Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Cytoduction as a new tool in studying the cytoplasmic heredity in yeast

Summary When crossing the haploid cells of genetically marked yeast strains we observed the appearance of both normal diploid zygotes and haploid nuclear cytoplasmic hybrids. The latter had the nuclear markers of one and the cytoplasmic marker (rho⁺) of the other parent. The autonomous cytoplasmic factor transfer was termed as cytoduction. Cytoduction is supposed to be the abortive form of yeast cell mating. Only about 1% of cytoductants is usually observed.Cytoduction can be used as a simple test on cytoplasmic determination of some characters. We observed the transfer into cytoductant cells of not only rho⁺ marker but of resistance factors to antibiotics (erythromycin, neomycin) and killer factor as well. Cytoduction can be applied towards constructing strains having the identical nucleus genotype with mitochondria and other cytoplasmic factors of different origin.In crossing strains with doubly marked mitochondria recombination of mitochondrial markers in cytoductant haploid cells was observed, the pattern of which was similar to that of mitochondrial recombination in normal zygotes.     

Le transfert autonome des facteurs mitochondriaux (la cytoduction) lors du croisement des cellules de levure Saccharomyces cerevisiae

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 ρ⁻ parent and the mitochondrial markers (rho⁺ E^R C^R) of the other parent. The frequency of such cytoduction was about 1 per cent. The mitochondrial markers E^R and C^R were transmited to cytoductants together and did not segregate. The possible mechanisms of the cytoduction and its significance are discussed.     

Genetic determination of ubiquinol-cytochromec reductase

Summary In order to find new genetic loci on the yeast mitochondrial DNA, especially mutations affecting the structure and function of ubiquinol-cytochrome c reductase, 45 independently arisen mutants resistant to mucidin have been isolated after MnCl₂ mutagenesis. The majority of the mutants exhibited increased sensitivity to chloramphenicol, diuron and antimycin A, respectively. it was shown by several criteria that all mutants resulted from mutations localized on the mitochondrial DNA.The allelism tests revealed that these mutations fall into three distinct loci muc1, muc2 and muc3. Mutations at a new locus muc3 were correlated with the changes in the binding or inhibitory sites on the inner mitochondrial membrane. Multifactorial crosses involving the mucidin resistance mutations and mitochondrial mutations conferring resistance to chloramphenicol, erythromycin, oligomycin and diuron revealed that the studied mutations at the loci muc1, muc2 and muc3 did not significantly influence the process of mitochondrial recombination and its control by the mitochondrial locus . The locus muc1 was found to be allelic to the locus diu2. The locus muc2 which was found to be allelic to cob1 locus appears to be linked to the locus oli1 but unlinked to the loci , cap1, ery1 and muc1. The new locus muc3 appears to be weakly linked to the locus diu1 but unlinked to the loci , cap1, ery1, oli1 and muc1.The results are consistent with the gene order oli1-muc2-muc3-diu1-muc1-oli2 and suggest the participation of at least three mucidin resistance loci and one diuron resistance locus in the biogenesis of the bc ₁ complex of the mitochondrial respiratory chain.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Circadian rhythms in Neurospora crassa: the effects of point mutations on the proteolipid portion of the mitochondrial ATP synthetase

Summary Five oligomycin-resistant (oli ^r) mutant strains of Neurospora crassa were analyzed for their growth rate and for the periodicity of their circadian rhythm. The most resistant strains had periods of 18–19 h while the least resistant strain had a normal period of 21.0 h. There was a rough correlation between the in vivo degree of oligomycinresistance and the amount of change in the period. Several of the oli ^r mutations have been previously described by Sebald et al. (1977) in terms of known amino acid changes in the primary structure of the proteolipid, or DCCD-binding protein, found in the F₀ membrane portion of the mitochondrial ATP synthetase. Amino acid changes in the structure of this protein are reported here for two other oli ^r mutations. The proteolipid isolation procedures were slightly modified to include a delipidation step, and an HPLC procedure was developed to separate the hydrophobic peptides of this protein. Analysis of heterocaryons carrying both the oli ^r and oli ^s markers indicated that the oli ^r and oli ^s mutations were codominant to each other in terms of period and growth rate. The changes in the primary structure of this DCCD-binding protein reported here are the first known examples of changes in the primary structure of a protein which alter the period of a circadian rhythm.     

The genetic map of the mitochondrial genome in yeast

Summary An approach for the screening of mit ^- mutants, the isolation and preliminary classification of a series of such mutants is reported. Loss and retention of 8 mit ^- and 6 drug ^r markers in mitDNA was analyzed in populations of rho^- clones derived from four yeast strains. The populations studied constitute a representative fraction of the rho^- petites formed during growth at 35° C under the influence of mutation tsp-25 which is in common to the four strains. The majority of the rho^- clones retained several of the markers studied. Depending on the marker regarded retention frequencies between 15% (oxi3) and 45% (oli1, cob) were observed. Loss of one and retention of the other of a pair of markers was determined in all rho^- clones of the four populations. The frequencies of marker separation by rho^- deletion thus obtained are assumed to reflect the distance between markers on the mitochondrial genome: the higher the frequency of separation the longer the distance between two markers. Based on these frequencies a unique order of markers on a circular map was determined. Positions of markers on a scale from 0 to 100 were found to be: cap/ery (0) — olil (16) — cob1-1354 (21) — ana101 (22) — cob2-1625 (24) — oli2 (35) — pho1 (40) — oxi3-2501 (44) — oxi3-3771 (47) — par (65) — oxi2 (79) — oxil (87) tms8 (93) —cap (100). The relevance of this map as to the faithful representation of the topology of gene loci on mitDNA is discussed. Correlation of retention frequencies of markers to their map positions reveals a pronounced polarity: mitDNA segments carrying the cob-oli1 segment prevail whereas segments retaining oxi3 are the least frequent.     

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.     

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.     

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.     

Extrachromosomal inheritance inSchizosaccharomyces pombe

Summary Spontaneous chloramphenicol (cap ^r)- and erythromycin (ery ^r)-resistant mutants were isolated from strain ade7–50 h ^- and the antimycin-resistant mutant ana ^r-8 ade 7–50 h^- of Schizosaccharomyces pombe (Sch. p.). By mitotic segregation analysis all 154 cap ^r- and 120 ery ^r-mutants derived from ade 7–50 h ^- proved to be recessive chromosomal, whereas all 108 cap ^r- and 200 ery ^r-mutants originating from ana ^r-8 were extrachromosomally inherited. The rate of spontaneous cap ^r- and ery ^r-mutants was about hundredfold in ana ^r-8 compared to ade 7–50 h ^-. Growth of cap ^r-and ery ^r-mutants was not inhibited by chloramphenicol or erythromycin, respectively, in glucose-medium and only slightly in glycerol-medium at concentrations which completely inhibited ana ^r-8. By mitotic segregation-, tetrad-, and mitotic haploidization-analysis the extrachromosomal inheritance of mutants derived from ana ^r-8 was established. Segregational patterns of cap ^r- and ery ^r-determinats during mitosis, meiosis, and mitotic haplidization of diploids are discussed.     

General organization of the genes specifically involved in the diaminopimelate-lysine biosynthetic pathway of Corynebacterium glutamicum

Summary To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a supersuppressor selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HAT^r) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT mutants — selected by their resistance to 6-thioguanine (TG^r) — and TK^- mutants — selected by their resistance to 5-bromodeoxyuridine (BrdUrd^r) — are HAT^s. Therefore, from HPRT^- TK^- double nonsense mutants, whose phenotype would be TG^r BrdUrd^r (HAT^s), simultaneous HPRT⁺ TK⁺ double phenotypic revertants could be obtained by selecting HAT^r (TG^s BrdUrd^s) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TG^r variants, 53 of which were HAT^s and the rest HAT^r. Among 36 TG^r (HAT^s) variants tested, 23 did not revert to HAT^r, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT^- nonsense mutants because they were very stringent (had less than 2% of wild-type [³H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrd^r) in 7 of these strains allowed us to isolate 29 TG^r BrdUrd^r (HAT^s) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HAT^r (TG^s BrdUrd^s) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.     

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.     

