HYDROXYLAMINE MUTAGENESIS OF HSV DNA AND DNA FRAGMENTS - INTRODUCTION OF MUTATIONS INTO SELECTED REGIONS OF THE VIRAL GENOME

Procedures for the induction and isolation of temperature-sensitive (ts) mutants by in vitro mutagenesis of purified HSV DNA and DNA fragments have been developed. In order to establish conditions for the mutagenesis of viral DNA fragments with hydroxylamine (HA), virion-associated and intact DNA were mutagenized first. Under the conditions of mutagenesis employed, HA was shown to be a more powerful mutagen of purified DNA than of virion-associated DNA. Mutagenesis of intact DNA resulted in the identification of three new complementation groups. A mixture of EcoRI fragments A and B was next mutagenized. Mutations in DNA fragments were recovered by cotransfection with intact, wild-type DNA. Six ts mutants were isolated from the progeny of the mixed infection. Five of these mutants were analyzed further. One mutant, ts508, failed to complement tsB2 which had previously been shown to lie in fragment B. Like tsB2 and other members of this group, ts508 is DNA-. The remaining four HA-induced mutants constitute three new DNA+ complementation groups. Marker rescue experiments indicate that mutants in two of these groups lie in fragment A. © 1979.