1887

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Volume 68, Issue 9

Effect of Mutations in a Vaccinia Virus Early Promoter Region Monitored by Herpes Simplex Virus Thymidine Kinase Expression in Recombinant Vaccinia Virus Free

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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): HSV thymidine kinase , in vitro mutations and vaccinia virus early promoter
© Society for General Microbiology 1987
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1987-09-01
2024-05-07
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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. 68, 2299 (1987); https://doi.org/10.1099/0022-1317-68-9-2299
/content/journal/jgv/10.1099/0022-1317-68-9-2299
/content/journal/jgv/10.1099/0022-1317-68-9-2299
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