Biochemical transformation of LM(TK −) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Abstract

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK −)cells to the TK + phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 m M thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK −)/TF pMH110 E2 and LM(TK −)/TF pMH110 Hc2] with [ 35S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK −) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.