1887

Abstract

Summary

The location of a promoter (PF) in the dIII F region of the vaccinia virus genome was mapped by introducing deletions into this region of the DNA. Modified promoters were fused to the herpes simplex virus (HSV) thymidine kinase (TK) gene in plasmids facilitating the construction of recombinant vaccinia viruses, and promoter function was monitored by the ability of such plasmids to rescue TK vaccinia viruses from cells infected with TK virus. Deletions from the 3′ end of the promoter region produced mutants for which function was either not inhibited or abolished, allowing the 3′ promoter boundary to be defined to within 13 nucleotides. As indicated by the presence of the PF transcript in early RNA and the kinetics of HSV TK expression in recombinant vaccinia viruses, transcription from PF occurred primarily at early times during infection. The major transcript was initiated at a site within 20 nucleotides of the 3′ end of the promoter and nine bases upstream of the probable translation initiation codon. In one mutant for which a small but reproducible increase in promoter function was detected, the transcription start site was deleted. Nevertheless, transcription still appeared to begin at the equivalent position with respect to the promoter, despite the altered nucleotide sequence. The location of the start site for the PF transcript indicated that the HSV TK gene, inserted at the HI site following the promoter, was preceded by an initiation codon which could potentially attenuate expression of the inserted gene. Conversion of this ATG codon to TAG did not significantly improve HSV TK expression.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-68-9-2299
1987-09-01
2022-07-06
Loading full text...

Full text loading...

/deliver/fulltext/jgv/68/9/JV0680092299.html?itemId=/content/journal/jgv/10.1099/0022-1317-68-9-2299&mimeType=html&fmt=ahah

References

  1. Bertholet C., Drillien R., Wittek R. 1985; One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proceedings of the National Academy of Sciences U.S.A.: 822096–2100
    [Google Scholar]
  2. Boyle D. B., Coupar B. E. H. 1986; Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus. Journal of General Virology 67:1591–1600
    [Google Scholar]
  3. Boyle D. B., Coupar B. E. H., Both G. W. 1985; Multiple-cloning-site plasmids for the rapid construction of recombinant poxviruses. Gene 35:169–177
    [Google Scholar]
  4. Boyle D. B., Coupar B. E. H., Gibbs A. J., Seigman L. J., Both G. W. 1987; Fowlpox virus thymidine kinase: nucleotide sequence and relationships to other thymidine kinases. Virology 156:355–365
    [Google Scholar]
  5. Chakrabarti S., Brechling K., Moss B. 1985; Vaccinia virus expression vector: coexpression of p-galactosidase provides visual screening of recombinant virus plaques. Molecular and Cellular Biology 5:3403–3409
    [Google Scholar]
  6. Cochran M. A., Puckett C., Moss B. 1985; In vitromutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals. Journal of Virology 54:30–37
    [Google Scholar]
  7. Coupar B. E. H., Andrew M. E., Both G. W., Boyle D. B. 1986; Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response. European Journal of Immunology 16:1479–1487
    [Google Scholar]
  8. Dales S., Pogo B. G. T. 1981 Biology of Poxviruses New York: Springer-Verlag;
    [Google Scholar]
  9. Hanggi M., Bannwarth W., Stunnenberg H. G. 1986; Conserved TAAAT motif in vaccinia late promoters: overlapping TATA box and site of transcription initiation. EMBO Journal 5:1071–1076
    [Google Scholar]
  10. Jamieson A. T., Subak-Sharpe J. H. 1974; Biochemical studies on the herpes simplex virus-specified deoxypyrimidine kinase activity. Journal of General Virology 24:481–492
    [Google Scholar]
  11. Kozak M. 1984; Selection of initiation sites by eucaryotic ribosomes: effect of inserting AUG triplets upstream from the coding sequence for preproinsulin. Nucleic Acids Research 12:3873–3893
    [Google Scholar]
  12. Kozak M. 1986; Point mutations define a sequence flanking the AUG initiation codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292
    [Google Scholar]
  13. Liu C. C., Simonsen C. C. 1984; Initiation of translation at internal AUG codons in mammalian cells. Nature; London: 30982–85
    [Google Scholar]
  14. Mackett M., Smith G. L. 1986; Vaccinia virus expression vectors. Journal of General Virology 67:2067–2082
    [Google Scholar]
  15. Mackett M., Smith G. L., Moss B. 1982; Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proceedings of the National Academy of Sciences U.S.A.: 797415–7419
    [Google Scholar]
  16. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  17. Maxam A. M., Gilbert W. 1977; A new method for sequencing DNA. Proceedings of the National Academy of Sciences U.S.A.: 74560–564
    [Google Scholar]
  18. Nakano E., Panicali D., Paloletti E. 1982; Molecular genetics of vaccinia virus: demonstration of marker rescue. Proceedings of the National Academy of Sciences U.S.A.: 791593–1596
    [Google Scholar]
  19. Nisbet I. T., Beilharz M. W. 1985; Simplified DNA manipulations based on in vitromutagenesis. Gene Analysis Techniques 2:23–29
    [Google Scholar]
  20. Panicali D., Paoletti W. 1982; Construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proceedings of the National Academy of Sciences U.S.A.: 794927–4931
    [Google Scholar]
  21. Panicali D., Davis S. W., Weinberg R. L., Paoletti W. 1983; Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin. Proceedings of the National Academy of Sciences U.S.A.: 80:5364–5368
    [Google Scholar]
  22. Paoletti E., Lipinskas B. R., Samsonoff C., Mercer S., Panicali D. 1984; Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D. Proceedings of the National Academy of Sciences U.S.A.: 81193–197
    [Google Scholar]
  23. Plucienniczak A., Schroeder E., Zettlmeissl G., Streeck R. E. 1985; Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome. Nucleic Acids Research 13:985–998
    [Google Scholar]
  24. Rhim J. S., Cho H. Y., Huebner R. J. 1975; Non-producer human cells induced BY murine sarcoma virus. International Journal of Cancer 15:23–29
    [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences U.S.A.: 745463–5467
    [Google Scholar]
  26. Shida H. 1986; Nucleotide sequence of the vaccinia virus hemagglutinin gene. Virology 150:451–462
    [Google Scholar]
  27. Vassef A., Mars M., Dru A., Plucienniczak A., Streeck R. E., Beaud G. 1985; Isolation of ds-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses. Journal of Virology 55:163–172
    [Google Scholar]
  28. Vieira J., Messing J. 1982; The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
    [Google Scholar]
  29. Wagner M. J., Sharp J. A., Summers W. C. 1981; Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type I. Proceedings of the National Academy of Sciences U.S.A.: 781441–1445
    [Google Scholar]
  30. Weir J. P., Moss B. 1983; Nucleotide sequence of the vaccinia virus thymidine kinase gene and the nature of spontaneous frameshift mutations. Journal of Virology 46:530–537
    [Google Scholar]
  31. Weir J. P., Moss B. 1987; Determination of the transcriptional regulatory region of a vaccinia virus late gene. Journal of Virology 61:75–80
    [Google Scholar]
  32. Weir J. P., Bajszar G., Moss B. 1982; Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA. Proceedings of the National Academy of Sciences U.S.A.: 791210–1214
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-68-9-2299
Loading
/content/journal/jgv/10.1099/0022-1317-68-9-2299
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error