Genetic analysis of the products of a cross involving a suppressive ‘petite’ mutant of S. cerevisiae

Summary A genetically defined highly suppressive petite yeast strain ( ^–cob⁺A^sE^oC^oO^oP^o) was crossed with a grande strain carrying a multiply marked mitochondrial genome ( ⁺A^rE^rC^rO rp^r). Petite diploid progeny, isolated from individual zygotic clones consisting either of wholly petite or mixtures of grande and petite cells, were characterised genetically by crossing to grande haploids. The diploid petites were found to closely resemble the petite parent and in general not to carry mitochondrial markers from the grande parent. In the petites from the mixed clones recombination was detected, but only within the region of homology between the genomes. These observations are inconsistent with models of suppressiveness based on destructive recombination and suggest that the petite genome eliminates the grande genome from zygotic progeny through being preferentially replicated. The most plausible model to explain the observed pattern of zygotic clones postulates a limited number of mDNA replication sites in zygotes, competition for sites between input mDNA molecules and an advantage in this competition for suppressive ^– mDNA.     

Does mitochondrial DNA length influence the frequency of spontaneous petite mutants in yeasts?

Summary Results from the theory of random walks applied to the random excision hypothesis for production of petite mutants in yeast suggest that frequency of excision should increase as a linear function of mitochondrial DNA length (see appendix). For a series of petite positive yeasts we have determined the spontaneous petite frequency (ranging from about 0.003% to 9%) and length of mtDNA (ranging from about 19 Kbp to c. 108 Kbp) and found that, while the frequency of petite mutants does generally increase with mtDNA length, the relationship is far from linear. Although these results are inconclusive concerning the random excision hypothesis they do indicate that amongst related yeasts other factors have a greater influence than mtDNA length in determining the frequency of petite mutants.     

The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene

Summary As part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to lie in its single G,C rich cluster, which differs in just one nucleotide from the standard ori ^s sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergencies (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.     

Cytoplasmic mixing in Saccharomyces cerevisiae: Studies on a grande X neutral petite mating and implications for the mechanism of neutrality

Summary We have studied zygotic cytoplasmic mixing in two neutral petite by grande matings. Cytoplasmic mixing in zygotes and zygotic buds was followed histochemically and genetically. Both techniques showed that in most zygotes rapid cytoplasmic mixing occurred after early zygote formation and before first bud completion. The progeny mitochondrial genotypes produced from each cross were compatible with a model assuming random segregation of petite and grande inputs between the zygote and its first bud.While both crosses produced only grande zygotic colonies and grande diploid progeny when assayed by classical genetic methods, a class of events producing petite zygotes, retaining petite mitochondrial DNA, with grande first diploid buds was discovered. The results show that the expression of neutrality in our strains requires efficient cytoplasmic mixing during zygote maturation. The results suggest that the expression of mitochondrial genome neutrality requires competition or interaction between petite and grande parental mitochondrial DNA molecules. The possible nature of these phenomena is discussed.     

A controversy concerning the structure of the mitochondrial genome of a yeast petite mutant: resolution by sequence analysis

Summary Sequence analysis was used to define the repeat unit that constitutes the mitochondrial genome of a petite (rho ^–) mutant of the yeast Saccharomyces cerevisiae. This mutant has retained and amplified in tandem a 2,547 by segment encompassing the second exon of the oxi3 gene excised from wild-type mtDNA between two direct repeats of 11 nucleotides. The identity of the mtDNA segment retained in this petite has recently been questioned (van der Veen et al., 1988). The results presented here confirm the identity of this mtDNA segment to be that determined previously by restriction mapping (Carignani et al., 1983).     

The organization of repeating units in mitochondrial DNA from yeast petite mutants

Summary We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ^– petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ^– petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.     

In-vivo selection of an azole-resistant petite mutant of Candida glabrata

Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.     

