Isolation of Escherichia coli mutants lacking methylcytosine-dependent restriction systems for cloning extensively methylated frog virus 3 DNA.

Many bacterial strains possess methylation-dependent restriction systems (MDRS) that demonstrate methylcytosine-dependent restriction endonuclease activity for the dinucleotide sequence, dCpdG. This makes these strains unsuitable for cloning methylated DNA. Some commercially available bacterial cells are recommended for cloning DNA fragments with methylated cytosines and adenines, e.g., Escherichia coli DH5-alpha MCR. Our attempts to clone frog virus 3 (FV3) DNA, which has the highest degree of cytosine methylation ever reported, using DH5-alpha MCR cells, were not successful. This and other observations suggested the existence of additional MDRS that have not yet been eliminated from DH5-alpha MCR cells. In order to isolate a mutant from this bacterial strain that is suitable to clone highly methylated FV3 DNA, we transformed these cells with a recombinant pUC19 plasmid containing a methylated 1.4-kb genomic DNA fragment from FV3, and selected for ampicillin (Ap) resistance. Three such attempts yielded only one colony that contained a fully methylated 1.4-kb FV3 genomic DNA fragment. Furthermore, plasmid-cured Ap-sensitive colonies originating from this clone were isolated and have been successfully employed to clone the highly methylated FV3 genomic DNA fragment.