Functional expression of the Herpes simplex virus thymidine kinase gene in Escherichia coli K-12

The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance ( tet r) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK −) cells to TK + but is only weakly expressed in TK − bacteria, pMH110 not only efficiently transforms LM(TK −) cells to TK + but also enables TK − Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluoro-deoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [ 3H] dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [ 125I]deoxycytidine.