FIG. 1.
Construction, Southern blot analysis, and one-step growth curve of KOS6β relative to wild-type strain KOS. (A) Construction of KOS6β. (i) Diagram of the HSV-1 genome (not drawn to scale) showing the unique long (UL) and unique short (US) regions of the genome, each flanked by inverted repeat sequences (ab and b′a′ [UL] and a′c′ and ca [US], respectively) shown as open boxes. (ii) Diagram of pUIC relative to the locations of the UL49 and UL50 genes in the cloned, subgenomic PvuII-PstI fragment. This plasmid contains a unique BglII site in the intergenic region between the UL49 and UL50 genes. Relevant restriction enzyme cleavage sites and map distances presented in base pairs are indicated. (iii) Diagram of pUIClacZ. The BamHI subfragment of plasmid pD6p containing the ICP6 promoter and E. coli lacZ protein coding sequence (open box) was cloned into the unique BglII site in pUIC. (B) Southern blot comparing KpnI digests of the UL49- and UL50-coding sequences of KOS and KOS6β DNA obtained from infected Vero cells by using 32P-labeled pUIClacZ as the probe (Fig. 1A, diagram iii). Expected sizes of fragments (in kilobases) are indicated to the left of the gel. (C) One-step growth curves of KOS6β and KOS in Vero cells at a multiplicity of 5 PFU per cell. Infectious virus was measured by standard plaque assay on Vero cell monolayers.