Mitochondrial and nuclear myxothiazol resistance in Saccharomyces cerevisiae

Summary Mitochondrial and nuclear mutants resistant to myxothiazol were isolated and characterized. The mitochondrial mutants could be assigned to two loci, myx1 and myx2, by allelism tests. The two loci map in the box region, the split gene coding for apocytochrome b. Locus myx1 maps in the first exon (box4/5) whereas myx2 maps in the last exon (box6). The nuclear mutants could be divided into three groups: two groups of recessive mutations and one of dominant mutations. Respiration of isolated mitochondria from mitochondrial mutants is resistant to myxothiazol. These studies support the conclusion that myxothiazol is an inhibitor of the respiratory chain of yeast mitochondria. The site of action of myxothiazol is mitochondrial cytochrome b.Abbreviations box mosaic gene coding for apocytochrome b - cyt b cytochrome b - MIC minimum inhibitory concentration - MNNG N-methyl-N'-nitro-N-nitrosoguanidine - Myx ^R/Myx ^S allelte forms of a locus conferring myxothiazol resistance - myx1, myx2 mitochondrial loci conferring myxothiazol resistance - rho ⁺/rho ^– grande/cytoplasmic petite - rho ⁰ cytoplasmic petite that is deleted of all mitochondrial DNA     

On the formation of rho? petites in yeast

Summary The inheritance of an extrakaryotic mutation conferring temperature-sensitive growth on nonfermentable substrates and a high frequency of mutation to rho^– has been studied. Multifactorial crosses (rho⁺xrho⁺) involving this mutation T ₈ ^S and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin or paromomycin revealed: a) Mutation T ₈ ^S is localized on the mitDNA, referring to a new gene locus TSM1. b) Locus TSM1 appears to be weakly linked to the locus PAR1 and to the loci RIB1 and RIB3 but unlinked to the locus OLI1. c) The position of TSM1 is between PAR1 and the two closely linked loci RIB1 and RIB3, OLI1 is outside and not linked to the segment PAR-TSM-RIB. d) Mutation T ₈ ^S does not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus .     

Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1

Summary Detailed mapping localized the PHO 1 mutation between the OLI 2 and OLI 4 loci on mitochondrial DNA of Saccharomyces cerevisiae.In its mitochondrially integrated form, the PHO 1-ATPase³ was difficult to identify either immunologically or by specific inhibitors like oligomycin and DCCD. Solubilization by Triton X-100 allowed unambiguuous identification of this enzyme as an authentic mitochondrial ATPase. However, Triton extraction produced a 2 to 3 fold enhancement of the PHO 1-ATPase activity which also became drastically cold-sensitive. The wild type ATPase was neither activated nor made cold-labile by solubilization, and retained full sensitivity to oligomycin and DCCD.Sucrose gradient analysis of the Triton-extracted ATPase from wild type, PHO 1 mutant and rho ^- strains showed a density difference between the solubilized PHO 1-and wild type ATPase, and similarity between solubilized PHO 1-and rho ^- ATPase (F₁).Whole cells of the PHO 1 mutant present considerably increased respiration rates.Comparison of oligomycin-sensitivity in whole cells, coupled isolated mitochondria and membrane-bound ATPase indicates a contrast between oligomycin-resistance of the ATPase and oligomycin-sensitivity of in vivo or in vitro coupling systems, which might characterize the products of this region of mitochondrial DNA.     

Biogenesis of Mitochondria: Analysis of Deletion of Mitochondrial Antibiotic Resistance Markers in Petite Mutants of Saccharomyces cerevisiae

Yeast strains carrying markers in several mitochondrial antibiotic resistance loci have been employed in a study of the retention and deletion of mitochondrial genes in cytoplasmic petite mutants. An assessment is made of the results in terms of the probable arrangement and linkage of mitochondrial genetic markers. The results are indicative of the retention of continuous stretches of the mitochondrial genome in most petite mutants, and it is therefore possible to propose a gene order based on co-retention of different markers. The order par, mik1, oli1 is suggested from the petite studies in the case of three markers not previously assigned an unambiguous order by analysis of mitochondrial gene recombination. The frequency of separation of markers by deletion in petites was of an order similar to that obtained by recombination in polar crosses, except in the case of the ery1 and cap1 loci, which were rarely separated in petite mutants. The deletion or retention of the locus determining polarity of recombination (ω) was also demonstrated and shown to coincide with deletion or retention of the ery1, cap1 region of the mitochondrial genome. Petites retaining this region, when crossed with rho⁺ strains, display features of polarity of recombination and transmission similar to the parent rho⁺ strain. By contrast a petite determined to have lost the ω⁺ locus did not show normal polarity of marker transmission. Differences were observed in the relative frequency of retention of markers in a number of strains and also when comparing petites derived spontaneously with those obtained after ultraviolet light mutagenesis. By contrast, a similar pattern of marker retention was seen when comparing spontaneous with ethidium bromide-induced petites.     

Mutants in yeast affecting ethidium bromide induced rho- formation and their effects on transmission and recombination of mitochondrial genes.

Summary A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the ^+– mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of ^– cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.Abbreviations E.B. Ethidium bromide - E.M.S. Ethyl méthane sulfonate - C^S/C^R Allelic forms of the rib 1 locus conferring chloramphenicol sensitivity/resistance - E^S/E^R Allelic forms of the rib 3 locus conferring erythromycine sensitivity/resistance - O^R/O^R Allelic forms of the oli 1 locus conferring oligomycin sensitivity/resistance - P^S/P^R Allelic forms of the par 1 locus conferring paromomycine sensitivity/resistance - D^S/D^R Allelic forms of the diu 1 or diu 2 loci conferring diuron sensitivity/resistance - C^S/C^R Allelic forms of the mitochondrial locus - ⁺ grande or respiratory competent cells - ^– petite or cytoplasmic respiratory deficient cells     

Biogenesis of mitochondria 49 identification and mapping of a new mitochondrial locus (tsr1) which maps within polar region of yeast mitochondrial genome.

Summary An enrichment procedure which facilitates the isolation of conditional respiratory-deficient mutants of Saccharomyces cerevisiae is reported. Detailed genetic analysis of one mutant which exhibits a respiratory deficient phenotype at low temperature (18°C) is also presented. The phenotype is due to a single lesion at a new locus, tsr1, located on the mitochondrial DNA. By analysis of locus retention patterns in a set of physically characterized petite strains, the tsr1 mutation has been mapped within the segment 0–5 map units on the physical map of the yeast mitochondrial genome. This segment of the mitochondrial DNA also contains the cap1 and ery1 loci and the cistron for the mitochondrial 21S rRNA. Studies of the frequencies of co-retention of markers in petite populations, and of the frequencies of recombination of markers in non-polar crosses (⁺ × ⁺), demonstrate linkage of the tsr1 locus to both the cap1 and ery1 loci. The degree of linkage indicates that tsr1 is closer to the ery1 locus. Comparison of pairwise recombination frequencies for these three markers indicate the order cap1-tsr1-ery1. The tsr1 locus lies within the segment of the mitochondrial genome which is influenced by the polarity locus , and analysis of transmission and recombination frequencies and polarities in a polar (⁺ × ^-) cross show that the behaviour of the tsr1 locus is similar to that of ery1. However striking features of this cross are that the recombination frequency between tsr1 and ery1 is comparable to that observed in non-polar crosses, and that the polarity for recombination between tsr1 and cap1 or ery1 is extremely low.     

Biogenesis of mitochondria. 52

Summary The characteristics of recombination of several petite (rho ^-) mutants of S. cerevisiae that retain the -influenced region of the mitochondrial genome, identified by the markers cap1-r, ery1-r and tsr1, are described. The petites were derived from an ^– grande (rho ⁺) strain and those petites which retain all three markers show recombination properties similar to those of the ^- parental strain. However, other rho ^- mutants that retain the cap1 and ery1 loci but have lost the tsr1 locus, which is located between cap1 and ery1, show markedly different properties of mitochondrial transmission and recombination, consistent with the presence of ⁺ alleles. The association of an internal deletion between the cap1 and ery1 loci with a change in phenotype provides additional evidence for the location of between these two loci.Although the petites deleted for the tsr1 locus exhibited the recombination properties of ⁺ strains, it was not possible to transmit this characteristic to rho ⁺ recombinant cells. Experiments on the kinetics of elimination by ethidium bromide of the cap1 and eryl markers from the petites and measurements of the buoyant densities of their mtDNA species did not indicate major changes (such as selective sequence repetition) in the sequences of the mtDNAs. The possible nature of the changes in the mtDNAs of these petites is discussed in light of recent studies on the physical nature of the alleles.     

