Allelism of pleiotropic drug resistance in Saccharomyces cerevisiae

Allelism of pleiotropic drug resistant (pdr) mutants was evaluated by complementation tests, linkage to chromosome-VII centromere markers and response to a partial suppressor (sur). Complementation tests were confounded by incomplete dominance and somatic segregation. Phenotypic suppression by sur was observed for all mutant and wild type alleles and thus could not be used to distinguish alleles. Five different alleles were tentatively identified by their close linkage to leul; 88 tetrads from three factor crosses produced the following linkages--leul (4.7) pdrl (17.0) trp5. Resistance of DRI 9/T7, a [cir o] strain of French origin, was not inherited as an allele of pdr but was controlled by a different pleiotropic centromere linked gene. An evaluation of published data suggest that antl, AMYl, till, cyh3, BOR2, and axe1 may be alleles of pdr. Thus pdr appears to be an allele that influences permeability to many inhibitors.     

Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae

We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in resistance to fluconazole, are favored. In this environment, diploid populations of defined size consistently became fixed for a resistance mutation sooner than haploid populations. Experiments manipulating population sizes showed that this advantage of diploids was due to increased mutation availability relative to that of haploids; in effect, diploids have twice the number of mutational targets as haploids and hence have a reduced waiting time for mutations to occur. Under conditions of high drug concentration, recessive mutations in ERG3, which result in resistance through altered sterol synthesis, are favored. In this environment, haploids consistently achieved resistance much sooner than diploids. When 29 haploid and 29 diploid populations were evolved for 100 generations in low drug concentration, the mutations fixed in diploid populations were all dominant, while the mutations fixed in haploid populations were either recessive (16 populations) or dominant (13 populations). Further, the spectrum of the 53 nonsynonymous mutations identified at the sequence level was different between haploids and diploids. These results fit existing theory on the relative abilities of haploids and diploids to adapt and suggest that the ploidy of the fungal pathogen has a strong impact on the evolution of fluconazole resistance.     

Ultracytochemical characterization of non-specific acid phosphatase activities in Saccharomyces cerevisiae.

Glutaraldehyde prefixation causes a considerable inactivation of the acid phosphatase of yeast protoplasts in dependence on the duration of aldehyde influence. Lead ions necessary for ultracytochemical demonstration effect a still stronger inhibition of enzymatic activity. Prefixation, however, protects the enzyme from further inhibition by lead. At pH 4.4 in intact cells acid phosphatase activities are mainly localized in the periplasmic space and in vesicles fused with the plasma membrane. The cell wall and cytoplasm usually remain free of reaction products. On the cell surface activities are found in form of globular lead deposits. At pH 5.2 and 6.3 the periplasmic activity appears decreased compared to that at lower pH values and the intracellular activity is increased. The plasma membrane of protoplasts is completely free of precipitates. The intracellular activity sites of protoplasts (cisternae of endoplasmic reticulum and/or Golgi-like system, small vesicles, central vacuole, nuclear envelope) are the same as for intact cells. The occurrence of at least two forms of acid phosphatase in S. cerevisiae id deduced.     

Folic acid utilisation related to sulfa drug resistance in Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants deficient in folate synthesis have been constructed and employed to study the utilisation of exogenous folates in yeast. One mutant specifically lacked dihydropteroate synthase while the second lacked dihydrofolate synthase. Exogenous folinic acid restored optimal growth to both strains. Folic acid did not generally rescue growth but spontaneous isolates capable of utilising folic acid were selected. The folic acid synthesis pathway in the folate utilising isolates was restored via transformation with FOL1 or FOL3 expression plasmids and transformants were tested for resistance to sulfamethoxazole (SMX). The presence of elevated levels of folic acid led to greatly reduced SMX sensitivity regardless of whether strains were folate utilisers or not.     

Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae

To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.     

Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae

We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.     

Spontaneous mutation of leu2 in Saccharomyces cerevisiae

The data obtained indicate that spontaneous mutations in Saccharomyces cerevisiae are formed during DNA replication. With no DNA replication in the lag-period, in the stationary growth phase, spontaneous mutations are not formed in cell culture during the G1 phase of cell cycle. Experimental data show the absence of primary spontaneously occurring DNA lesion accumulation in the cell G1 phase. Spontaneous mutations of yeasts are formed in the S phase of cell cycle, apparently as DNA replication errors. It is established that the frequency of spontaneous reversions of the leu2 gene in Saccharomyces cerevisiae strain NA3-24 increases when the cells are cultivated on the culture medium with different concentrations of leucine.     

