Experimental studies of deleterious mutation in Saccharomyces cerevisiae

Yeast has proven to be a very useful model organism for studying eukaryotic cell functions. Its applicability for population and quantitative genetics is less well known. Among its advantages is the ease of screening for mutants. The present paper reviews experiments aimed at estimating the parameters of spontaneous mutations deleterious to fitness. The rate of deleterious mutation was found to be moderately high. A large fraction of detectable mutants were lethal; among the non-lethal mutants, the least harmful ones dominated. Deleterious mutations, and especially the lethal ones, were generally very well masked by wild-type alleles when in heterozygous loci. The negative effects of mutations were much stronger under stressful than under benign conditions. Interactions between loci with deleterious mutations did alter their fitness, but no strong overall effect of synergism or antagonisms was observed.     

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.     

A direct study of the relative synthesis of petite and grande mitochondrial DNA in zygotes from crosses involving suppressive petite mutants of Saccharomyces cerevisiae

Summary Work in recent years has produced indirect evidence to support the view that the phenomenon of suppressiveness in yeast is the result of the ability of the petite mtDNA to out-replicate the wild-type genome. We have developed a method, based on fluorography of gels containing restriction fragments of radioactively labelled zygotic mtDNA, by which it has been possible to follow directly the incorporation of label into the two mtDNA species and hence their relative synthesis. Four petite isolates of 70%, 43%, 23% and 12% suppressiveness were tested by this method in crosses with a grande strain. Only the mtDNA from the 70% suppressive petite showed a replicative advantage over the grande mtDNA. The mtDNA from the 43% and 23% suppressive actually appeared to undergo, if anything, less replication in the zygote than the grande mtDNA. It is concluded that while some petites may exhibit suppressiveness as a result of enhanced replicative efficiency of their mtDNA, this cannot be the explanation for all suppressive petite strains.     

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.     

Ultrastructural modifications of vesicular and Golgi elements in the Saccharomyces cerevisiae sec21 mutant at permissive and non-permissive temperatures

Background: The secretory protein transit between cisternae of endoplasmic reticulum (ER) and Golgi elements is blocked when the yeast Saccharomyces cerevisiae sec21 mutant is shifted from the permissive (24°C) to a non-permissive (37°C) temperature, but 30–50 nm vesicles accumulate in the cytoplasm. At the semi-permissive temperature of 33°C there is no complete block but rather a slowdown of the protein transport between ER and Golgi. The purpose of the present investigation is to analyze the structural expression of these events. Methods: S. cerevisiae sec21 mutants were maintained for 90 min at semi-restrictive (33°C) or restrictive (37°C) temperatures and then progressively returned to 24°C. Following fixation in glutaraldehyde and a postfixation in potassium ferrocyanide reduced osmium, 0.08 to 0.2 μm thick sections were cut from Epon embedded yeasts. Using the thicker sections, stereopairs of electron microscope photographs were prepared and used to visualize the three-dimensional configuration of the organelles. Results: At permissive temperature, the Golgi elements appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm. The diameter of these membranous tubules varied considerably from one Golgi element to another. Larger tubules showed at their intersections distensions with size and staining intensity comparable with that of the secretory granules seen at proximity of the Golgi networks or at the cell periphery. Small vesicles in the 30–50 nm size range were rarely if ever observed in cells grown at permissive temperature. Golgi networks and secretion granules were less conspicuous in mutant cells maintained at 33°C and completely disappeared at 37°C. In both cases, the main structural feature was the presence in the cytoplam of numerous small vesicles and of short membranous tubules with a diameter identical to that of the small vesicles. As soon as 5 minutes after shifting mutants from 33°C to 24°C, the small vesciles disappeared from the cytoplasm, while secretory granules were actively produced in extensively developed Golgi network. When mutants were returned from 37°C to 24°C, the disappearance of small vesicles was more progressive and concomitant with the progressive reconstruction of Golgi networks. Conclusions: It is thus postulated that, in the above mentioned conditions, the small vesicles of the sec21 mutant did not act as intermediate carriers between the endoplasmic reticulum and a pre-existing Golgi apparatus, but rather fused together to produce newly formed Golgi networks. © 1994 Wiley-Liss, Inc.     

A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae

Summary A novel type of regulatory mutation for galactose metabolism in Saccharomyces cerevisiae is described. The mutation named gal11 was recessive, non-allelic to GAL4, GAL80, GAL2, or GAL3, and unlinked to the gene cluster of GAL1, GAL10, and GAL7. It caused a coordinate reduction of galactokinase, galactose-1-P uridylyl transferase, and UDP-glucose 4-epimerase by a factor of more than 5, rendering the mutant cells galactose-nonfermenting. The effect of the mutation was manifested not only in cells grown on galactose but also in cells constitutively synthesizing the galactose-metabolizing enzymes.This work was supported in part by a grant (Project No. 410712) from the Ministry of Education, Culture, and Science or Japan     

Suppression of temperature sensitive mutations in oncogene-related CDC genes in Saccharomyces cerevisiae by catabolite repression resistance and cytoplasmic petite mutations

Summary The start cell division control genes CDC36 and CDC28 have been reported to contain a certain sequence homology to tissue oncogenes (ets and some protein kinase encoding oncogenes respectively). Here we report that temperature sensitive mutations in these genes are suppressed in cytoplasmic petite mutants and catabolite repression resistant mutants.     

Identification of extragenic suppressors of the cif1 mutation in Saccharomyces cerevisiae

The cif1 mutation of Saccharomyces cerevisiae causes inability to grow on glucose and related fermentable carbon sources. We have isolated two different suppressor mutations that allow growth on glucose of yeasts carrying the cif1 mutation. One of them, sci1-1, is recessive and caused inability to grow on non-fermentable carbon sources and to de-repress fructose-1,6-bisphosphatase. The other suppressor mutation, SCI2-1, is dominant and diminished the capacity to phosphorylate glucose or fructose. The SCI2-1 mutation decreased sporulation efficiency by 70% in heterozygosis and by more than 90% in homozygosis. In a CIF1 background, cells carrying the mutation SCI2-1 accumulated trehalose during the logarithmic phase of growth and hyperaccumulated it during the stationary phase. Genetic tests showed that SCI2 was either allelic, or else closely linked, to HXK2. The concentrations of the glycolytic metabolites measured during growth on glucose in cells carrying the cif1 mutation and any of the suppressor mutations were similar to those of a wild-type. Both types of suppressor mutations restored the transient cAMP response to glucose to cif1 mutants.This paper is dedicated to Prof. J. R. Villanueva on the occasion of his 65th birthday     

Effects of chloramphenicol on the thermal profile of Saccharomyces cerevisiae

Chloramphenicol decreased the maximum temperature for growth of a petite mutant of Saccharomyces cerevisiae, shifted the Arrhenius plot of thermal death to lower temperatures and shortened correspondingly, the Arrhenius plot of growth, while an associative thermal profile was maintained. At saturating concentrations (about 5 mg per ml) of chloramphenicol in liquid mineral medium with vitamins and glucose the final maximum temperature for growth was depressed from about 40°C to about 37°C. The results suggested that chloramphenicol acted in the mutant on targets other than mitochondrial ribosomes and that these targets are identical or associated with the death and T_max sites of the yeast.     

A mutation affecting sexual agglutinability in MATα locus of Saccharomyces cerevisiae

Summary A temperature-sensitive non-agglutinative mutant of Saccharomyces cerevisiae was isolated and characterized. The mutation, sag2, affected sexual agglutination, conjugation and production of -mating pheromone at a restrictive temperature, but not the response to -mating pheromone. Genetic analyses showed that the mutation was recessive and in the MAT locus. The sag2 mutation complemented with mat2 but not with mat1 These results suggest that sag2 is in the MAT1 gene and that at a restrictive temperature the mutation, sag2, inactivated the MAT1 product, a positive regulator of -mating functions. The sag2 mutation is like mat1-5 in its retention of response to -mating pheromone. However, at 25 °C, sag2 cells were competent to mate, whereas mat1-5 cells were not. Hence, sag2 is regarded as a new allele in the MAT1 gene, which we designate mat1-11.     

Ribosome depurination is not sufficient for ricin-mediated cell death in Saccharomyces cerevisiae

The plant toxin ricin is one of the most potent and lethal substances known. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved alpha-sarcin/ricin loop in the large rRNA. Very little is known about how ricin interacts with ribosomes and the molecular mechanism by which it kills cells. To gain insight to the mechanism of ricin-induced cell death, we set up yeast (Saccharomyces cerevisiae) as a simple and genetically tractable system to isolate mutants defective in cytotoxicity. Ribosomes were depurinated in yeast cells expressing the precursor form of the A chain of ricin (pre-RTA), and these cells displayed apoptotic markers such as nuclear fragmentation, chromatin condensation, and accumulation of reactive oxygen species. We conducted a large-scale mutagenesis of pre-RTA and isolated a panel of nontoxic RTA mutants based on their inability to kill yeast cells. Several nontoxic RTA mutants depurinated ribosomes and inhibited translation to the same extent as wild-type RTA in vivo. The mutant proteins isolated from yeast depurinated ribosomes in vitro, indicating that they were catalytically active. However, cells expressing these mutants did not display hallmarks of apoptosis. These results provide the first evidence that the ability to depurinate ribosomes and inhibit translation does not always correlate with ricin-mediated cell death, indicating that ribosome depurination and translation inhibition do not account entirely for the cytotoxicity of ricin.     

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.     

Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae

Summary Ten nuclear suppressors (nam mutations) of the mitochondrial oxi1-V25 ochre mutation are characterized. They restore to some extent the functional form of cytochrome oxidase, as judged by the results of growth tests, cytochrome spectra, cytochrome oxidase activities, and electrophoresis of the products of mitochondrial translation. The nam mutants can suppress some mit ^– mutations mapping in four mitochondrial genes. They act on a number of chain-terminating mit ^– mutations. When grown on glycerol medium some double mutants nam _x-V25 show an increased sensitivity to paromomycin, while the growth of others is stimulated by the drug. The nam mutants are probably omnipotent suppressors resulting from mutations in nuclear gene(s) specifying mitoribosomal protein(s).     

Molecular cloning of the PEL1 gene of Saccharomyces cerevisiae that is essential for the viability of petite mutants

The PEL1 gene of Saccharomyces cerevisiae is essential for the cell viability of mitochondrial petite mutants, for the ability to utilize glycerol and ethanol on synthetic medium, and for cell growth at higher temperatures. By tetrad analysis the gene was assigned to chromosome III, centromere proximal of LEU2. The PEL1 gene has been isolated and cloned by the complementation of a pel1 mutation. The molecular analysis of the chromosomal insert carrying PEL1 revealed that this gene corresponds to the YCL4W open reading frame on the complete DNA sequence of chromosome III. The putative Pel1 protein is characterized by a low molecular weight of approximately 17 kDa, a low codon adaptation index, and a high leucine content.     

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.     

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae

Summary DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or Pstl. This cloned fragment hybridized with the total DNA from C. utilis and from Leu⁺ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.Journal Article No. 11669 from the Michigan Agricultural Experiment Station     

Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae

We have studied the influence of a temperature-sensitive cdc2-1 mutation in DNA polymerase on the selection-induced mutation occurring at the LYS-2 locus in the yeast Saccharomyces cerevisiae. It was found that in cells plated on synthetic complete medium lacking only lysine, the numbers of Lys⁺ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number. When cdc2-1 mutant cells, after selective plating, were incubated at the restrictive temperature of 37°C for 5 h daily for 7 days, the frequency of an adaptive reversion of lys ^- Lys⁺ was significantly higher than the frequency in cells incubated only at the permissive temperature, or in wild-type cells incubated either at 23°C or 37°C. Therefore, when the proof-reading activity of DNA polymerase is impaired under restrictive conditions, the frequency of adaptive mutations is markedly enhanced.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene

Summary Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc⁺ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc⁺ colonies by UV irradiation. It is postulated that these UV-induced cdc⁺ colonies arise as the result infidelity in DNA replication.