Physical mapping of temperature-sensitive mutations of herpes simplex virus type 1 using cloned restriction endonuclease fragments.

Abstract

Sequences from the whole of the HSV-1 strain 17 genome were cloned into bacterial plasmid vectors, with the exception of part of BamHI v which was deleted in all cloned DNAs spanning this region of the virus DNA. The cloned DNAs were used in intratypic marker rescue experiments to map temperature-sensitive (ts) mutations on to the virus genome. Since the sequences of these DNAs overlapped, any mutation could be rapidly assigned a physical map position. This approach is particularly useful for mapping spontaneous mutations and lesions induced by mutagenesis of whole virus DNA. In this study, we mapped ten ts mutations comprising eight different complementation groups. Five lesions, representing three different cistrons, were located within BglII k (map units 0.098 to 0.166), and three mapped within EcoRIf (map units 0.321 to 0.414), two of which were in previously unidentified cistrons of HSV-1 strain 17. One mutation analysed had a defect within the short repeat region and another had a mutation within EcoRI i (map units 0.632 to 0.720).