The optional group I intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae contains a 235-codon-long open reading frame the translation product of which (the omega transposase) catalyzes the formation of a double-strand break within the intron-minus (omega-) copies of the same gene. Purified omega transposase generates in vitro a 4-base-pair staggered cut with 3' hydroxyl overhangs at the exact position where the intron eventually inserts in the gene. Using randomly mutagenized synthetic oligonucleotides, single-base mutants were produced at 21 positions around the cleavage site. Experiments with these oligonucleotides show that the recognition site extends over an 18-base pair-long sequence within which minimal sequence degeneracy is tolerated. The intron-encoded omega transposase is, therefore, one of the most specific restriction endonucleases known to date.The group I intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (rl intron), which is optional among different laboratory strains, shows the interesting property of propagating itself during crosses between intronplus strains (omega') and intron-minus ones (omega-) (1-4). The phenomenon, which is formally equivalent to a gene conversion, causes rapid spreading of this particular intron within yeast populations undergoing random mating. Insertion ofthe intron is associated with a coconversion offlanking exons extending over a few hundred base pairs (bp) on each side (5). Analysis of mtDNA in young zygotes of omega + x omega-crosses has revealed the transient formation of a double-strand break occurring at or near the recipient site of the intron-minus gene (omega -site) (6, 7). Mutants that have an altered omega -site do not show the double-strand break in similar crosses.The rl intron contains a 235-codon-long open reading frame (rl ORF) that is conserved in a variety of yeast species belonging to the Saccharomyces and Kluyveromyces genera and shares distinctive features with ORFs of other group I introns (8, 9). Mutants within the rl ORF easily demonstrate that its translation product must exist and is required for the gene conversion of the intron and its flanking exons (5, 10). Thus, the overall organization of the rl intron is reminiscent of a transposable element encoding its own transposase.To characterize the omega transposase, which, like the other intron-encoded proteins, remains undetectable in normal mitochondria, we have previously engineered the rl ORF by modifying its nonuniversal codons (11). The resulting universal code equivalent directs, from expression plasmids, the synthesis of a protein with all characteristic properties expected of the genuine mitochondrial omega transposase. This protein exhibits a specific endonuclease activity, generating in Escherichia coli a double-strand break at omegasites carried on plasmids.The omega transposase has already been partially purified from E. coli cells, and the fractions containing the partially purified substance can be shown to generate a double-strand...
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