Effects of methylation of the beta-galactosidase genome upon in vitro synthesis of beta-galactosidase.

A template DNA from phage lambdah80dlacp5 coding for the in vitro synthesis of beta-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 mM concentration in the incubation medium led to an increase of DNA methylation. When incubated for 10 min at 37 degrees 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 degrees C incubation temperature than at 20 degrees C. Methylation of lambdah80dlacp5 DNA alone without methylation of other factors in the incubation mixture caused inhibition of the synthesis of beta-galactosidase in vitro. Increasing levels of DNA methylation caused greater inhibition of the newly synthesized enzyme activity. Total protein and RNA synthesis was inhibited by the methylated DNA to a much lesser extent than the inhibition of enzyme activity. When the level of nucleotide methylation was 0.74%, only 2% of enzyme activity remained, but total protein and RNA synthetic activities were found to be 72% and 44%, respectively.