A mutant rho ATPase from Escherichia coli that is temperature-sensitive in the presence of RNA

Summary The Escherichia coli mutant rho-115 suppresses lac operon polarity conferred by the lacZ::IS1 insertion MS319. The ATPase activity of purified rho-115 protein was maximal at 40°C, in contrast to 45°C for rho ⁺. At higher temperatures (50°C, 55°C), the fractions of activities at maximal temperature were consistently lower for rho-115 compared to rho ⁺. The 30-minute time course of rho-115 ATP hydrolysis was linear at 37°C but at 45°C the linear kinetics of hydrolysis reached a plateau between 10 and 15 minutes. The 30-minute time courses for rho ⁺ were linear at both 37°C and 45°C. The rho-115 and rho ⁺ ATPase activities were equally heat-stable during preincubation at 45°C in buffer. Inclusion of ATP during preincubation protected these rho proteins from inactivation to the same extent. The presence of polyC during preincubation protected rho ^- activity but produced substantial inactivation of rho-115 ATPase. The presence of polyU during preincubation gave similar results. Concentrations of polyC between 625 ng/ml and 100 g/ml yielded the same extent of rho-115 ATPase inactivation during preincubation at 45°C. Thermal inactivation of rho-115 ATPase by polyC was halted by shifting preincubation temperature from 45°C to 35°C, indicating that polyC-induced destabilization of rho-115 was irreversible.     

Extrachromosomal inheritance of nalidixic acid resistance in the petite negative yeast Schizosaccharomyces pombe

Summary Spontaneous mutants resistant to nalidixic acid (NAL) were isolated from the petite negative yeast Schizosaccharomyces pombe (S. pombe). One of these mutants, resistant to 200 g/ml NAL, nal ^r–Y13, was characterized both genetically and biochemically. The extrachromosomal inheritance of this mutation was demonstrated both by mitotic segregation and by mitotic haploidization analysis. In the wild-type, NAL at a concentration of 100 g/ml almost completely inhibits incorporation of [¹⁴C]adenine in total DNA as well as in mitochondrial DNA. In the NAL-resistant mutant both total DNA synthesis and mitochondrial DNA synthesis were resistant to the drug. These results are discussed in view of previously published findings on the close interaction between the two DNA synthesizing systems in S. pombe.     

News & Notes: Correlation of Resistance to the Alkaloid Lycorine with the Degree of Suppressiveness in Petite Mutants of Saccharomyces cerevisiae

In previous papers (Del Giudice et al. Curr Genet 8:493–497, 1984; Massardo et al. Curr Genet 17:455–457, 1985) we have shown that strains of Saccharomyces cerevisiae that are devoid of mitochondrial DNA (rho ^o) are resistant to the alkaloid lycorine isolated from Amaryllis plants, whereas strains containing mitochondrial DNA (rho ⁻ , mit ⁻ , or rho ⁺ ) are sensitive to this drug. In addition, we were able to show that the so-called hypersuppressive petites, whose mitochondrial genomes consist of short regions of DNA containing an ori sequence, show intermediate resistance. In this paper, we demonstrate that the degree of suppressiveness of a rho ⁻ mutant correlates with the degree of resistance to lycorine. Received: 20 September 1996 / Accepted: 10 January 1997     

Petite deletion map of the mitochondrial oxi3 region in Saccharomyces cerevisiae.

Summary Fifty eight mitochondrial mutants (p ⁺ mit^- mutants), all deficient in cytochrome oxidase activity and previously assigned to the genetic region oxi3 on the mitochondrial DNA, were mapped by the method of petite deletion mapping.This procedure resulted in the identification of at least twenty one different classes of oxi3 mutants, which could be arranged in a linear order.Moreover, it provided a set of twenty three p ^- petite mutants, each containing a differentially deleted mit DNA segment included in the oxi3 region. The two sets of mutants, p ⁺ oxi3 ^- and p ^- oxi3 ⁺, will be of interest for a further genetic and physical analysis of this mitochondrial DNA segment which spans over about ten thousand base pairs and controls the subunit I of cytochrome oxidase.     

