Tetracycline-mediated regulation of gene expression within the human cytomegalovirus genome.
To evaluate the utility of tetracycline gene regulation in the study of human cytomegalovirus gene functions, expression of luciferase under the control of tetracycline-regulatable promoters was studied following transient plasmid transfections and from within recombinant human cytomegalovirus genomes. The tetracycline-regulatable promoter PhCMV*-1 contains sequences from the human cytomegalovirus ie1/ie2 promoter and seven upstream tet operator sites which bind the activator protein tTA only in the absence of tetracycline (Gossen and Bujard (1992). Proc. Natl. Acad. Sci. USA 89, 5547-5551). Two modifications of PhCMV*-1 were also studied: P1129, in which the tet operator sites were reduced from seven to one; and P1125, in which human cytomegalovirus sequences were replaced by adenovirus major late promoter and terminal deoxynucleotidyltransferase initiator sequences. In transient assays, PhCMV*-1 and P1125 exhibited modest differential regulation but were strongly activated by viral infection. P1129 exhibited less viral activation and narrower regulation. In the viral genome, PhCMV*-1 exhibited regulation up to 7-fold during late times of infection, whereas P1125 displayed nearly 100-fold regulation. Regulation of P1125 was fully reversed within 12 to 24 h of adding or removing tetracycline. These results suggest that P1125 may provide sufficient conditional expression to effectively regulate human cytomegalovirus late genes.
McVoy
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Department of Pediatrics, Medical College of Virginia Campus of Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, Virginia, 23298-0163, USA. mmcvoy@hsc.vcu.edu
Mocarski
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D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics*; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral / Q000187:drug effects*; D016679:Genome, Viral; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators
D015534:Trans-Activators; D008156:Luciferases; D013752:Tetracycline