Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro

摘要

When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5′ flanking and intragenic sequences are deleted. The other set is deleted at the 3′ end of the gene, By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5′ and 3′ termini of the HSV tk gene.