Nuclear and mitochondrial revertants of a yeast mitochondrial tRNA mutant

We isolated revertants capable of respiration from the respiratory deficient yeast mutant, FF1210-6C/170, which displays greatly decreased mitochondrial protein synthesis due to a single base substitution at the penultimate base of the tRNAAsp gene on mitochondrial (mt) DNA. Three classical types of revertant were identified: (1) same-site revertants; (2) intragenic revertants which restore the base pairing in the acceptor stem of the mitochondrial tRNAAsp; and (3) extragenic suppressors located in nuclear DNA. In addition a fourth type of revertant was identified in which the mutant tRNAAsp is amplified due to the maintenance of both the original mutant mtDNA and a modified form of the mutant mtDNA in which only a small region around the tRNAAsp gene is retained and amplified. The latter form resembles the mtDNA in vegetative petite (rho-) strains which normally segregates rapidly from the wild-type mtDNA. Each revertant type was characterized genetically and by both DNA sequence analysis of the mitochondrial tRNAAsp gene and analysis of the quantity and size of RNA containing the tRNAAsp sequence. These results indicate that the mitochondrial tRNAAsp of the mutant retains a low level of activity and that the presence of the terminal base pair in tRNAAsp is a determinant of both tRNAAsp function and the maintenance of wild-type levels of tRNAAsp.