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To study the effects of in vivo DNA methylation, we have developed an inducible system to control the intracellular concentration of S-adenosyl-L-methionine (AdoMet). The product of the bacteriophage T3 AdoMet hydrolase-encoding gene (amh), which degrades AdoMet to L-homoserine and 5'-methylthioadenosine, was employed to lower AdoMet concentrations in vivo. The amh gene was placed downstream from the inducible tetA promoter of the Tn10 tetracycline regulon substituting for most of the tetA gene. Unlike in the original isolates [Hughes et al., J. Bacteriol. 169 (1987) 3625-2632], this promoter allows controlled expression. These constructs are stable and can be induced in a dose-dependent manner. The system is maximally induced 2-3 h after addition of the inducer, autoclaved chlortetracycline (cTc). DNA methylation in vivo was assessed 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 which 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 replication, gene repair and other cellular phenomena affected by methylation. |
