Nuclear and mitochondrial revertants of a yeast mitochondrial tRNA mutant

Abstract

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.

Structure and transcription of the mitochondrial genome in heteroplasmic strains ofSaccharomyces cerevisiae

“…We have recently characterized a number of respiring revertants from a respiratory deficient strain harboring a mutation in the mitochondrial tRNAAsP gene. In these revertants a small region in the tRNA gene cluster is amplified and maintained along with the full-length mtDNA (20). Maintenance of the amplified region containing the mutated tRNAASP gene results in an overproduction of the mutated tRNAASP which allows respiration in the otherwise respiratory deficient strain.…”

“…These revertants were derived by MnCl2 mutagenesis from the respiratory-deficient S. cerevisiae strain, FF1210-6C/170 (MATa ural ura2 rho+ syn- [21]), which in turn was derived from the respiratory-sufficient strain FF1210-6C (MATa ural ura2 rho+). The revertants carry two tpes of mtDNA, a full-length genome and a deleted and amplified 'petite' genome (20). The strain designation and the extent of the repeating units of the amplified genome are shown in Fig.…”

“…RNAs produced from the repeating unit of the amplified DNAs are selectively enriched and high molecular weight transcripts with homology to tRNAs accumulate (20). These transcripts are useful for identifying the processing sites of mitochondrial tRNA precursors.…”