Suppressor of yeast mitochondrial ochre mutations that maps in or near the 15S ribosomal RNA gene of mtDNA.

Abstract

A polypeptide chain-terminating mutation in the yeast mitochondrial oxi 1 gene has been shown to be an ochre (TAA) mutation by DNA sequence analysis. Mitochondrially inherited revertants of this mutation include two types: In the first, the ochre codon has been changed to a sense codon by further mutation in the oxi 1 gene while, in the second, the ochre codon is still present, indicating the occurrence of an extrageneic ochre suppressor mutation. This mitochondrial ochre suppressor, termed MSU1, has been "cloned" in rho-strains ofyeast and tested against other oxi 1 mutations. Several additional mutations are also suppressible, and those examined so far are also ochre mutations. MSU1 does not suppress known frameshift or missense mutations at oxi 1. Isoelectric focusing of the gene product (cytochrome oxidase subunit II) from a suppressed-mutant strain indicates that suppression does not involve insertion of charged amino acids. Physical mapping of the mtDNA retained in the MSUl-carrying rhoF clones localizes the suppressor mutation to the gene coding the 15S rRNA or a site not more than 300 base pairs from it. No known tRNA genes occur this close to the 15S rRNA gene, and mtDNA from a suppressor-carrying rho-does not hybridize detectably to mitochondrial tRNAs. These results suggest that MSU1 may be an alteration in the 15S rRNA.Nonsense mutations and their suppressors have been of great value for the study of both protein-coding structural genes and the genetic systems that express them. Although the mitochondrial genetic system of yeast (Saccharomyces cerevisiae) has been intensively studied over the past few years (1, 2), no defined set ofnonsense mutations and suppressors for them has been described. Suppressors of yeast mitochondrial mutations have indeed been reported. They arise as a result of either mutations in nuclear genes (3-6) or second-site mutations in mtDNA (4, 5, 7). However, in the previously described cases, the nature of the suppressible mutations was not determined precisely.We have been engaged in a study ofmutants at the oxi 1 gene in mtDNA (8-10), a simple uninterrupted gene that codes for the polypeptide cytochrome c oxidase subunit II (9,11,12). To examine suppression in this gene, we have now analyzed the DNA sequence alteration in the mutant oxi l-V25, which has been reported to be a chain-terminating mutation (13) that can be suppressed by several recessive nuclear mutations (A. Putrament and T. Zoladek, personal communication). We report here that oxi l-V25 is an ochre (TAA) mutation.Among the revertants isolated from this mutant, we have identified a strain that carries an extrageneic ochre suppressor mutation in mtDNA, termed MSUI. MSUI Table 3 were obtained from B. Weiss-Brummer (16), with the exception of M13-249 (9). Media and Genetic Techniques. YPEG medium contained 1% yeast extract, 2% Bactopeptone, 3% ethanol, and 3% glycerol. Glucose-containing medium (YPD) and genetic techniques were as described (17).Isolation of mtDNA and Sequence Analysis. The procedures ...

Gene expression and survival changes in Saccharomyces cerevisiae during suspension culture

“…These genes are involved in a variety of cellular processes, which provides a picture of the cells response to shear. For example, the increased expression of genes such as PLB1, RHO4, and SMC4 indicate that the structure of the cell is affected by the increased shear as these genes code for proteins involved in lipid homeostasis, the establishment of cell polarity, and chromosome organization, respectively (Dong et al, 2003;Fox and Staempfli, 1982;Gao et al, 1997;Lee et al, 1994;Levin, 2005). The shear stress on the cell may also result in an increase in transcriptional errors and hence the upregulation of MSU1, an RNase known to be involved in the turnover of aberrant mRNAs (Freeman et al, 2000).…”

NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

“…The suppressor NAM9-1 was isolated by Smolinska (57) after strong ethyl methanesulfonate treatment of the mit mutant which carries the V25 ochre mutation (sequenced by Fox and Staempfli [16]) in the oxil gene coding for subunit II of cytochrome oxidase. The NAM9-1 mutant strain was selected as a glycerol-positive colony on glycerol medium at 28°C.…”

Alteration of the Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader by mitochondrial gene replacement and functional interaction with the translational activator protein PET111.

“…Transformants containing replicating plasmids in their mitochondria were identified by their ability to produce respiring diploids by recombination (marker rescue) when mated to the mutant tester strain, M461 1, carrying an ochre allele in the COX2 coding sequence (Fox and Staempfli, 1982). To determine whether the 5'-UTL mutations affected expression of COX2, we tested their ability to complement a complete deletion, cox2-17 , by Figure 1.…”