Association between defects of karyogamy and translation termination in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mutants capable of a high frequency of cytoduction (Hfc⁺) were obtained in a haploid strain of Saccharomyces cerevisiae, suggesting impaired cytogamy. Nine of the 68 Hfc⁺ mutants showed the antisuppressor effect with respect to mutations of the SUP35 and SUP45 genes, which code for translation termination factors, or to the [PSI ⁺] factor, which is the prion form of Sup35. Cosegregation of the characters higher frequency of cytoduction and antisuppression was demonstrated for three Hfc⁺ mutants. One (HFC12-2) of the Hfc⁺ mutations exerted a dominant antisuppressor effect with respect to [PSI ⁺] and had no effect on [PSI ⁺] maintenance. On the strength of the results, an interaction was assumed for translation termination components and cytoskeleton proteins, which play a role in karyogamy in yeasts.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 178–186.Original Russian Text Copyright © 2005 by Borchsenius, Repnevskaya, Kurischko, Inge-Vechtomov.     

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.     

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.     

Effects of tellurite on growth of Saccharomyces cerevisiae

The effects of potassium tellurite on growth and survival of rho⁺ and rho⁰ Saccharomyces cerevisiae strains were investigated. Both rho⁺ and rho⁰ strains grew on a fermentable carbon source with up to 1.2 mM K₂TeO₃, while rho⁺ yeast cells grown on a non-fermentable carbon source were inhibited at tellurite levels as low as 50 μM suggesting that this metalloid specifically inhibited mitochondrial functions. Growth of rho⁺ yeast cells in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture, a phenomenon not observed with rho⁰ cultures. Transmission electron microscopy of S. cerevisiae rho⁺ cells grown in the presence of tellurite showed that blackening was likely due to elemental tellurium (Te⁰) that formed large deposits along the cell wall and small precipitates in both the cytoplasm and mitochondria.     

Mitochondrial genes inPodospora anserina: Recombination and linkage

Summary A fifth cytoplasmic mutation (cap ^r 1) obtained inPodospora anserina is described. In addition to chloramphenicol resistance it confers a strong deficiency in cytochrome aa₃ and impairs the germination of ascospores. Genetic analysis shows: 1) strict maternal inheritance of (cap ^r 1) allele; 2) selection against the (cap ^r 1) allele as well in sexual crosses as during vegetative growth; 3) complete reversion of this selection by even low concentration of CAP. On the basis of their cytoplasmic inheritance and altered cytochrome spectra the five cytoplasmic mutations are assumed to be mitochondrial. Analysis of crosses between them allows to class them in 3 loci, 2 of which being closely linked.     

Study of the Solute Flows of Multicomponent and Heterogeneous Non-Ionic Solutions in Double-Membrane System

The solute flows were studied in a double-membrane osmotic-diffusive cell, in which two membranes mounted in horizontal planes separate three compartments (l,m,r) containing the non-homogeneous, non-electrolytic binary and ternary solutions. The volume of inter-membrane compartment (m), which is the infinitesimally layer of solution, and volume of external compartments (l and r) fulfill the conditions V _m 0 and V _l =V _r , respectively. In an initial moment, the solution concentrations satisfy the condition (C ^o _s ) _l < (C ^o _s ) _m >(C ^o _s ) _r. The double-membrane osmotic-diffusive cell is composed of two complexes: boundary layer/membrane/boundary layer, mounted in horizontal planes. In the cell, solute flux was measured as a function of concentration and gravitational configuration. The linear dependencies of the solute flux on concentration difference in binary solutions and nonlinear – in ternary solutions were obtained. It was shown that the double-membrane osmotic-diffusive cell has rectifying and amplifying properties of solute flows.     

Assembly of the mitochondrial membrane system: Sequence analysis of a yeast mitochondrial ATPase gene containing the oli-2 and oli-4 loci

The mitochondrial DNA (mtDNA) segments of several ρ^? mutants carrying the oli-2, oli-4 and pho-1 loci have been sequenced. The segments contain a common structural gene sequence that has been identified to include all three genetic markers. The gene codes for a protein with a molecular weight of 28,257. This new gene is located between 61.5 and 62.6 units on the wild-type map of Saccharomyces cerevisiae and is transcribed from the same DNA strand as most other yeast mitochondrial genes sequenced to date. The amino acid composition and sequence deduced from the DNA sequence indicate that the protein is very hydrophobic, with three long domains (>30 residues) consisting of nonpolar amino acids. Based on its molecular weight, the gene product is tentatively proposed to be either subunit 3 or 6 of the oligomycin-sensitive ATPase.