A procedure is proposed that will isolate genes for type II restriction enzyme genes without advance knowledge of the protein or DNA sequence or recognition site. The protocol is based on the idea that restriction enzyme genes will be enriched in arrays of gene cassettes called integrons or superintegrons. These cassette arrays offer an efficient method for obtaining intact genes, with a high likelihood that the orientation of the gene is known in advance. Together with a cloning vector that allows tight regulation and a cloning host that will report which genes are capable of damaging DNA, it will be possible to rapidly identify a small subset of cassettes that are candidate restriction endonuclease genes. Survey of this subset for DNA cleavage properties and sequence similarities will allow efficient identification of type II restriction enzyme genes. Limited available data suggest that the genes obtainable in this way are more likely to cut rarely than frequently. Rare-cutting restriction enzymes are of great value in biotechnology, and efficient strategies for creating or discovering new ones are needed.
Rare-cutting restriction enzymes are of great value in biotechnology, yet only a limited number of different specificities are available. Two of the six sequence enzymes with recognition sites of 8 base-pairs are present in arrays of the kind described. The proposed technique should provide an efficient strategy for discovering new restriction specificities, particularly rare-cutters.
