Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

Isolation of glutathione-deficient mutants of the yeast Saccharomyces cerevisiae

Glutathione-deficient (gsh-) mutants of the yeast Saccharomyces cerevisiae were isolated after UV treatment using MNNG as selective agent. For genetic and biochemical characterization 5 mutant strains were chosen which exhibited considerably decreased residual GSH contents varying from 2 to 6% of the wild-type levels. All 5 isolates showed a 2:2 segregation of the gsh-:GSH+ phenotypes alluding to a monogenic recessive mutation. Complementation analysis indicates that all gsh- mutants belong to one complementation group.     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.     

Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae were isolated which have decreased ability to hydrolyze leucine beta-naphthylamide, a chromogenic substrate for amino-peptidases. The mutations were shown by starch gel electrophoresis to affect one of four different aminopeptidases. Mutations affecting a given enzyme belong to a single complementation group. The four genes were symbolized lap1, lap2, lap3, and lap4, and the corresponding enzymes LAPI, LAPII, LAPIII, and LAPIV. Both lap1 and lap4 were mapped to the left arm of chromosome XI, and lap3 was mapped to the left arm of chromosome XIV. Strains which possessed only one of the four leucine aminopeptidases (LAPs) were constructed. Crude extracts from these strains were used to study the properties of the individual enzymes. Dialysis against EDTA greatly reduced the activity of all the LAPs except for LAPIII. Of the cations tested, Co2+ was the most effective in restoring activity. LAPIV was the only LAP reactivated by Zn2+. LAPI was purified 331-fold and LAPII was purified 126-fold from cell homogenates. Both of the purified enzymes had strong activity on dipeptides and tripeptides. The activity levels of the LAPs are strongly dependent on growth stage in batch culture, with the highest levels in early-stationary phase. Strains lacking all four LAPs have slightly lower growth rates than wild-type strains. The ability of leucine auxotrophs to grow on dipeptides and tripeptides containing leucine is not impaired in strains lacking all four LAPs.     

Isolation and characterization of Metolachlor-resistant mutants of Saccharomyces cerevisiae

The herbicide Metolachlor (-chloroacetamide group) inhibits the growth of Saccharomyces cerevisiae on complete, minimal, and non-fermentative media. Spontaneous and induced resistant mutants showed monogenic segregation patterns. Among the resistant clones, 70% were recessives, 16.4% were partially dominants and 13.4% were dominants. The spontaneous partially dominant mutation Mtc1 was mapped on linkage group XV at 33.3cM from ade2 and 31.7cM from his3, in a region that is characterized by the presence of several resistant genes. The recessive mutation mtc2 was located on chromosome IV. Although all the mutants had the ability to grow in the presence of the herbicide, they remained affected in their respiration efficiency, indicating two different mechanism of action of Metholachor on yeast cells.     

Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae

The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation. Evidence suggests that it is also involved in osmohomeostasis of the cell. We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium. Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and alpha-mannosidase. The mutants fall into at least 17 complementation groups, termed ssv for salt-sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others. We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.     

Isolation and characterization of mevinolin resistant mutants of Saccharomyces cerevisiae

Mutant of Saccharomyces cerevisiae resistant to mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A (HMGCoA) reductase (EC1.1.1.34) were isolated and one mutant (MV71) was extensively characterized. While growth of resistant strains in the presence of mevinolin was growth. Diploids produced by mutant/wild-type matings showed levels of mevinolin resistance which indicated incomplete dominance. Sterol synthesis in the presence of mevinolin was inhibited in strain MV71 but to a lesser degree than seen in the wild-type strain. All mevinolin resistant mutants also demonstrated a slight resistance to the antibiotic nystatin. The subcellular location of HMGCoA reductase activity in MV71 and the wild-type strain were determined and it was shown that yeast HMGCoA reductase is not regulated by a dephosphorylation mechanism as has been shown for mammalian reductases. In vivo and in vitro studies of strain MV71 and the wild-type indicated that mevinolin resistance did not result in changes in HNGCoA reductase activity as has been demonstrated in mammalian systems. Based on growth data, sterol analysis, and the lack of detection of HMGCoA reductase activity differences between strain MV71 and the wild-type, mevinolin resistance is concluded to result possibly from a mutation in HMG2, one of the two functional yeast HMGCoA reductase genes, which accounts for a minor (up to 17%) amount of total cellular reductase activity.     

Isolation and characterization of Saccharomyces cerevisiae glycolytic pathway mutants.

Yeast strains carrying recessive mutations representing four different loci that cause defects in pyruvate kinase, pyruvate decarboxylase, 3-phosphoglycerate kinase, and 3-phosphoglycerate mutase were isolated and partially characterized. Cells carrying these mutations were unable to use glucose as a carbon source as measured in turbidimetric growth experiments. Tetrad analysis indicated that these mutations were not linked to each other; one of the mutations, that affecting phosphoglycerate kinase, was located on chromosome III.     

Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis.

A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase. Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1. Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C. The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots. Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium. Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants. cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts.     

Isolation and characterization of temperature-sensitive mak mutants of Saccharomyces cerevisiae.

The K1 killer plasmid of Saccharomyces cerevisiae is a 1.5-megadalton linear double-stranded ribonucleic acid molecule. Using simplified screening and complementation procedures, we have isolated mutants in three chromosomal genes that are temperature sensitive for killer plasmid maintenance or replication. One of these genes, mak28-1, was located on chromosome X. Two of the temperature-sensitive mutants rapidly lost the wild-type killer plasmid of A364A during spore germination and outgrowth at nonpermissive temperatures, but during vegetative growth, they only lowered the plasmid copy number. These two mutants did not lose two other wild-type K1 killer plasmids, indicating a heterogeneity of the killer plasmids in laboratory yeast strains.     

Isolation of new nonconditional Saccharomyces cerevisiae mutants defective in asparagine-linked glycosylation

We describe the isolation and partial characterization of Saccharomycescerevisiae nonconditional mutants that show defects in N-glycosylationof proteins. The selection method is based on the reductionof affinity for the ion exchanger QAE-Sephadex as a consequenceof the decrease in the negative charge of the cell surface.This characteristic reflects a decrease in the incorporationof mannosyl-phosphate units into the N-linked oligosaccharidesof the mannoproteins. The mutants exhibit low affinity for thebasic dye alcian blue and for that reason we have called themldb (low dye binding) mutants. Eight of the complementationgroups seem to be new as shown by complementation studies withpreviously isolated mutants of similar phenotype. Four of thegroups showed a significant reduction in the number and/or sizeof the N-linked oligosaccharides attached to secreted invertase.We have analyzed the N-linked oligosaccharides of ldb1 and ldb2,the mutants that show the most drastic reduction in the affinityfor the alcian blue dye. In both cases, the purified endo H-releasedoligosaccharides from the mannoproteins lacked detectable amountsof phosphate groups as shown by ion exchange chromatographyand the ¹H NMR spectra. In addition, ldb1 synthesizes a truncatedand unbranched outer chain lacking any     

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae

Laboratory strains of yeast ( Saccharomyces cerevisiae ) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys⁺ strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.     

Isolation of temperature-sensitive mutants for mRNA capping enzyme in Saccharomyces cerevisiae

The guanylyltransferase activity of mRNA capping enzyme catalyzes the transfer of GMP from GTP to the 5′ terminus of mRNA. In Saccharomyces cerevisiae, the activity is carried on the α subunit of capping enzyme, the product of the CEG1 gene. We have isolated 10 recessive, temperature-sensitive mutations of CEG1; nine (cegl-1 to cegl-9) were isolated on a single-copy plasmid and the remaining one (cegl-10) on a multicopy plasmid. The presence of cegl-10 in multiple copies is essential for the viability of cells carrying the mutation, and a shift to the restrictive temperature resulted in rapid growth arrest of cegl-10 cells, while growth rates of other mutants decreased gradually upon temperature upshift. Intragenic complementation was not observed for pairwise combinations of the mutations. Although the majority of the mutations occurred at the amino acid residues conserved between Cegl and the Schizosaccharomyces pombe homologue, none were located in the regions that are also conserved among viral capping enzymes and polynucleotide ligases. Guanylyltransferase activity of the mutant proteins as measured by covalent Ceg1-GMP complex formation was heat-labile. The availability of these mutants should facilitate studies of the structure-function relationships of capping enzyme, as well as the roles and regulation of mRNA capping.     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress.     

Isolation of secretory vesicles from Saccharomyces cerevisiae

Purification of secretory vesicles from Saccharomyces cerevisiae has been hindered because these organelles normally represent a small proportion of cellular membranes. In the yeast secretory mutant sec1, secretory vesicles accumulate intracellularly in large quantities. Using a sec1 strain we have devised a procedure for the partial purification of these vesicles. The purification employs differential and density gradient centrifugations and an electrophoretic separation of membranes. The fractions obtained from this procedure are enriched for secretory vesicles at least fivefold over other cellular membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane fractions reveals a distinct set of polypeptides associated with secretory vesicles.