Genetic analysis of mitochondrial rho-mutability in Saccharomyces. IV. Relation between spontaneous rho-mutability and mitotic stability in disomic Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae the disomy for chromosome XIV resembles the previously described disomy for chromosome IV in that it leads to a significant decrease in spontaneous rho- mutability. The nuclear srm1 mutation, reducing spontaneous rho- mutability, diminishes significantly the mitotic disome stability. So, the mechanisms of spontaneous rho- mutagenesis and mitotic disome stability seem to compete for the function affected by the srm1 mutation.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces. III. Comparative analysis of the effect of various nuclear srm mutations and disomy for chromosome IV on rho-mutagenesis

Different combinations of modifying genes which enhance the rho- mutability of haploid yeast cells are shown to be suppressible by the srm1, srm2, srm3 mutations and by the disomy for chromosome IV. The srm1 mutation leads to dramatic decrease in both the spontaneous and ethidium-bromide induced rho- mutability. Other srm mutations studied and the disomy appear to cause relatively moderate quantitative changes in the spontaneous rho- mutation rate and to have no significant effect on mutation induction by ethidium bromide. Neither additivity nor synergism was revealed by the analysis of the interaction between the srm mutations. We suggest that in Saccharomyces an efficient mechanism of the rho- mutagenesis operates which can be directly affected by the srm1 mutation and more or less modified by other srm mutations under study and by the disomy for chromosome IV.     

Genetic analysis of the mitochondrial rho-mutability in Saccharomyces. II. Disomy for chromosome IV and spontaneous rho-mutability

The disomy for chromosome IV in the strains studied led to: reduction in the red pigmentation of ade1 mutant colonies; a decrease in spontaneous rho- mutant frequency, and impairment of sporulation in hybrids descended from disomic parents. The nuclear srm1 mutation decreasing the spontaneous rho- mutability promoted the spontaneous extra chromosome loss in the disomics for chromosome IV. This result suggests a close connection between the spontaneous rho- mutability and mitotic chromosome stability.     

Genetic analysis of mitochondrial rho-mutability in Saccharomyces. I. Polygenic determination of spontaneous rho-mutability: gene-modifiers and srm mutation

The phenotypic trait "starry colony" in Saccharomyces is associated with a high spontaneous rho- petite mutability. Genetic analysis of this trait has shown the high rho- mutability to be caused by several modifying genes present together in the cell genome. Every single modifying gene only produces a relatively small enhancement in the rho- mutability. Mutations in four nuclear srm (spontaneous rho- mutability) loci were isolated after mutagenic treatment of highly rho- mutable haploid cells. In contrast to the modifying genes, each of these mutations has a pronounced effect on the spontaneous rho- mutability, causing significant decrease in it.     

New mutation in Saccharomyces cerevisiae SRM genes and some features of their phenotypic effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17 on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiae cells and on cell sensitivity to ionizing radiation were studied. The srm8 mutation caused instability of chromosome maintenance in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srm mutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17 also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8 mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17 were shown to increase cell sensitivity to gamma-ray irradiation. The SRM8 gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.     

New Mutations of SRMGenes in Saccharomyces cerevisiae and Certain Characteristics of Their Phenotypic Effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiaecells and on cell sensitivity to ionizing radiation were studied. The srm8mutation caused an increase in spontaneous chromosome loss in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srmmutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17were shown to increase cell sensitivity to -rays. The SRM8gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.     

The SRM12/ADA1 gene acts as a cis-active factor when inserted into recombinant plasmids and makes them more stable in Saccharomyces

A DNA fragment containing the SRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted fragment in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.     