Spontaneous and induced rho mutants of Saccharomyces cerevisiae: patterns of loss of mitochondrial genetic markers.

The deletion which leads to spontaneous rho mutants occurs preferentially at a unique region covering genes oxi3, pho1/OII, and mit175. The frequency of loss of genetic markers in this region was significantly higher than in other regions as determined with a 15- marker system. When various mutagenic treatments were applied, this specific pattern of deletion was also observed, but it was dramatically amplified. This suggests that the basic mechanism of rho production is the same in yeast mitochondrial genomes in both spontaneous and induced mutants.     

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     

DNA damage induced mating type switching in Saccharomyces cerevisiae.

Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the alpha state. The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion. It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction. Because X-rays induce switching immediately after irradiation and because the DNA double-strand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break. Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks. Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.     

Extra-chromosomal inheritance of rhodamine 6G resistance in Saccharomyces cerevisiae.

Summary Rhodamine 6G was found to be a specific inhibitor of aerobic growth of yeast, having no effect on fermentative growth. A single step spontaneous mutant of S. cerevisiae resistant to rhodamine 6G was isolated, which showed cross-resistance to the ATPase inhibitors venturicidin and triethyltin, to the uncoupler 1799, to bongkrekic acid and to cycloheximide, but not to oligomycin or to the inhibitors of mitochondrial protein synthesis, chloramphenicol and erythromycin. The genetic analysis of this mutant showed that both nuclear and cytoplasmic (but apparently not mitochondrial) factors may be involved in the determination of the mutation. The behaviour is discussed as a possible function for 2 micron circular (omicron) DNA.     

Nuclear inheritance of resistance to antimycin A in Saccharomyces cerevisiae.

Summary A group of 30 independent mutants of Saccharomyces cerevisiae, resistant to the respiratory inhibitor antimycin A, was investigated from a genetical and biochemical point of view. All the mutants can be grouped into two nuclear loci: AMY1 maps on the VII chromosome, between leu 1 and trp 5; AMY2 is close to its centromere on either chromosome XVIII or XIX. Both genes do not affect mitochondrial structures or functions.     

Spontaneous chromosome loss in Saccharomyces cerevisiae is suppressed by DNA damage checkpoint functions.

Genomic instability is one of the hallmarks of cancer cells and is often the causative factor in revealing recessive gene mutations that progress cells along the pathway to unregulated growth. Genomic instability can take many forms, including aneuploidy and changes in chromosome structure. Chromosome loss, loss and reduplication, and deletions are the majority events that result in loss of heterozygosity (LOH). Defective DNA replication, repair, and recombination can significantly increase the frequency of spontaneous genomic instability. Recently, DNA damage checkpoint functions that operate during the S-phase checkpoint have been shown to suppress spontaneous chromosome rearrangements in haploid yeast strains. To further study the role of DNA damage checkpoint functions in genomic stability, we have determined chromosome loss in DNA damage checkpoint-deficient yeast strains. We have found that the DNA damage checkpoints are essential for preserving the normal chromosome number and act synergistically with homologous recombination functions to ensure that chromosomes are segregated correctly to daughter cells. Failure of either of these processes increases LOH events. However, loss of the G2/M checkpoint does not result in an increase in chromosome loss, suggesting that it is the various S-phase DNA damage checkpoints that suppress chromosome loss. The mec1 checkpoint function mutant, defective in the yeast ATR homolog, results in increased recombination through a process that is distinct from that operative in wild-type cells.     

Spontaneous mutations in diploid Saccharomyces cerevisiae: more beneficial than expected

We performed a 1012-generation mutation-accumulation (MA) experiment in the yeast, Saccharomyces cerevisiae. The MA lines exhibited a significant reduction in mean fitness and a significant increase in variance in fitness. We found that 5.75% of the fitness-altering mutations accumulated were beneficial. This finding contradicts the widely held belief that nearly all fitness-altering mutations are deleterious. The mutation rate was estimated as 6.3 x 10(-5) mutations per haploid genome per generation and the average heterozygous fitness effect of a mutation as 0.061. These estimates are compatible with previous estimates in yeast.