Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts

A systematic investigation covering a wide diversity of yeast species was made on the appearance of respiratory deficient (petite) mutants after treatment with acriflavine. Petite mutants were obtained from certain species only, but in these species all strains were found to have in common the property of giving rise to petite mutants; such species were designated as petite positive. Species failing to give rise to petite mutants were accordingly called petite negative. The primary action of acriflavine, namely the inhibition of the synthesis of the respiratory system, was shown to occur not only in petite positive yeasts, but also in petite negative ones. Some implications of the results are discussed.     

A possible relationship between the fatty acid composition of yeasts and the ‘petite’ mutation

A number of Crabtree-positive and Crabtree-negative yeasts were tested for their ability to yield respiratory-deficient (petite) mutants on treatment with acriflavine. Crabtree-positive species produced petite mutants but did not contain the polyunsaturated linoleic (C 18.2) and linolenic (C 18.3) fatty acids. Crabtree-negative species contained these fatty acids and were resistant to acriflavine. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

Ability for anaerobic growth is not sufficient for development of the petite phenotype in Saccharomyces kluyveri

Saccharomyces cerevisiae is a petite-phenotype-positive ("petite-positive") yeast, which can successfully grow in the absence of oxygen. On the other hand, Kluyveromyces lactis as well as many other yeasts are petite negative and cannot grow anaerobically. In this paper, we show that Saccharomyces kluyveri can grow under anaerobic conditions, but while it can generate respiration-deficient mutants, it cannot generate true petite mutants. From a phylogenetic point of view, S. kluyveri is apparently more closely related to S. cerevisiae than to K. lactis. These observations suggest that the progenitor of the modern Saccharomyces and Kluyveromyces yeasts, as well as other related genera, was a petite-negative and aerobic yeast. Upon separation of the K. lactis and S. kluyveri-S. cerevisiae lineages, the latter developed the ability to grow anaerobically. However, while the S. kluyveri lineage has remained petite negative, the lineage leading to the modern Saccharomyces sensu stricto and sensu lato yeasts has developed the petite-positive characteristic.     

Induction of petite yeast mutants by membrane-active agents

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Isolation of a petite mutant from a histidine auxotroph of Candida albicans and its characterization

Respiration-deficient (petite) mutations have been induced in various yeasts, which are categorized as petite-positive. Candida albicans was classified among the petite-negative yeasts. Since then, a few reports have appeared, describing the isolation of petite mutants in C. albicans. We report in the present study on the isolation of a petite mutant of C. albicans-SAR1. This mutant was isolated from a histidine auxotroph of C. albicans after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, thus our petite mutant carries a double mutation. SAR1 was characterized morphologically, biochemically and ultrastructurally. The results revealed differences from the wild type in respect to morphological, physiological and biochemical characteristics. Electron microscopy showed that the cells of the petite mutant contain only very few mitochondria that looked ‘thread like’ without any cristae. The significance of the mutation in the virulence of the mutant vs. that of the wild-type is being assessed. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Effect of the petite mutation on maltose and alpha-methylgucoside fermentation inSaccharomyces cerevisiae.

Summary Several hundred petite mutants were isolated from yeast strains of different genotype to examine the effect of the petite mutation on maltose and alpha-methylglucoside fermentation. In most cases petite mutants isolated retain the ability to ferment maltose and alpha-methylglucoside, although at a slower rate. In one strain (1403-7A), however, the ability to ferment alpha-methylglucoside is completely lost in all petite mutants isolated from this strain. It is suggested that mitochondrial factors may be involved in the utilization of alpha-methylglucoside in strain 1403-7A.     

Partial duplication of the large ribosomal RNA sequence in an inverted repeat in circular mitochondrial DNA from Kloeckera africana: Implications for mechanisms of the petite mutation

The 27,100 base-pair circular mitochondrial DNA from the yeast Kloeckera africana has been found to contain an inverted duplication spanning 8600 base-pairs. Sequences hybridizing to transfer RNAs and the large ribosomal RNA are present in the duplication; however, one end of this segment terminates in the large mitochondrial ribosomal RNA sequence so that at least 1000 base-pairs of the gene are not repeated. The large and small mitochondrial ribosomal RNAs have been shown to have lengths of 2700 and 1450 bases, respectively, and genes for these sequences are separated by a minimum of 1300 base-pairs and a maximum of 1750 base-pairs. Consequences of the large inverted duplication to mechanisms of the petite mutation are discussed in terms of previous hypotheses centred on intramolecular recombination in yeast mitochondrial DNA at sequences of homology or partial homology. Despite the long inverted duplication in K. africana mitochondrial DNA, this yeast has one of the lowest frequencies of spontaneous petite mutants amongst petite positive yeasts. One implication of these findings is that in this yeast intra-molecular mitochondrial DNA sequence homology may not be an important factor in the excision process leading to petite formation.     

Mutants of yeast specifically resistant to petite induction by fluorinated pyrimidines

Induction of the cytoplasmic petite mutation in yeast by 5-fluorouracil (5FU) and 5-fluorocytosine (5FC) is known to depend on the incorporation of 5FU into some species of RNA; 5FC is active only following deamination to 5FU. Several mutants have now been isolated which are resistant to petite mutagenesis by 5FU but remain sensitive to growth inhibition by this analogue. They fall into two classes: those in class I are also resistant to mutagenesis by 5FC, while class II mutants retain partial sensitivity to the latter agent. The growth of both classes is sensitive to 5FC. The behavior of class II mutants requires that exogenous 5FU is specifically excluded from the site of synthesis of the target RNA involved in petite mutagenesis, while 5FC has access to it. The most likely explanation is that the RNA concerned is synthesized in the mitochondria, and that the mitochondrial membranes of class II mutants are impermeable to 5FU but not 5FC. This is supported by the finding that the membrane-active agent dimethylsulfoxide restored 5FU sensitivity to this class of mutants. No such effect was observed with class I mutants, and these are thought to have altered mitochondrial RNA-synthesizing systems which are unable to recognize fluorinated nucleotides.During the course of this work, S. G. O. was supported by a Medical Research Council Scholarship.     

Differential effects of nalidixate on the cell growth of respiratory competent strains and cytoplasmic petite mutants of Saccharomyces cerevisiae

Summary Sodium nalidixate inhibited the cell growth and division of several respiratory competent strains of Saccharomyces cerevisiae. A number of cytoplasmic petite strains (both spontaneous and induced by ethidium bromide) were shown to be more resistant to sodium nalidixate than the wild-type strains from which they were derived. There was considerable variation in sensitivity of different petites derived from the same wild-type. Usually petite strains which were induced by ethidium bromide were more resistant than spontaneously arising petites. The susceptibility of a wild-type strain to nalidixate was found to be least when the mitochondrial respiratory system was maximally repressed. It was also noted that sodium nalidixate (100 g/ml) induced petite mutants.Dr. Carnevali is a Senior Research Worker of the Centro di Studio per gli Acidi Nucleici of the National Research Council of Rome and is on leave of absence at the above address     

Biogenesis of mitochondria: XXV. Studies on the mitochondrial genomes of petite mutants of yeast using ethidium bromide as a probe

This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of Saccharomyces cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (ρ ° cells).The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondrial DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones.It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (ρ^?ER^r) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two ρ^?ER^r clones, and in both the gene elimination rate is slower than in the parent ρ⁺ ER^r strain. It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is the possible purification of erythromycin resistance genes.     

Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA

Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.     

Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts

Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative.     

Unstable petite and grande variants of Candida shehatae

Summary Two strains of Candida shehatae (ATCC 22984 and CSIR Y492) exhibit marked variability in colony size (petite, grande) and respiratory activity (tetrazolium reaction) when grown on glucose, xylose, and--especially--xylitol agar. The transitions occur in both directions at high frequency. Strains showing a negative or weak tetrazolium reaction on xylitol ferment xylose better than those showing a strong tetrazolium reaction. The type strain (ATCC 34887) shows stable colonial morphology with moderate respiratory and fermentative activities. The objective of this report is to demonstrate these variations.     

Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae

Ethidium bromide is known to be a powerful mutagen for the induction of cytoplasmically inherited petite mutations in yeast. The effect of ethidium bromide on the degree of suppressiveness of the induced mutants as a function of exposure time is described. The mitochondrial DNA of 20 ethidium bromide-induced petite mutants has been studied to determine its absence or presence and its buoyant density. Ten mutants, in which we were not able to detect any mitochondrial DNA, were neutral petites. The 10 remaining mutants with mitochondrial DNA simultaneously showed a measurable degree of suppressiveness. It was not possible to correlate the buoyant density of the mutant mitochondrial DNA with the degree of suppressiveness.This study was supported in part by USPHS grant GM 10017. G.M. received a Fulbright Travel Grant.     

Studies on the induction of petite mutants in yeast by analogues of berenil

Summary Compound Hoe 15 030 is an analogue of berenil which is as effective as berenil in inducing petite mutants in Saccharomyces cerevisiae. Hoe 15 030 has greater stability than berenil in aqueous solution, and is less toxic to yeast at high drug concentrations. Mutants of S. cerevisia strain J69-1B have been isolated which are resistant to the petite inducing effects of Hoe 15 030. Three mutant strains (HR7, HR8 and HR10) were characterized and each was shown to carry a recessive nuclear mutation determining resistance to Hoe 15 030. The degree of resistance to Hoe 15 030 is different for each mutant, and each was found to be co-ordinately cross-resistant both to berenil and to another analogue of berenil, Hoe 13 548. However, the three mutants show no cross-resistance to other unrelated petite inducing drugs, including ethidium bromide, euflavine and 1-methyl phenyl neutral red.Further studies on the mutants revealed that each strain exhibits characteristic new properties indicative of changes in mitochondrial membrane functions concerned with the replication (and probably also repair) of mitochondrial DNA. Thus, mutant HR7 is hypersensitive to petite induction by the detergent sodium dodecyl sulphate under conditions where the parent J69-1B is unaffected by this agent. Mutant HR8 is even more sensitive to sodium dodecyl sulphate than is HR7, and additionally shows a markedly elevated spontaneous petite frequency. Isolated mitochondria from strains HR8 and HR10 (but not HR7) show resistance to the inhibitory effects of Hoe 15 030 on the replication of mitochondrial DNA in vitro.     

Mitochondrial DNA replication in petite mutants of yeast: Resistance to inhibition by ethidium bromide, berenil and euflavine

Summary Mitochondrial DNA (mtDNA) replication in petite mutants ofSaccharomyces cerevisiae is generally less sensitive to inhibition by ethidium bromide than in grande (respiratory competent) cells. In every petite that we have examined, which retain a range of different grande mtDNA sequences, this general phenomenon has been demonstrated by measurements of the loss of mtDNA from cultures grown in the presence of the drug. The resistance is also demonstrable by direct analysis of drug inhibition of mtDNA replication in isolated mitochondria. Furthermore, the resistance to ethidium bromide is accompanied, in every case tested, by cross-resistance to berenil and euflavine, although variations in the levels of resistance are observed.In one petite the level of in vivo resistance to the three drugs was very similar (4-fold over the grande parent) whilst another petite was mildly resistant to ethidium bromide and berenil (each 1.6-fold over the parent) and strongly resistant (nearly 8-fold) to inhibition of mtDNA replication by euflavine. The level of resistance to ethidium bromide in several other petite clones tested was found to vary markedly. Using genetic techniques it is possible to identify those petites which display an enhanced resistance to ethidium bromide inhibition of mtDNA replication.It is considered that the general resistance of petites arises because a product of mitochondrial protein synthesis is normally involved in facilitating the inhibitory action of these drugs on mtDNA synthesis in grande cells. The various levels of resistance in petites may be modulated by the particular mtDNA sequences retained in each petite.     

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.     

Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants

The T4 gene 23 product (gp23) encodes the major structural protein of the mature capsid. Mutations in this gene have been described which disrupt the normal length-determining mechanism (A.H. Doermann, F.A. Eiserling, and L. Boehner, J. Virol. 12:374-385, 1973). Mutants which produce high levels of petite and giant phage (ptg) are restricted to three tight clusters in gene 23 (A.H. Doermann, A. Pao, and P. Jackson, J. Virol. 61:2823-2827, 1987). Twenty-six of these ptg mutations were cloned, and their DNA sequence alterations were determined. Each member of this set of ptg mutants arose from a single mutation, and the set defined 10 different sites at which ptg mutations can occur in gene 23. Two petite (pt) mutations in gene 23 (pt21-34 and ptE920g), which produce high frequencies of petite particles but no giants, were also sequenced. Both pt21-34 and ptE920g were shown to include multiple mutations. The phenotypes attributed to both pt and ptg mutations are discussed relative to the mechanism of capsid morphogenesis. A site-directed mutation (SD-1E) was created at the ptgNg191 site, and its phenotypic consequences were examined. Plaque morphology revertants arising from a gene 23 mutant derivative of pt21-34 and from SD-1E were isolated. A preliminary mapping of the mutation(s) responsible for their revertant phenotypes suggested that both intra- and extragenic suppressors of the petite phenotype can be isolated by this method.