Isolation of five type IIG restriction modification (RM) enzyme genes with different DNA recognition sites from a single environmental DNA sample

Abstract

A new method of screening type IIG restriction modification (RM) enzyme has been developed using REBASE, a database of all known and putative restriction enzymes and methyltransferases found throughout the bacterial genome sequences available in GENBANK. The in silico analysis of a group of putative type IIG RM enzymes in Microcystis aeruginosa showed a high sequence homology at both ends. This peculiarity allows for primers designing that can be used in polymerase chain reaction (PCR) to amplify the corresponding genes out of one environmental DNA extracted from a cyanobacteria-rich sample. PCR products were cloned into the pSAPV6 vector. Among eight recombinant DNA sequenced, five showed different sequences in the protein regions that interact specifically with DNA.". These five recombinant proteins expressed type IIG RM enzyme activity. Their specificities were determined, and all correspond to new DNA recognition sites.