Transactivation of the HIV promoter by Tat can be estimated by a bacterial blue-white color system.
We describe an assay system which allows easy quantitation of transactivation of the human immunodeficiency virus (HIV) promoter by the viral Tat protein. We make use of the novel expression assay for the study of transcriptional activators [Rusconi et al., Gene 89 (1990) 211-221]. After transfection of a reference plasmid, a Tat expression plasmid, a plasmid in which the expression of simian virus 40 (SV40) large T antigen is driven by the HIV promoter and a replicator plasmid containing an SV40 ori into mammalian cells, low Mr DNA is shuttled back into Escherichia coli. Transactivation is quantitated by comparing the number of white colonies (due to the replicator plasmid) in presence and absence of Tat to the number of blue colonies (due to the reference plasmid). At high copy numbers of transfected reporter plasmid the system was saturated with respect to large T antigen and not accessible to transactivation by the viral Tat protein. Gradual decrease of the concentration of the HIV-promoter-containing plasmid resulted in continuous improvement of transactivation of this promoter. The demonstration of a 200-fold stimulation of the HIV-1 promoter indicates the sensitivity of the assay and its general applicability to analyse the interplay between a transacting factor and the responsive DNA/RNA sequences.
Wöllisch
E M
EM
Boehringer Mannheim GmbH, Abteilung für DNS-Neukombination, Penzberg, F.R.G.
Rusconi
S
S
Weidle
U H
UH
D000818:Animals; D015139:Blotting, Southern; D004247:DNA / Q000096:biosynthesis*; D015967:Gene Expression Regulation, Viral; D015756:Genes, tat / Q000502:physiology*; D006678:HIV / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D066298:In Vitro Techniques; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004247:DNA