Methylation of mouse DNA in vivo: Di- and tripyrimidine sequences containing 5-methylcytosine

Abstract

1. Highly labeled pyrimidine isopliths from DNA of newborn mice and mouse embryo cell cultures treated with l-[ Me- 3H]methionine were separated by column chromatography on DEAE-cellulose. 2. The dipyrimidines were isolated by high-voltage electrophoresis followed by chromatography on paper and hydrolyzed by snake venom phosphodiesterase. All of the nine theoretically possible combinations of thymidine (T), deoxycytidine (C), and 5-methyldeoxycytidine (M) were shown to be represented in mouse DNA. Of these sequences, TpM occurred with the greatest frequency: it contained 40% of the radioactivity appearing in the dipyrimidine fraction. By contrast, in bacterial DNA which was hypermethylated by a Bacillus subtilis extract, the sequence TpM was not found at all, while MpT was found to contribute 70% of the radioactivity recovered in the dipyrimidines containing 5-methylcytosine. 3. The 27 theoretically possible tripyrimidine sequences of mouse DNA were separated by high-voltage electrophoresis into eight groups. By treating these with snake venom and spleen phosphodiesterases and subsequent paper chromatography, four sequences (CpTpM, TpMpC, MpTpT, TpTpM) were unequivocally identified out of the 19 possible combinations containing 5-methylcytosine. The existence of at least three more tripyrimidine sequences was established. The sequence MpTpT contributed 38% of the radioactivity in the tripyrimidine fraction. 4. The results establish that methylation of mammalian DNA is not confined to the sequence MpG as previously reported. Radiomethylation and quantitative analysis of pyrimidine sequences containing 5-methylcytosine may thus provide a sensitive tool for discriminating between closely related DNA species.