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. 1981 Sep;39(3):733–745. doi: 10.1128/jvi.39.3.733-745.1981

Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs.

J A Cooper, R Wittek, B Moss
PMCID: PMC171307  PMID: 6270348

Abstract

Physical, transcriptional, and translational maps of an EcoRI fragment located between 15,800 and 20,600 base pairs from the left end of the vaccinia virus genome were prepared. Major polypeptides with molecular weights of 14,000 (14K polypeptide), 32,000 and 38,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA made in the presence of cycloheximide and selected by hybridization to lambda recombinant DNA containing the EcoRI fragment. With early RNA made in the presence of cytosine arabinoside, an inhibitor of DNA replication, the polypeptide pattern was similar except for quantitative differences in which less 38K polypeptide was detected as a translation product. With late RNA, isolated 6 h after infection without inhibitors, only traces of the early translation products were found and a new 40K polypeptide was detected. The size of the mRNA's for the 14K, 32K, and 38K polypeptides were determined to be approximately 760,880, and 1,150 nucleotides, respectively, by several independent procedures. Several large early RNAs not shown to code for any additional translation products were also detected. The size of the late message for the 40K polypeptide varied from 920 to 3,100 nucleotides. This heterogeneity appeared to be a general property of vaccinia virus late mRNA's. No evidence of RNA splicing was obtained by analysis of RNA-DNA hybrids after nuclease S1 treatment. Further analyses using separated recombinant DNA strands and restriction fragments indicated that all mRNA's were encoded by the leftward-reading DNA strand and at least two were overlapping. Since early and late mRNA's were encoded by the same DNA strand, the possibility of temporal regulation by transcriptional strand switching was eliminated. In conjunction with previous studies, a transcriptional map of the left 20,600 base pairs of the vaccinia virus genome was derived.

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Selected References

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

  1. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boone R. F., Parr R. P., Moss B. Intermolecular duplexes formed from polyadenylylated vaccinia virus RNA. J Virol. 1979 Apr;30(1):365–374. doi: 10.1128/jvi.30.1.365-374.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colby C., Jurale C., Kates J. R. Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro. J Virol. 1971 Jan;7(1):71–76. doi: 10.1128/jvi.7.1.71-76.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooper J. A., Moss B. In vitro translation of immediate early, early, and late classes of RNA from vaccinia virus-infected cells. Virology. 1979 Jul 30;96(2):368–380. doi: 10.1016/0042-6822(79)90095-3. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A., Wittek R., Moss B. Hybridization selection and cell-free translation of mRNA's encoded within the inverted terminal repetition of the vaccinia virus genome. J Virol. 1981 Jan;37(1):284–294. doi: 10.1128/jvi.37.1.284-294.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garon C. F., Barbosa E., Moss B. Visualization of an inverted terminal repetition in vaccinia virus DNA. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4863–4867. doi: 10.1073/pnas.75.10.4863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gershowitz A., Boone R. F., Moss B. Multiple roles for ATP in the synthesis and processing of mRNA by vaccinia virus: specific inhibitory effects of adenosine (beta,gamma-imido) triphosphate. J Virol. 1978 Aug;27(2):399–408. doi: 10.1128/jvi.27.2.399-408.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harper J. M., Parsonage M. T., Pelham H. R., Darby G. Heat inactivation of vaccinia virus particle-associated functions: properties of heated particles in vivo and in vitro. J Virol. 1978 Jun;26(3):646–659. doi: 10.1128/jvi.26.3.646-659.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lowery-Goldhammer C., Richardson J. P. An RNA-dependent nucleoside triphosphate phosphohydrolase (ATPase) associated with rho termination factor. Proc Natl Acad Sci U S A. 1974 May;71(5):2003–2007. doi: 10.1073/pnas.71.5.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nevins J. R., Joklik W. K. Poly (A) sequences of vaccinia virus messenger RNA: nature, mode of addition and function during translation in vitra and in vivo. Virology. 1975 Jan;63(1):1–14. doi: 10.1016/0042-6822(75)90365-7. [DOI] [PubMed] [Google Scholar]
  14. Oda K. I., Joklik W. K. Hybridization and sedimentation studies on "early" and "late" vaccinia messenger RNA. J Mol Biol. 1967 Aug 14;27(3):395–419. doi: 10.1016/0022-2836(67)90047-2. [DOI] [PubMed] [Google Scholar]
  15. Paoletti E. High molecular weight virion-associated RNA of vaccinia. A possible precursor to 8 to 12 S mRNA. J Biol Chem. 1977 Feb 10;252(3):872–877. [PubMed] [Google Scholar]
  16. Paoletti E., Lipinskas B. R. The role of ATP in the biogenesis of vaccinia virus mRNA in vitro. Virology. 1978 Jun 15;87(2):317–325. doi: 10.1016/0042-6822(78)90137-x. [DOI] [PubMed] [Google Scholar]
  17. Smith H. O., Birnstiel M. L. A simple method for DNA restriction site mapping. Nucleic Acids Res. 1976 Sep;3(9):2387–2398. doi: 10.1093/nar/3.9.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Venkatesan S., Gershowitz A., Moss B. Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus. J Biol Chem. 1980 Feb 10;255(3):903–908. [PubMed] [Google Scholar]
  19. Venkatesan S., Moss B. In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites. J Virol. 1981 Feb;37(2):738–747. doi: 10.1128/jvi.37.2.738-747.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wittek R., Barbosa E., Cooper J. A., Garon C. F., Chan H., Moss B. Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs. Nature. 1980 May 1;285(5759):21–25. doi: 10.1038/285021a0. [DOI] [PubMed] [Google Scholar]
  22. Wittek R., Cooper J. A., Barbosa E., Moss B. Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition. Cell. 1980 Sep;21(2):487–493. doi: 10.1016/0092-8674(80)90485-7. [DOI] [PubMed] [Google Scholar]
  23. Wittek R., Cooper J. A., Moss B. Transcriptional and translational mapping of a 6.6-kilobase-pair DNA fragment containing the junction of the terminal repetition and unique sequence at the left end of the vaccinia virus genome. J Virol. 1981 Sep;39(3):722–732. doi: 10.1128/jvi.39.3.722-732.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wittek R., Menna A., Müller H. K., Schümperli D., Boseley P. G., Wyler R. Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA. J Virol. 1978 Oct;28(1):171–181. doi: 10.1128/jvi.28.1.171-181.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wittek R., Moss B. Tandem repeats within the inverted terminal repetition of vaccinia virus DNA. Cell. 1980 Aug;21(1):277–284. doi: 10.1016/0092-8674(80)90135-x. [DOI] [PubMed] [Google Scholar]

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