The 5′-cytosine in CCGG1is methylated in two eukaryotic DNAS and Msp I is sensitive to methylatlon at this site

Novikoff rat hepatoma and bovine liver DNAs were digested with Msp I or Hpa II. Restriction fragments were end-labeled using alpha-32P-dCTP and the Klenow fragment of E. coli DNA polymerase I and then digested to 2'-deoxyribonucleoside-3'-monophosphates using micrococcal nuclease and spleen phosphodiesterase. Mononucleotides were separated by two-dimensional thin layer chromatography, localized by radioautography, and the 32P-label quantitated by scintillation spectrometry. This method, based on known specificities of Msp I and Hpa II, shows that CCGG, CMGG, and MCGG (M refers to 5-methylcytosine) occur at frequencies of 89.6%, 1.4%, and 9.0%, respectively, in the rat DNA and at 41.6%, 48.3%, and 10.0%, respectively, in the bovine DNA. 32P recovery in 3'-5-MedCMP from end-labeled Msp I digests was negligible compared to recovery from Hpa II digests. Hence, Msp I is sensitive to methylation at the 5' cytosine in the sequence CCGG.