Reversion of mutations in the thymidine kinase gene in herpes simplex viruses resistant to phosphonoacetate

Abstract

Mutations in the DNA polymerase locus of phage, bacteria, and eukaryotic may change the mutation rates at other loci of the genome. We used resistance to phosphonoacetate to select mutants of herpes simplex virus with mutated DNA polymerase and then determined the reversion frequency of viral thymidine kinase mutation in mutants and recombinants. The results obtained indicate that mutations causing resistance to phosphonoacetate do not affect the mutation rate of the viral genes. This finding is consistent with the existence of two functional regions in the DNA polymerase molecule, one involving the pyrophosphate acceptor site and responsible for resistance to phosphonoacetate and another involved in the editing ability and recognition specificity of the enzyme.

Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA

“…The optimal amount of carrier DNA was determined to be 20 Time of DNA addition. Only small differences were found when the time ofplasmid DNA addition was varied from 30 lowest value obtained at 30 min and the maximum obtained at 2 hr was no more than 2-fold. Little or no effect on the yield of TKV virus was noted when a 2-min boost with 20% glycerol was given 4 hr after transfection (31).…”

“…TK assays were performed essentially as described by Hruby and Ball (23 (30), that these cells cannot tolerate methotrexate for more than two days. Nevertheless, a vaccinia virus plaque assay could be completed within this time period provided a liquid overlay was used.…”