Expression of Barstar as a selectable marker in yeast mitochondria.
We describe a new and potentially universal selection system for mitochondrial transformation based on bacterial genes, and demonstrate its feasibility in Saccharomyces cerevisiae. We first found that cytoplasmically synthesized Barnase, an RNase, interferes with mitochondrial gene expression when targeted to the organelle, without causing lethality when expressed at appropriate levels. Next, we synthesized a gene that uses the yeast mitochondrial genetic code to direct the synthesis of the specific Barnase inhibitor Barstar, and demonstrated that expression of this gene, BARSTM, integrated in mtDNA protects respiratory function from imported barnase. Finally, we showed that screening for resistance to mitochondrially targeted barnase can be used to identify rare mitochondrial transformants that had incorporated BARSTM in their mitochondrial DNA. The possibility of employing this strategy in other organisms is discussed.
Mireau
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Station de Génétique et d'Amélioration des Plantes, INRA, Route de Saint-Cyr, 78026 Versailles, France. mireau@versailles.inra.fr
Arnal
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N
Fox
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D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D005819:Genetic Markers; D008928:Mitochondria / Q000235:genetics*; D012260:Ribonucleases / Q000037:antagonists & inhibitors* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004272:DNA, Mitochondrial; D005819:Genetic Markers; C057438:barstar protein, Bacillus amyloliquefaciens; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease