Use of restriction enzymes to study eukaryotic DNA methylation

Restriction enzymes HpaII, AvaI, HhaI and HaeII have been found to distinguish Xenopus laevis somatic (erythrocyte) rDNA† from amplified rDNA. Amplified rDNA is cleaved at many sites by each enzyme whilst somatic rDNA is relatively resistant to digestion. The difference is attributed to the presence of 5-methylcytosine rendering many restriction sites in somatic rDNA resistant to nuclease attack. The major methylated sequence in eukaryotic DNA is CpG, and recognition sites for all four enzymes contain this sequence. The “ enzymes” thus identified have been used to study the pattern of methylation in erythrocyte rDNA. Unmethylated CpGs were located by mapping vulnerable restriction sites and the distribution and level of CpG methylation was determined. For most detected CpGs the level of methylation is high (about 99%). However there is one site which is unmethylated in 30 to 60% of rDNA repeat units. There is also a region in the non-transcribed “spacer” within which unmethylated sites for HpaII and AvaI are concentrated, though it is not yet certain whether this is due to undermethylation.