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. 1988 Nov 11;16(21):10267–10282. doi: 10.1093/nar/16.21.10267

The use of beta-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses.

J P Weir 1, P R Narayanan 1
PMCID: PMC338851  PMID: 2848220

Abstract

The expression of Herpes Simplex Virus 1 (HSV-1) glycoprotein C (gC), a well defined herpesvirus late gene, was studied by linking the promoter-regulatory region of this gene to the coding sequences for the bacterial enzyme, beta-galactosidase (beta-gal). A chimeric gene, containing the beta-gal gene under the control of gC sequences from -1350 to +30 relative to the mRNA start site, was inserted by homologous recombination into the thymidine kinase (TK) locus of the HSV-1 genome. Selection of the TK- recombinant virus by plaque assay was facilitated by addition of a beta-gal indicator to the agarose overlay. Recombinant virus containing the gC promoter-beta-gal chimeric gene faithfully expressed beta-gal as a viral late gene, as shown by the absence of beta-gal expression when viral DNA replication was inhibited with phosphonoacetic acid. In contrast, the inhibition of viral DNA replication had no effect on the expression of beta-gal when the beta-gal gene was under the control of the early HSV-1 TK promoter in a separate recombinant virus. Analysis of recombinant viruses containing 5' to 3' deletions in the gC regulatory region revealed no apparent difference in beta-gal expression as deletions extended from -1350 to -109 base-pairs (bp) before the RNA start site, demonstrating that sequences between -109 and +30 are sufficient for regulated gC expression in the viral genome. Analysis of the mRNA made by these recombinant viruses confirmed the results of the beta-gal assays, and demonstrated that the transcriptional start sites of the gC promoter-beta-gal chimeric genes were the same as the start site of the gC gene.

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Selected References

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