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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 under 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-1(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 to lie 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 rescue Ltk- cells. With the exception of cleavage at the StuI site at 0.303 in HSV-2, which reduced activity only eightfold, all cleavages that affected TK transformation reduced the efficiency at least 50-fold. A direct comparison of the HSV-1 and HSV-2 minimal transforming regions with the nucleotide sequence of the structural HSV-1 TK gene indicates that the HSV-2 StuI site lies 30 bp beyond the poly(A) addition site at the 3'-end of TK mRNA. On the other hand, cleavage at the SmaI site in HSV-1 TK, located 80 bp in front of the poly(A) addition point, abolishes colony formation. Comparison of the putative 5'-end of the HSV-2 TK gene defined by transfection assays, with a 250 bp non-transcribed region at the front of the HSV-1 TK gene, suggests that the promoter regions contain a much higher frequency of conserved cleavage sites than do the coding portions of the two genes. Direct nucleotide sequencing of the 5'-flanking sequences for HSV-2 TK confirmed that large portions of the two promoters possess greater than 95% sequence homology. At least 140 bp, but no more than 200 bp, of this 5'-promoter region are essential for efficient transfer and expression of the viral TK gene. Combining the results from HSV-1 and HSV-2, we conclude that a contiguous sequence of 1480 to 1540 bp is necessary to achieve at least 10% of the maximum transformation efficiency.

Transfection with the Isolated Herpes Simplex Virus Thymidine Kinase Genes. I. Minimal Size of the Active Fragments from HSV-1 and HSV-2