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. Key words: Environmental DNA, polymerase chain reaction (PCR), recombinant protein, Type IIG RM enzyme screening, uncultured bacteria.
Highlights
The type II restriction enzymes discovered in 1968 (Smith and Welcox, 1970) are endonucleases that cut DNA at specific 4-8 nucleotide sequences which mainly exist in prokaryotes
Screening in REBASE genes coding for type IIG restriction modification (RM) putative enzymes from M. aeruginosa
Three putative genes coding for type IIG RM putative enzymes were found in M. aeruginosa: Mae2549ORF1146, Mae843ORF8180 and
Summary
The type II restriction enzymes discovered in 1968 (Smith and Welcox, 1970) are endonucleases that cut DNA at specific 4-8 nucleotide sequences which mainly exist in prokaryotes. Each restriction enzyme always pairs with a methyltransferase which modifies host DNA at the same site. The two enzymes form a restriction modification (RM) system that probably contributes to protecting bacteria against foreign DNA (Raleigh and Brooks, 1998). 4000 restriction enzymes have been characterized, which recognize 365 different sites (Pingoud et al, 2014), representing a statistically minor fraction of all the possible DNA sequences.
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