Effects of T-2 toxin on induction of petite mutants and mitochondrial function in Saccharomyces cerevisiae

Summary The influence of the trichothecene mycotoxin T-2 on the mitochondria of Saccharomyces cerevisiae was studied. T-2 is a cytotoxic molecule inhibiting growth and macromolecular synthesis in S. cerevisiae. At low concentrations, T-2 toxin arrested yeast growth on glycerol medium and at higher concentrations, it arrested growth on glucose medium. The toxin was not capable itself of inducing petite mutations. Its inhibitory effect on the growth of petite strains, of both chromosomally isogenic and nonisogenic strains was less than that of grande strains. One exception to this was equally low susceptibility of psi ⁺ SUP4-3 strain in both rho ⁺ and rho ^– state. T-2 toxin was also capable of retarding the petite inducing activity of the mutagen, ethidium bromide. T-2 toxin inhibited the polymerization of P-ribosylaminoimidazole in an ade2 strain of S. cerevisiae. These results show that T-2 toxin is capable of interfering with the activity of the mitochondria in addition to its well studied effects on cytoplasmic protein synthesis.     

Crosses between Saccharomyces cerevisiae and Saccharomyces bayanus generate fertile hybrids

Crossings between strains of Saccharomyces cerevisiae and Saccharomyces bayanus were carried out. Genetic, molecular and electrophoretic karyotyping data indicated that interspecific hybrids were obtained. The hybrid cells segregated "grande" and "petite" colonies, and the latter ranged between 20 and 50%; unlike "grande" colonies, "petite" colonies did not sporulate and did not ferment maltose. In the hybrids, the extent of sporulation varied between 10 and 20%; only very rare asci (around 10(-4)) held viable ascospores. Clones from the viable ascospores sporulated and produced asci with viable ascospores able to give mating with spores from both hybrid derivatives and parental species. Fertile asci could derive from allotetraploid cells generated by endomitotic events in allodiploid cells, a mechanism that enables overcoming the species barrier between S. cerevisiae and S. bayanus.     

Amphimeric mitochondrial genomes of petite mutants of yeast. II. A model for the amplification of amphimeric mitochondrial petite DNA

  A model for the recombination-directed replication and amplification of the mtDNA of amphimeric petite mutants of S. cerevisiae is proposed. Replication of an amphimeric master basic unit datA would be initiated in the inverted components a and A. The initiation of replication should be associated with the amphimeric structure of the master basic unit itself, but could be promoted by the presence of ori sequences or of sequences facilitating the initiation of replication in the inverted duplications. The amplification unit of amphimeric genomes is considered to be the double-stranded circular hetero-diamphimer datA-DaTA. Amplification of both diamphimeric strands involves an invasion of the 3′ ends of the newly synthesized strands into symmetrical homologous duplex DNA regions promoting the continuation of replication, and leads to the accumulation of two (``flip' and ``flop') types of multi-amphimers. We consider that this mode of amplification represents a modified rolling-circle mechanism. By analogy, we propose to call our model of amplification the ``rocking-circle model'. This model is likely to apply to other genomes organized as amphimeric structures. Received: 2 November 1995     

Effects of the kar gene on cytoplasmic mixing and mitochondrial genome suppressiveness,and consequences for cytoduction of petite DNA in Saccharomyces cerevisiae

Summary During a series of cytoduction experiments to transfer Saccharomyces cerevisiae mitochondrial genomes from one nuclear background to another, using the karl-1 nuclear fusion mutation, one of the five petite genomes used proved difficult to transfer. This genome, ^- F13, was highly suppressive (90%) in its original nuclear background. Molecular and genetic studies on the putative karl-1 ^–F13 cytoductant were done to discover the nature of this difficulty. They showed that while the ^–F13 was maintained in a karl-l background, zygotes from a mating with a ⁰ strain showed poor cytoplasmic mixing and therefore inefficient ^–F 13 DNA transfer into first zygotic buds. This also caused a reduction of ^–F13 suppressiveness to 20–30% in crosses with different ⁺ strains. The effect was genome specific since another highly suppressive petite in the karl-l background did not show suppressiveness reduction when crossed to ⁺. The nature of suppressiveness modulation is discussed. Since the ^–F13 genome was eventually transferred using a modification of the original scheme, the problems were not caused by the inability of the acceptor nuclear background to maintain the ^–F13 genome.     

Distribution of ultraviolet light irradiated mitochondrial genomes during meiosis in yeast

Summary Clones derived from ascospores from ultraviolet irradiated diploid cells were examined for the genetic determinants or respiratory properties. Approximately 10% of the cells produced petites of mitochondrial origin at the dose applied. Among 13 asci which produced mitochondrial petites with high frequencies, 6 asci of uniparental type, 0 grandes : 4 petites, were observed. Furthermore, most of the petite spore clones from each individual uniparental ascus showed similar levels of suppressiveness and of mitochondrial gene retention. From these results it is suggested that a single mitochondrial genome participates meiosis in yeast.     