Transfer of cytoplasmic organelles from an oligomycin-resistant Nicotiana cell suspension into tobacco protoplasts yielding oligomycin-resistant cybrid plants

Summary Oligomycin-resistant lines were derived from a Nicotiana sylvestris cell suspension, after N-nitroso-N-methylurea mutagenesis followed by selection in the presence of 0.4 g/ml oligomycin, a specific mitochondrial ATPase inhibitor. One of the lines, oli ^R38 was further analyzed to investigate the role of mitochondria in this resistance. The oli ^R38 line proved to be also highly resistant to venturicidin, another specific inhibitor of mitochondrial ATPase. By the donor-recipient protoplast-fusion procedure the cytoplasmic organelles of oli ^R38 were transferred to protoplasts of Line 92, a line of tobacco plants which contain the cytoplasmic organelles of N. undulata. Cell suspensions prepared from several cybrid plants, containing the cytoplasmic organelles of oli ^R38, exhibited the same level of oligomycin resistance as the oli ^R38 line.     

Effect of auxotrophic starvation on mitochondrial marker transmission in the cdc8 mutant of Saccharomyces cerevisiae

Summary Crosses were made using strains of S. cerevisiae which carried mitochondrial markers conferring resistance to erythromycin and chloramphenicol. The effect of auxotrophic starvation of one parent prior to mating on the transmission of its mitochondrial markers was studied in different crosses relative to the presence of the cdc8 nuclear mutation (a temperature-sensitive DNA replication).In crosses between two cdc8 mutant strains, auxotrophic starvation of one of the haploid parental strains prior to mating caused a marked decrease of its mitochondrial marker transmission to the diploid progeny of the cross. The transmission decreased as a function of the time of starvation. This effect was not observed in the cross between two wild type strains and in crosses of starved cdc8 phenotypic revertants with cdc8 mutant strains. Only a small, if any, effect of starvation on mitochonrial marker transmission was observed when starved cdc8 mutant strains were crossed either with their phenotypic revettants or with the wild-type strains.In one of the haploid parental strains the starvation increased the frequency of petites as a function of starvation time, while in the other this effect was not observed.In the progeny of cdc8xcdc8 crosses (both in starvation experiments and in control crosses) an increased frequency of diploid petite cells accompanied by a decreased frequency of recombination between mitochondrial markers was noticed.The influence of the cdc8 mutation on the transmission of mitochondrial markers is discussed in terms of high frequency of ^– molecule formation in cdc8 strains.     

Cytoduction: A tool for mitochondrial genetic studies in yeast

Summary A study has been made of the general applications of the nuclear-fusion mutation, kar1, to mitochondrial genetic research. Procedures were developed which are suitable for constructing new strains by transfer (cytoduction) of mitochondrial genomes containing drug ^r, mit ^-, syn ^- or rho ^- mutations.Several examples of crosses of the type KAR rho ⁺ drug ^r xkarl rho ^o drug ^s were carried out and the resulting zygotes and their first buds separated by micromanipulation. Clones derived from these were in most cases homogeneous for any of the following nuclear types: heterokaryon (a+), diploid (a/), haploid a nucleus or haploid nucleus. The term cytoductant is given to the haploid rho ⁺ segregant having the same type of nucleus as the rho ^o strain employed in the cross.As examples of cytoduction of mit ^- mutations, two different pho mitochondrial genomes were studied. Transfer of pho to rho ^o acceptors was achieved with little difficulty.Haploid segregants containing recombinant mitochondrial genomes were obtained by crossing a KAR rho ⁺ cap ^r oli ^j par ^r strain with a karl rho ⁺ cap ^s oli ^s par ^s strain. Studies of intraclonal distribution of alleles indicate that cytoplasmic mixing is restricted when the dikaryotic state is maintained. Haploid cytoductant clones are usually comprised of cells retaining the mitochondrial genome of only one parent, and commonly both first-bud and residual-zygote clones are homogeneous for the same parental genome (apparent uniparental inheritance). Less frequently mixed clones are found where the number of classes of mitochondrial recombinant type is limited compared to the progeny of zygotes in which nuclear fusion takes place.These observations are discussed in terms of number and form of mitochondria forming the chondriome in the yeast cell, together with its association with the nucleus.     

Synergistic effects of ethanol and temperature on yeast mitochondria

At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho ⁺ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho ^– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho ⁺ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho ^– cells in the population is very high. This does not result in an increase in the frequency ofrho ^– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho ^– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho ⁺ andrho ^– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho ^– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho ^– mutants are less viable thanrho ⁺ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho ^– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol.     

A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation

Summary In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD ⁺) strains carrying mitochondrial DNA or not (rho ⁰). This was also true for the following DNA-repair deficient (rad ^-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae

Summary In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H⁺-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to man the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu⁺ and Pma⁺ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5 end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.Publication n^o 2456 from the Biology Directorate of the Commission of European Communities