Comparative analysis of vaccinia virus promoter activity in fowlpox and vaccinia virus recombinants.
A quantitative and qualitative comparison of vaccinia virus (VV) promoter activity in fowlpox virus (FPV) and VV recombinants was performed. The VV PL11 late promoter was used to express beta-galactosidase from the E. coli LacZ gene in FPV (FPV-LacZ) and VV (VV-LacZ) recombinants. Time courses of FPV-LacZ beta-galactosidase expression in chicken embryo skin (CES) cells demonstrated temporal regulation of the PL11 promoter with maximum enzyme activity nine- and four-fold lower than those obtained in VV-LacZ infected 143B and CES cells, respectively. The level of beta-galactosidase activity per LacZ DNA gene copy was determined for each recombinant and found to be greater for VV-LacZ than FPV-LacZ. The VV P7.5 early/late promoter was used to express the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene in FPV and VV recombinants. Northern blot analysis showed early Ecogpt RNA transcripts to be of defined lengths. Transcript size estimations mapped the termination sites to regions containing sequences associated with VV early transcript termination, providing supportive evidence for a common poxvirus early transcript termination signal. Late LacZ and Ecogpt transcripts were heterogeneous in length. S1 nuclease mapping of the 5'-ends of early and late Ecogpt RNA transcripts produced by FPV and VV recombinants showed transcription initiation occurred at the same sites in both poxviruses and corresponded to the regions previously identified as the early and late start sites of the P7.5 promoter. These results would indicate a high level of conservation in the expression and regulation of genes by poxviruses.
Prideaux
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Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra.
Kumar
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Boyle
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D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004274:DNA, Recombinant; D005587:Fowlpox virus / Q000201:enzymology / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D007763:Lac Operon; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004274:DNA, Recombinant; D012367:RNA, Viral; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001616:beta-Galactosidase