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N6-METHYLADENINE (6-MeAde) and 5-methylcytosine occur as minor bases in bacterial and phage DNA1–7 and seem to result from the selective methylation of adenine and cytosine residues by specific DNA methylases8. Methylation is the final stage in DNA synthesis and is essential for the phenomenon of host modification of phages9–11; it is one of the mechanisms controlling DNA replication in the cell12, 13. A study of the distribution of minor bases in DNA is therefore important not only for the elucidation of the specificity and mechanism of action of DNA methylases but also for an understanding of the purpose of this methylation. We believe that in Escherichia coli, DNA methylase exerts its action on adenine residues in chain terminating triplets: 6-MeAde may serve as a signal for gene termination in this system.

The biological function of the majority of methylated bases in DNAs is still unknown [ 1,2]. Recently Roy and Smith purified and characterized four DNA-adenine methylases from Hemophilus injluensue Rd [3]. Evidently, the bacterial cell also contains several DNA-cytosine methylases which methylate cytosine in several nucleotide sequences. The present study in an attempt to elucidate the possible multiplicity of DNA-cytosine methylases in bacteria. To this end, a study was made of the pattern of cytosine methylation in heterologous DNAs methylated by a cell-free extract and different fractions of DNAcytosine methylases from Escherichia coli MRE 600 obtained in the course of its purification. The present work describes the procedure used to separate two DNA-cytosine methylase activities. The specificity of cytosine methylase activities was studied by analysis the SmeCyt* (formed in vitro) distribution in pyrimidine isopliths of heterologous DNAs. The data obtained show the existence of at least two different nucleotide sequences containing SmeCyt and some cytosine methylase fractions from E. coli MRE 600 producing modifications of cytosine in these sequences.