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It is obvious that the availability of rare cutter restriction endonucleases would facilitate large scale genome mapping, cloning and sequencing projects. Restriction endonucleases of bacterial origin have recognition sites of 8 bp long, at most, and even when used in combination with methylases they can produce cleavage specificities of up to 12 bp only (1). Artificial endonucleases have been made by chemical modifications of either DNA binding proteins (2) or synthetic oligodeoxynucleotides (3). Although this last strategy can, in principle, be generalized to many sequences, cleavage occurs at low efficiency. Cleavage of yeast and E. coli genomic DNAs at a single predetermined site using a frequent cutter bacterial endonuclease has recently been achieved by the Achille's heel method, an elegant three step procedure combining the action of the lac repressor on artificially inserted sites with that of a methylase (4). We present here the first evidence for cleavage of total yeast genomic DNA at a single predetermined site in a one step procedure using a new endonuclease, l-Sce I, encoded by a mobile group I intron of yeast mitochondria. We further show that, although l-Sce I is extremely specific, cleavable sites can also be found in natural DNA.

Cleavage of yeast and bacteriophage T7 genomes at a single site using the rare cutter endonuclease I-SceI