A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae.
Pp LSU3 is a mobile group I intron in the extrachromosomal nuclear ribosomal DNA (rDNA) of Physarum polycephalum. As found for other mobile introns, Pp LSU3 encodes a site-specific endonuclease, I-Ppo, which mediates "homing" to unoccupied target sites in Physarum rDNA. The recognition sequence for this enzyme is conserved in all eucaryotic nuclear rDNAs. We have introduced this intron into a heterologous species, Saccharomyces cerevisiae, in which nuclear group I introns have not been detected. The expression of Pp LSU3, under control of the inducible GAL10 promoter, was found to be lethal as a consequence of double-strand breaks in the rDNA. However, surviving colonies that are resistant to the lethal effects of I-Ppo because of alterations in the rDNA at the cleavage site were recovered readily. These survivors are of two classes. The first comprises cells that acquired one of three types of point mutations. The second comprises cells in which Pp LSU3 became inserted into the rDNA. In both cases, each resistant survivor appears to carry the same alterations in all approximately 150 rDNA repeats. When it is embedded in yeast rDNA, Pp LSU3 leads to the synthesis of I-Ppo and appears to be mobile in appropriate genetic crosses. The existence of yeast cells carrying a mobile intron should allow dissection of the steps that allow expression of the highly unusual I-Ppo gene.
Muscarella
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Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Vogt
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D000818:Animals; D001483:Base Sequence; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005804:Genes, Lethal; D007438:Introns; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016839:Physarum polycephalum / Q000235:genetics*; D010957:Plasmids; D017354:Point Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D004706:Endodeoxyribonucleases; C064579:I-Ppo endonuclease; D005690:Galactose