Cloning of mitochondrially determined oligomycin resistance, oli-1 in yeast

An Escherichia coli-yeast shuttle vector pOL 221 is described, which consists of pBR 322 and a yeast mitochondrial fragment. The mitochondrial insert in pOL 221 possessed a mitochondrial autonomously replicating sequence (ARS) and a single Sst II site. The ARS function of pOL 221 was demonstrated by insertion of LEU2 gene into pOL 221 (giving pOL 2211) followed by the transformation of leu- yeast cells. By use of this vector, chimeric plasmids pOL 37 and pOL 379 (a derivative of pOL 37 inserted with LEU2 gene), carrying the yeast mitochondrial oli-I gene were constructed. Marked instability was observed on the maintenance of pOL 37 in E. coli under non-selective conditions. pOL 37 can, however, be maintained in E. coli in the presence of tetracycline. pOL 2211, pOL 379 and other pOL plasmids showed mitotic instability in yeast. Transformants of oligomycin sensitive yeast cells with pOL 37 and pOL 379 did not show oligomycin resistance. The absence of recombination between the cloned mitochondrial gene and the host mitochondrial gene is discussed.