Direct detection of methylated cytosine in DNA by use of the restriction enxyme Mspl

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction en-zyme Mspl known to be specific for this sequence. The methylation.of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with Mspl and its isoschizomer Hpall and analyzed by gel electrophoresis. A direct method for detection of 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with Mspl were radioactively labeled at their 5 ' ends and subsequently degraded to the corresponding 5'-deoxyTibonucleoside mono-phosphates. 5 methylcytidylic acid has been found in most of the 5 ' ends of Mspl fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric nethylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence