A NOVEL TN10 TETRACYCLINE REGULON SYSTEM CONTROLLING EXPRESSION OF THE BACTERIOPHAGE-T3 GENE ENCODING S-ADENOSYL-L-METHIONINE HYDROLASE

To study the effects of in vivo DNA methylation, we have developed an inducible system to control the intracellular concentration of S-adeno syl-L-methionine (AdoMet). The product of the bacteriophage T3 AdoMet hydrolase-encoding gene (amh), which degrades AdoMet to L-homoserine a nd 5'-methylthioadenosine, was employed to lower AdoMet concentrations in vivo. The amh gene was placed downstream from the inducible tetA p romoter of the Tn10 tetracycline regulon substituting for most of the tetA gene. Unlike in the original isolates [Hughes et al., J. Bacterio l. 169 (1987) 3625-2632], this promoter allows controlled expression. These constructs are stable and can be induced in a dose-dependent man ner. The system is maximally induced 2-3 h after addition of the induc er, autoclaved chlortetracycline (cTc). DNA methylation in vivo was as sessed in this model system by BamHI cleavage of plasmid DNA isolated from cells cotransformed by two compatible plasmids, one carrying the inducible amh gene, the other M.BamHII methyltransferase encoding gene . The induction of amh decreased the intracellular pool of AdoMet whic h M.BamHII requires as a cofactor. Under these conditions, there is a decrease in DNA methylation. The unmethylated DNA is assayed by BamHI cleavage. This system will be useful for studying transcription, DNA r eplication, gene repair and other cellular phenomena affected by methy lation.