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 syste ms (MDRS) that demonstrate methylcytosine-dependent restriction endonu clease activity for the dinucleotide sequence, dCpdG. This makes these strains unsuitable for cloning methylated DNA. Some commercially avai lable bacterial cells are recommended for cloning DNA fragments with m ethylated cytosines and adenines, e.g., Escherichia coli DH5-alphaMCR. Our attempts to clone frog virus 3 (FV3) DNA, which has the highest d egree of cytosine methylation ever reported, using DH5-alphaMCR cells, were not successful. This and other observations suggested the existe nce of additional MDRS that have not yet been eliminated from DH5-alph aMCR cells. In order to isolate a mutant from this bacterial strain th at is suitable to clone highly methylated FV3 DNA, we transformed thes e cells with a recombinant pUC19 plasmid containing a methylated 1.4-k b genomic DNA fragment from FV3, and selected for ampicillin (Ap) resi stance. Three such attempts yielded only one colony that contained a f ully 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.