Restriction analysis of actinomycetes chromosomal DNA

Abstract

Actinomycetes DNAs were digested with restriction enzymes to study the presence of methylated bases. Analysis showed that the enterobacterial Dam and Dcm systems are absent. Methylation at the internal cytosine in CCGG sequences, typical of eukaryotes, was also absent. We also tested 18 restriction endonucleases recognizing six base pair sequences (all of which were inhibited by methylation). Results showed a higher number of restriction sites for enzymes recognizing CG-rich sequences (CG endonucleases) than for enzymes recognizing AT-rich sequences (AT endonucleases). Restriction patterns with CG endonucleases were quite uniform, with the remarkable exception of XhoI, which yielded a small number of DNA bands. The study performed with AT endonucleases allowed differentiation of three groups of enzymes based on different degrees of chromosomal sensitivity. One group (BelI and BglII) produced restriction patterns with more abundant restriction sites than expected, a second group (ClaI, EcoRI, and EcoKV) yielded the predicted number of DNA fragments, and the third group (HpaI, HindIII, XbaI, and DraI) produced an unexpectedly low number of fragments. Some individual cases of resistance to particular enzymes could be explained by the presence of restriction-modification systems with the same specificity.Key words: Streptomyces, DNA methylation, restriction modification, sequence counterselection, pulsed-field gel electrophoresis.