Transfection with the isolated herpes simplex virus thymidine kinase genes. I. Minimal size of the active fragments from HSV-1

We have defined the minimal size and physical map locations in the genomes of both herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) for DNA sequences capable of conferring stable biochemical transformation u der thymidine kinase (TK) selection. The experiments involved transfection of Ltk- cells with either isolated virus DNA fragments or cloned pBR322 plasmids containing the 3.5 kilobase (kb) BamHI-O fragment from HSV-I(MP) or the 5.6 kb SalI-G fragment from HSV-2(333). Mapping of restriction enzyme sites within these cloned DNAs, followed by assays for colony formation in HAT medium after transfection with cleaved DNA, localized the biologically active TK-transforming sequences tolie between coordinates 0.300 and 0.313 in HSV- 1 and between 0.303 and 0.315 in HSV-2. Experiments with a series of cloned plasmids containing deletions of the BamHI-O fragment towards either the 3'- or 5'-ends of the TK gene indicated that the sequences required for stable HSV-1 TK transformation lay within a 1600 base pair (bp) region at 0.303 to 0.313 map units. An internal deletion mutant plasmid, selected by a novel bacterial transfection assay for the absence of the KpnI site at 0.308, also failed to rescu