Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1982 Aug;43(2):403–409. doi: 10.1128/jvi.43.2.403-409.1982

Mapping and identification of the vaccinia virus thymidine kinase gene.

D E Hruby, L A Ball
PMCID: PMC256142  PMID: 6287024

Abstract

The thymidine kinase gene of vaccinia virus (VV) was mapped on the viral genome by using cloned fragments of the viral DNA to hybridize to early viral mRNA. Individual DNA fragments that represented about half of the viral genome were assayed, both for their ability to arrest the cell-free synthesis of active VV thymidine kinase and for their ability to select functional mRNA for the viral enzyme. Both activities were located in HindIII fragment J, which maps near the middle of VV DNA and contains about 2.6% of the genome (4,800 base pairs). This DNA fragment encodes four known early polypeptides, and to determine which of these was thymidine kinase, early VV mRNA was fractionated by sucrose gradient centrifugation and used to direct cell-free synthesis of the active enzyme. The thymidine kinase mRNA cosedimented with several species that encoded polypeptides in the molecular weight range 15,000 to 25,000. Hybridization of these mRNAs to HindIII-J DNA selected a message that directed the synthesis of thymidine kinase and a single polypeptide with an apparent molecular weight of 19,000. The native molecular weight of VV thymidine kinase is about 80,000, so these data indicate that, unlike thymidine kinase from several other sources, the active VV enzyme is probably a tetramer of 19,000-molecular-weight subunits.

Full text

PDF
409

Images in this article

407Image
on p.407
408Image
on p.408

Selected References

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

  1. Boone R. F., Moss B. Sequence complexity and relative abundance of vaccinia virus mRNA's synthesized in vivo and in vitro. J Virol. 1978 Jun;26(3):554–569. doi: 10.1128/jvi.26.3.554-569.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cabrera C. V., Esteban M., McCarron R., McAllister W. T., Holowczak J. A. Vaccinia virus transcription: hybridization of mRNA to restriction fragments of vaccinia DNA. Virology. 1978 May 1;86(1):102–114. doi: 10.1016/0042-6822(78)90011-9. [DOI] [PubMed] [Google Scholar]
  3. Chipchase M., Schwendimann F., Wyler R. A map of the late proteins of vaccinia virus. Virology. 1980 Aug;105(1):261–264. doi: 10.1016/0042-6822(80)90176-2. [DOI] [PubMed] [Google Scholar]
  4. DUBBS D. R., KIT S. ISOLATION AND PROPERTIES OF VACCINIA MUTANTS DEFICIENT IN THYMIDINE KINASE-INDUCING ACTIVITY. Virology. 1964 Feb;22:214–225. doi: 10.1016/0042-6822(64)90006-6. [DOI] [PubMed] [Google Scholar]
  5. Gallione C. J., Greene J. R., Iverson L. E., Rose J. K. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus N and NS proteins. J Virol. 1981 Aug;39(2):529–535. doi: 10.1128/jvi.39.2.529-535.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Geshelin P., Berns K. I. Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J Mol Biol. 1974 Oct 5;88(4):785–796. doi: 10.1016/0022-2836(74)90399-4. [DOI] [PubMed] [Google Scholar]
  7. Grady L. J., Paoletti E. Molecular complexity of vaccinia DNA and the presence of reiterated sequences in the genome. Virology. 1977 Jun 15;79(2):337–341. doi: 10.1016/0042-6822(77)90361-0. [DOI] [PubMed] [Google Scholar]
  8. Hardies S. C., Patient R. K., Klein R. D., Ho F., Reznikoff W. S., Wells R. D. Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter. J Biol Chem. 1979 Jun 25;254(12):5527–5534. [PubMed] [Google Scholar]
  9. Honess R. W., Watson D. H. Herpes simplex virus-specific polypeptides studied by polyacrylamide gel electrophoresis of immune precipitates. J Gen Virol. 1974 Feb;22(2):171–185. doi: 10.1099/0022-1317-22-2-171. [DOI] [PubMed] [Google Scholar]
  10. Hruby D. E., Ball L. A. Cell-free synthesis of enzymatically active vaccinia virus thymidine kinase. Virology. 1981 Sep;113(2):594–601. doi: 10.1016/0042-6822(81)90187-2. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Jungwirth C., Joklik W. K. Studies on "early" enzymes in HeLa cells infected with vaccinia virus. Virology. 1965 Sep;27(1):80–93. doi: 10.1016/0042-6822(65)90145-5. [DOI] [PubMed] [Google Scholar]
  14. Kit S., Jorgensen G. N., Liav A., Zaslavsky V. Purification of vaccinia virus-induced thymidine kinase activity from [35S]methionine-labeled cells. Virology. 1977 Apr;77(2):661–676. doi: 10.1016/0042-6822(77)90490-1. [DOI] [PubMed] [Google Scholar]
  15. MCAUSLAN B. R. THE INDUCTION AND REPRESSION OF THYMIDINE KINASE IN THE POXVIRUS-INFECTED HELA CELL. Virology. 1963 Nov;21:383–389. doi: 10.1016/0042-6822(63)90199-5. [DOI] [PubMed] [Google Scholar]
  16. Mackett M., Archard L. C. Conservation and variation in Orthopoxvirus genome structure. J Gen Virol. 1979 Dec;45(3):683–701. doi: 10.1099/0022-1317-45-3-683. [DOI] [PubMed] [Google Scholar]
  17. Paterson B. M., Roberts B. E., Kuff E. L. Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4370–4374. doi: 10.1073/pnas.74.10.4370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pellicer A., Robins D., Wold B., Sweet R., Jackson J., Lowy I., Roberts J. M., Sim G. K., Silverstein S., Axel R. Altering genotype and phenotype by DNA-mediated gene transfer. Science. 1980 Sep 19;209(4463):1414–1422. doi: 10.1126/science.7414320. [DOI] [PubMed] [Google Scholar]
  19. Pennington T. H. Vaccinia virus polypeptide synthesis: sequential appearance and stability of pre- and post-replicative polypeptides. J Gen Virol. 1974 Dec;25(3):433–444. doi: 10.1099/0022-1317-25-3-433. [DOI] [PubMed] [Google Scholar]
  20. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rose J. K., Gallione C. J. Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions. J Virol. 1981 Aug;39(2):519–528. doi: 10.1128/jvi.39.2.519-528.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  23. Thouless M. E., Wildy P. Deoxypyrimidine kinases of herpes simplex viruses types 1 and 2: comparison of serological and structural properties. J Gen Virol. 1975 Feb;26(2):159–170. doi: 10.1099/0022-1317-26-2-159. [DOI] [PubMed] [Google Scholar]
  24. 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]

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

RESOURCES