Analysis of Calf‐Thymus Satellite DNA: Evidence for Specific Methylation of Cytosine in C‐G Sequences

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Abstract
Digestion of purified calf thymus satellite I (.phi. = 1.714 g/cm3) with a series of restriction enzymes shows that modification in this satellite occurs preferentially in the sequence C-G. This was also the case in the other satellites and in bulk chromosomal calf thymus DNA. Cloning of purified satellite I DNA in Escherichia coli makes sites, previously modified, available for cutting with certain restriction enzymes. All these new sites contain the sequence C-G. High-resolution mass spectroscopy establishes that the satellites contain a low concentration of 5-methylcytosine. This infers that methylation which inhibits restriction enzyme cutting must occur preferentially in the sequence C-G. Hybridization of c[complementary]RNA of cloned satellite I DNA with the satellites III (.phi. = 1.706 g/cm3) and IV (.phi. = 1.710 g/cm3) shows that there is no or little sequence homology between these satellites. Digestion of calt thymus satellite I DNA with endoR .cntdot. EcoRI and subsequent hybridization studies with the fragments shows 2 EcoRI fragments in addition to the usual 1400 base pair EcoRI repeat unit.