The SRM12/ADA1 Gene Sequence Inserted into Recombinant Plasmids Makes Them More Mitotically Stable in Budding Yeast Cells

TheSRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted sequence in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

A nuclear mutation decreasing rho- production modifies the unstable nitrous-acid sensitivity of yeast

The nuclear mmgl mutation, which reduces rho- mutability in Saccharomyces cerevisiae, renders the rho+ cells less sensitive to inactivation by nitrous acid (NA) but has little or no effect on the NA sensitivity of the rho0 cells devoid of mitochondrial (mt) DNA. Therefore the cells' NA sensitivity seems to be influenced by an interaction of the mmgl mutation and the mt genome rather than the mmgl mutation itself. The clonal variation of NA sensitivity is high in MMG+ yeast and significantly reduced in rho0 mutants and mmgl cells. The results presented suggest that frequent spontaneous heritable changes of the mt genome occur in MMG+ cells, which, (i) unlike rho- mutations, do not damage the respiratory capacity, and (ii) manifest themselves in a high clonal variation of NA sensitivity.     

Genetic interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

 The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2. Received: 20 March 1996/Accepted: 1 July 1996     

Genetic interaction of DED1encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homolog in Saccharomyces cerevisiae

The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35°?C, but not at 37°?C. A revertant of srm1-1 ded1-21 that became able to grow at 37°?C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 ^- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 ^- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.     

Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase

Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of mitochondrial ATP synthase. A series of mutants was isolated in which the temperature-sensitive phenotype resulting from the aep2-ts1 mutation was suppressed. Three strains were classified as containing a mitochondrial suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial genome was replaced with wild-type mitochondrial DNA (mtDNA). Many other isolates were classified as containing dominant nuclear suppressors. The three mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides upstream of the oli1 reading frame. It is inferred that the suppressing mutation in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation (shown here to cause the substitution of proline for leucine at residue 413 of Aep2p). This mode of mitochondrial suppression is contrasted with that mediated by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a nuclear gene product in expression of a particular mitochondrial gene. In the present study, direct RNA-protein interactions are likely to form the basis of suppression.     

Deletion of mitochondrial genetic markers in yeast by ethidium and the photoaffinity probe, ethidium azide.

Induction of petite (cytoplasmic-respiration-deficient, rho-,rho-) mutations in yeast and deletion of mitochondrial drug-resistance genetic markers were compared after after treatment with ethidium and the corresponding photoaffinity probe, ethidium azide. Deletion of mitochondrial drug-resistance markers for chloramphenicol, erythromycin and oligomycin in these petite mutants was observed during prolonged treatment times with ethidium and with ethidium azide in the dark. A similar loss of drug-resistance markers was also observed in petites produced by photolytic treatment with the azide analogue, although the rate of loss appeared to be somewhat less. These results confirmed the usefulness of photoaffinity labeling with ethidium monoazide for studies of mitochondrial mutations.     

Study of a yeast mutant with modified mitochondrial translocation of adenine nucleotides

The effect of ethidium bromide on the growth of a yeast mutant with an impaired mitochrondrial translocation system of adenine nucleotides (op-1 mutant) was investigated. It was found that the op-1 mutant stops growing both under growing and non-growing conditions after treatment with ethidium bromide and that the growth cannot be restored by adding low-molecular compounds to the growth medium. It was the aim of the experiments to clarify whether the cessation of growth of the op-1 mutant after induction of the rho- mutation can be simulated by inhibitors phenotypically changing the mitochondrial function. It appears likely that the op-1 mutant stops growing only after the rho- mutation has been induced, because the phenotypic simulation of the rho- mutation does not lead the cessation of growth of the op-1 mutant.     

Direct estimation of the mitochondrial DNA mutation rate in Drosophila melanogaster

Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10⁻⁸ per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of the mitochondrial genome of Drosophila.     

The autonomic transfer of mitochondrial factors (cytoduction) during crossing of Saccharomyces cerevisiae cells]

When crossing the genetically marked yeast strains obtained from the Gif collection we observed the appearance of haploid nucleo-cytoplasmic hybrids carrying the 3 nuclear markers of the rho- parent and the mitochondrial markers (rho+ ER CR) of the other parent. The frequency of such cytoduction was about 1 per cent. The mitochondrial markers ER and CR were transmited to cytoductants together and did not segregate. The possible mechanisms of the cytoduction and its significance are discussed.