Characterization of eukaryotic transcriptional control signals by assay of herpes simplex virus type 1 thymidine kinase.
We describe the characteristics of a general assay for eukaryote transcription-control sequences using the herpes simplex virus (HSV) thymidine kinase (tk) gene. After transfection of cultured cells with tk-containing recombinant plasmids, two assays were used to measure gene expression: short term or transient levels of tk mRNA and TK enzyme activity, and the rate of biochemical transformation from a TK- to a TK+ phenotype in selective growth medium (HAT). Deletion of the endogenous tk promoter results in 500-fold inactivation of gene expression. Replacement with exogenous transcription-control sequences from the human epsilon globin, mouse beta major globin, simian virus 40 and Moloney murine sarcoma virus (MoMuSV) genomes results in reactivation of gene expression. The presence of enhancers or activators of gene expression can also be conveniently measured. The transient expression assay ranged over two orders of magnitude while the transformation assay was almost two orders of magnitude more sensitive using the same recombinants. Analysis of the transcription-control domains in the MoMuSV LTR sequences shows the presence of both an enhancer and a promoter whose activity equalled that of the tk endogenous promoter. Insertion of the LTR promoter between the LTR enhancer and the tk promoter had little effect on modulating gene expression, suggesting no absolute preference for proximal promoters by this element. The different levels of gene expression obtained appears to be mediated by transcriptional control of full-length tk mRNA. There was an apparent correlation between the results obtained with the transient expression and transformation assays. However, cultured transformed cells all contained roughly the same levels of tk DNA, tk mRNA and tk enzyme activity. We propose that initial expression levels have a major effect in determining the transformation efficiency but that additional genetic controls are superimposed in cells grown in selective HAT medium.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004274:DNA, Recombinant; D005786:Gene Expression Regulation; D005814:Genes, Viral; D005914:Globins / Q000235:genetics; D006801:Humans; D051379:Mice; D009876:Operon; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D012333:RNA, Messenger; D012367:RNA, Viral; D005914:Globins; D013937:Thymidine Kinase