Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.     

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences ¹. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington''s, Parkinson''s, and Alzheimer''s diseases ¹⁰. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme ¹². Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 ¹³, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system ⁵, to protein refolding to validate this approach.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.     

Genetic recombination of homologous plasmids catalysed by cell-free extracts of topo-isomerase mutant strains of Saccharomyces cerevisiae

Cell-free extracts of the yeast Saccharomyces cerevisiae can be used to catalyse the recombination of bacterial plasmids in vitro. Recombination between homologous plasmids containing different mutations in the gene encoding tetracycline resistance is detectable by the appearance of tetracycline-resistance following transformation of the recombinant plasmid DNA into Escherichia coli DH5. This in vitro recombination system was used to determine the involvement of eukaryotic topo-isomerases in genetic recombination. Cell-free extracts prepared from a temperature-sensitive topo-isomerase II mutant (top2-1) of S. cerevisiae yielded tetracycline-resistant recombinants, when the recombination assays were performed at both a non-restrictive temperature (30°C) and the restrictive temperature (37°C). This result was obtained whether or not ATP was present in the recombination buffer. Extracts from a non-conditional topo-isomerase I mutant (top1-1) of S. cerevisiae yielded tetracycline-resistant recombinants, as did a temperature-sensitive double mutant (top2-1/top1-8) at the restrictive temperature. The results of this study indicate that neither topo-isomerase I nor topo-isomerase II was involved in the recombinational activity examined.     

On the Prospects of Whole-Genome Association Mapping in Saccharomyces cerevisiae

Advances in sequencing technology have enabled whole-genome sequences to be obtained from multiple individuals within species, particularly in model organisms with compact genomes. For example, 36 genome sequences of Saccharomyces cerevisiae are now publicly available, and SNP data are available for even larger collections of strains. One potential use of these resources is mapping the genetic basis of phenotypic variation through genome-wide association (GWA) studies, with the benefit that associated variants can be studied experimentally with greater ease than in outbred populations such as humans. Here, we evaluate the prospects of GWA studies in S. cerevisiae strains through extensive simulations and a GWA study of mitochondrial copy number. We demonstrate that the complex and heterogeneous patterns of population structure present in yeast populations can lead to a high type I error rate in GWA studies of quantitative traits, and that methods typically used to control for population stratification do not provide adequate control of the type I error rate. Moreover, we show that while GWA studies of quantitative traits in S. cerevisiae may be difficult depending on the particular set of strains studied, association studies to map cis-acting quantitative trait loci (QTL) and Mendelian phenotypes are more feasible. We also discuss sampling strategies that could enable GWA studies in yeast and illustrate the utility of this approach in Saccharomyces paradoxus. Thus, our results provide important practical insights into the design and interpretation of GWA studies in yeast, and other model organisms that possess complex patterns of population structure.     

Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7

The term 'sake yeast' is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.     

Multiple functions of the vacuolar sorting protein Ccz1p in Saccharomyces cerevisiae

The CCZ1 (YBR131w) gene encodes a protein required for fusion of various transport intermediates with the vacuole. