Effects of methylation of the β-galactosidase genome upon in vitro synthesis of β-galactosidase

A template DNA from phage λh80dlacp s coding for the in vitro synthesis of β-galactosidase was used to study the effect of DNA methylation by the alkylating agent, dimethyl sulfate (DMS). Increasing the levels of DMS up to 50 M concentration in the incubation medium led to an increase of DNA methylation. When incubated for 10 min at 37°C, 3–4% of nucleotides were methylated. The increase was linear to about 0.6% nucleotide methylation level. A higher yield was obtained at 37°C incubation temperature than at 20‡C. Methylation of λh80dlacp s DNA alone without methylation of other factors in the incubation mixture caused inhibition of the systhesis of β-galactosidase in vitro. Increasing levels of DNA methylation caused greater inhibition of the newly synthesized enzyme activity. Total protem and RNA synthesis was inhibited by the methylated DNA to a much lesser extent than the inhibition of enzyme activity. When the level of nueleotide methylation was 0.74%, only 2% of enzyme activity remained, but total protem and RNA synthetic activities were found to be 72% and 44%, respectively.