Assay of Herpes Simplex Virus Thymidine Kinase Allows Analysis of Eukaryotic Transcriptional Control Signals: The characteristics of a general assay for eukaryote transcription- control sequences using the herpes simplex virus (HSV) thjmiidine kinase (tk) gene is described. After transfection of cultured cells with tk-(tk) containing recombinant plasmids, two assays were used to measure gene expression: short term or transient levels of TK enzyme activity, and the frequency 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, simian virus 40 and Moloney murine sarcoma virus (MoMuSV) genomes results in reactivation of gene expression. The presence of enhancers 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 further promoter sequence whose activity equalled that of the endogenous promoter. Insertion of the LTR promoter between the LTR enhancer and the 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 may be mediated by transcriptional control of 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 enzyme activity. It is proposed 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.

Characterisation of Transcriptional Control Signals within the Short Repeat Region of the HSV-1 Genome by Assay of HSV-1 TK: The promoter sequences controlling expression of the herpes virus immediate early (IE) genes Vmw 12 (IE mRNA 5) and Vmw 175 (IE mRNA 3) have been identified using the assay system described. The Vmw 12 promoter has been Located within a Sau 3A fragment which extends approximately 430 bp 5' to the IE mRNA 5 cap site. IE mRNA 5 is spliced (Watson et al 1981a; Rixon and Clements 1982), however, removal of the splice signals does not affect the level of gene expression when the Vmw 12 promoter is tested in a expression assay. The Vmw 17 5 promoter has been located within a Sma I - Bam HI fragment which extends 331 bp 5' to the IE mRNA 3 cap site. While residual promoter activity is contained within 110 bp of the cap site, DNA sequences essential for efficient gene expression have been located further upstream within the contiguous 221 bp sequence. An enhancer type activity has been identified within this region of the Vmw 175 promoter. The enhancer activity is stimulated by up to tenfold after super infection with tk, a temperature sensitive mutant of HSV-1. Recent experiments show the induction of enhancer activity is independent of de novo protein synthesis showing that stimulation or 'trans activation' is effected by a component of the virion. It is proposed that trans-regulation of IE mRNA 3 is mediated through an enhancer type sequence. Identification of Promoter Signals within the Human Genome Hind III cleaved total human DNA was cloned at random into a promoter- less containing vector. Two reccmbinant clones were subsequently isolated which reactivate tk gene expression and therefore appear to contain human promoter signals. Insertion of Chimeric DNA Containing the TK Gene into the TK Locus of HSV-1 by Homologous Recombination A chimeric gene containing a molecular linker insertion into the untranslated leader of the gene was inserted by non-selective homologous recombination into the tk locus of HSV-1.