Assembly of the mitochondrial membrane system: isolation of mitochondrial transfer ribonucleic acid mutants and characterization of transfer ribonucleic acid genes of Saccharomyces cerevisiae

Abstract

A method is described for isolating cytoplasmic mutants of Saccharomyces cerevisiae with lesions in mitochondrial transfer ribonucleic acids (tRNA's). The mutants were selected for slow growth on glycerol and for restoration of wild-type growth by cytoplasmic "petite" testers that contain regions of mitochondrial deoxyribonucleic acid (DNA) with tRNA genes. The aminoacylated mitochondrial tRNA's of several presumptive tRNA mutants were analyzed by reverse-phase chromatography on RPC-5. Two mutant strains, G76-26 and G76-35, were determined to carry mutations in the cysteine and histidine tRNA genes, respectively. The cysteine tRNA mutant was used to isolate cytoplasmic petite mutants whose retained segments of mitochondrial DNA contain the cysteine tRNA gene. The segment of one such mutant (DS504) was sequenced and shown to have the cysteine, histidine, and threonine tRNA genes. The structures of the three mitochondrial tRNA's were deduced from the DNA sequence.