Enzymatic analysis of 5-methylcytosine content in eukaryotic DNA. Study of intracellular Simian Virus 40 DNA

An enzymatic method is described for the analysis of the content of 5-methylcytosine in the DNA of eukaryotic cells. The conventional acid hydrolysis procedure was found to result in variable deamination of 5-methylcytosine into thymine, making the exact quantitation difficult. We applied the enzymatic method for the analysis of a simple eukaryotic viral genome. Viral and cell DNA in Simian Virus 40-infected monkey kidney cells was labeled with [methyl-3H]methionine in vivo and their 5-methylcytosine content was analyzed. The content of 5-methylcytosine per thymine in the total intracellular viral DNA was found to be less than 10(-4) of the host DNA. The implication of this result with regard to eukaryotic gene expression is discussed.