts B. E. Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules. Methods Enzymol. 1983;101:650–674. doi: 10.1016/0076-6879(83)01046-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Morgan J. R., Roberts B. E. Organization of RNA transcripts from a vaccinia virus early gene cluster. J Virol. 1984 Aug;51(2):283–297. doi: 10.1128/jvi.51.2.283-297.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  31. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Shaffer R., Traktman P. Vaccinia virus encapsidates a novel topoisomerase with the properties of a eucaryotic type I enzyme. J Biol Chem. 1987 Jul 5;262(19):9309–9315. [PubMed] [Google Scholar]
  34. Slabaugh M. B., Mathews C. K. Hydroxyurea-resistant vaccinia virus: overproduction of ribonucleotide reductase. J Virol. 1986 Nov;60(2):506–514. doi: 10.1128/jvi.60.2.506-514.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Slabaugh M. B., Mathews C. K. Vaccinia virus-induced ribonucleotide reductase can be distinguished from host cell activity. J Virol. 1984 Nov;52(2):501–506. doi: 10.1128/jvi.52.2.501-506.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sridhar P., Condit R. C. Selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive DNA polymerase. Virology. 1983 Jul 30;128(2):444–457. doi: 10.1016/0042-6822(83)90269-6. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Traktman P., Baltimore D. Protease bypass of temperature-sensitive murine leukemia virus maturation mutants. J Virol. 1982 Dec;44(3):1039–1046. doi: 10.1128/jvi.44.3.1039-1046.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Traktman P., Sridhar P., Condit R. C., Roberts B. E. Transcriptional mapping of the DNA polymerase gene of vaccinia virus. J Virol. 1984 Jan;49(1):125–131. doi: 10.1128/jvi.49.1.125-131.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  43. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weaver R. F., Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Yuen L., Moss B. Multiple 3' ends of mRNA encoding vaccinia virus growth factor occur within a series of repeated sequences downstream of T clusters. J Virol. 1986 Oct;60(1):320–323. doi: 10.1128/jvi.60.1.320-323.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES