Physical mapping and characterization of the mitochondrial DNA and RNA sequences from mit^- mutants defective in cytochrome oxidase peptide 1 (OXI 3)

Yeast mitochondrial mutants defective in cytochrome c oxidase have been isolated and genetically characterized by Slonimski and Tzagoloff (1976). Three genetic loci, designated OXI 1, OXI 2, and OXI 3, each affect the synthesis of a separate mitochondrially coded subunit of cytochrome oxidase (Cabral et al., 1977). We have analyzed the mitochondrial DNA and RNA of mutants with defects at the locus which impair the synthesis of the 43,000 subunit I peptide, in order to map the structural gene and identify the RNA species transcribed from this region. Mitochondrial DNA (mtDNA) from nine OXI 3 mutants derived from the grande strain D273-10B was digested with the endonucleases Eco RI, Hpa I, Hha I, Hine II, Xba I, and Hpa. II. Two mutants, M10-150 and M5-16, were found to have large contiguous deletions corresponding, respectively, to 7500 and 5400 base pairs (bp). In strain M11-125, two mutations separated by 8500 bp were detected; one results in the deletion of a Hha I site and the other introduces a new Hpa I (Hinc II) site. Based on the size of the lesion present in M5-16, the OXI 3 gene is mapped within 47.5 to 57.0 map units. No detectable changes in the restriction patterns of fragments in the OXI 3 region were observed in the remaining seven mutants. Mutant M11-82, in which no deletionwas detected, is genetically similar to the 5400 bp deletion mutant M5-16 (Slonimski and Tzagoloff, 1976). Since our gel resolves fragments that differ by 10-50 bp, the data indicate that a deletion present in this strain must be smaller than 50 bp. Mitochondrial RNA was isolated from OXI 3 mutants and analyzed by gel electrophoresis containing the denaturing agent methylmercury hydroxide. Three species, corresponding to 2800, 2550, and 620 nucleotides, are absent in mitochondrial RNA from M5-16 and M10-150. These three transcripts appear to be specific to the sequences absent in the deletion mutants M5-16 and M10-150, since they are present in a OXI 3 point mutant, M11-224, and in mit^- mutants affecting cytochrome b or subunits II and III of cytochrome oxidase.