Structural and replicative forms of mitochondrial DNA in tissues from adult and senescent BALB/c mice and Fischer 344 rats

Age-related changes in the structure and replication of mitochondrial DNA (mtDNA) were investigated in different organs from young adult (9–10 months' old) and senescent (28–29 months' old) BALB/c mice and Fischer 344 rats. Total mtDNA from brain, heart, kidney and liver was isolated by centrifugation in ethidium bromide—CsCl gradient and examined for the occurrence of complex forms and replicative intermediates by electron microscopy. The frequency of catenated mtDNA (interlinked molecules containing two or more circular units) varied from about 2.5% to 5% in adult tissues and showed a small increase in the majority of senescent organs. The frequency of double-sized circular molecules, or circular dimers, was very low in adult tissues, with an average of about 0.04% in mice and 0.1% in rats. The frequency of circular dimers increased with aging to 1.9% in mouse brain and 1.5% in rat kidney, with smaller increases (0.4% and 0.7%) in heart mtDNA from both species; there was no significant increase in the other organs. It is suggested that the increase in the frequency of circular dimer mtDNA reflects an overall deterioration of tissue physiology rather than intrinsic senescent changes in the mitochondria. The frequencies and types of the various replicative forms of mtDNA varied significantly according to tissue but not according to species or donor age. The only exception was a significant increase in the frequency of larger replicative forms in senescent mouse liver, to about 20% compared with 12% in adult liver, suggesting an age-related change in the rate of mtDNA replication and/or turnover in this organ.     

Correlation of the frequency of petite formation by isolates of Saccharomyces cerevisiae with virulence.

In previous studies on the colony phenotype switching of Saccharomyces cerevisiae, we observed that the least virulent isolates formed greater numbers of petite colonies when grown at body temperature, 37 degrees C. To determine if there is a link between virulence and petite formation, we examined the frequency of spontaneous petite formation for virulent clinical isolates (YJM128, YJM309), an intermediate virulent segregant of YJM128 (YJM145) and avirulent clinical (YJM308) and nonclinical S. cerevisiae (Y55, YJM237) after growth at 37 degrees C. The rank order of increasing frequency of petite formation was YJM128 = YJM145 < YJM309 < Y 55 < YJM308 = YJM237, which is similar to the rank-order of virulence in CD-1 mice. To assess the virulence of petites in vivo, two mouse models, CD-1 and DBA/ 2N, were infected i.v. with 10(7) cfu of either the parental grand or a spontaneously derived petite from one of four isolates previously classified with differing degrees of virulence: YJM128, YJM309, YJM145 and Y55. In both CD-1 and DBA/2N, the mean log10 cfu of grands recovered from the brain was significantly higher than that of the petites (P<0001). Overall, petites were significantly less virulent than the parental strains. However, death of some DBA/2N mice caused by YJM128 petite 1 showed that petites are not totally avirulent. To see if S. cerevisiae isolates form petite colonies in vivo, both mouse models were infected with parental grands of YJM128 and Y55. Recovered colonies were counted and confirmed as grand or petite, and the frequency of petite colonies in the brain, the target organ, correlated with the in vitro results. Overall, these studies show an inverse correlation between the frequency of petite-colony formation and the previously determined virulence of S. cerevisiae in CD-1 mice. Furthermore, petites were significantly less virulent than the parental grands, in most cases, and petites are spontaneously formed in vivo at a frequency inversely correlated to the virulence of the strain.     

Non-Utilization of sucrose by the petite mutant of a distiller's yeast

Summary A number of yeast strains are known to be unable to metabolize several sugars (galactose, maltose, -methylglucoside) when converted to their petite mutants. The basis of this phenomenon is considered to be the loss of the ability to transport the sugars across the cell membrane. However, sucrose is believed to be hydrolyzed before the products are transported into the cell, and the enzyme responsible (invertase) is thought to be either present in the periplasmic space or to be bound to the outer surface of the cytoplasmic membrane. Hence the loss of the ability to metabolize sucrose may infer the impairment of the mechanism for transport of invertase to its normal location outside the cytoplasm. We have found a distiller's yeast strain which has lost the ability to metabolize sucrose when it is converted to the petite mutant, and we report here some of its properties. We have shown that the cell produces invertase, which is present in the cell-free extract, but not in the pellet of cell walls and unbroken cells, though we have not determined whether the enzyme is present in the cytoplasm in the glycosylated or the unglycosylated form. The ability of the strain to ferment sucrose is also impaired in respiratory-competent cells, when the determination is made under anaerobic conditions.     

Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates

Candida infections are a permanent threat to immunocompromised individuals such as cancer patients, and Candida glabrata has emerged as a major problem in recent years. Resistance may develop during lengthy antifungal therapies and is often mediated by upregulation of fungal drug efflux pumps. During chemotherapy the yeast cell is also exposed to cytotoxic agents that may affect its drug susceptibility. Four C. glabrata isolates, three susceptible and one resistant to fluconazole (FLU), were incubated with 20 μg/ml of doxorubicin (DOX) for 90 min. In a second experiment, the isolates were cultured with DOX for ten days. Samples were taken on subsequent days to determine the minimal inhibitory concentration (MIC) of FLU and to analyze expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Samples were also used to assess the petite phenotype. Short-term DOX exposure did not induce efflux pump gene expression, but genes were consistently overexpressed in FLU-susceptible isolates during long-term exposure. An increase in MIC values on day 6 in two of the isolates coincided with the first occurrence of petite mutants in all susceptible isolates. The respiratory deficiency of selected petite mutants was confirmed by culturing mutants on agar containing glycerol as the sole carbon source. FLU MIC values for respiratory-deficient clones were ≥64 μg/ml, and efflux pump gene expression was greatly increased. The resistant isolate did not develop mitochondrial dysfunction. In summary, the cytotoxic agent DOX selects for FLU-resistant respiratory-deficient C. glabrata mutants, which may affect antifungal therapy.     

Repair properties in yeast mitochondrial DNA mutators

Summary After ethy1methanesulfonate mutagenesis of the strain Saccharomyces cerevisiae D273-1013, out of 100,000 survivors, 1,000 were selected for their high production of petite mutants at 36 °C. Among these 1,000 mutators, 5 also showed an increased frequency of spontaneous point mutations measured at 25 °C. Further analysis revealed that in all mutators, except 2, petite accumulation proceeded at 25 °C as well as 36 °C. In these 2 mutants, the production of petite mutants was much higher at 36 °C than at 25 °C. In one of them, however, the mutator and the thermosensitive petite phenotypes were due to mutations in two unlinked nuclear genes. In the other mutants, both traits were the result of a mutation in a single nuclear gene. The mutators fell into three complementation groups (tpm1, tpm2, mup1). No complementation was observed between tpm1 mutants and the gam4 mutant previously described by Foury and Goffeau (1979). From the latter and the present works, only four complementation groups (gam1, gam2, gam4 or tpml, mupl) have been identified and it is likely that the number of genes controlling specifically the spontaneous mutability of the mtDNA is low. The mutators exhibited a variety of responses to damaging agents such as UV light and ethidium bromide; especially in a representative mutant from the complementation group tpm1, the induction of ^– mutants was sensitive to UV light and resistant to ethidium bromide. In addition, we found that in the mutants from this complementation group, the synthesis of mtDNA in isolated mitochondria was low; however their mitochondrial DNA polymerase activity was similar to that of the wild type strain. A relationship might exist between the mutator phenotype and the low mtDNA synthesis in the tpm1 mutants.     

Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae

Summary Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glyccrol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

Kinetics of petite mutation and thermal death in Saccharomyces cerevisiae growing at superoptimal temperatures

Mass formation of petite mutants took place in a strain of Saccharomyces cerevisiae when grown at superoptimal temperatures. After an initial period of exponential growth, a second period followed during which exponential death and net exponential petite mutation concurred with exponential growth. The specific rates of the three exponential processes were of the same order of magnitude and varied with the temperature. Net exponential petite mutation did not occur during the deathless first period of growth at superoptimal temperatures nor at any time during growth at suboptimal temperatures. Mitochondria are discussed as possible targets of thermal death in mesophilic yeasts.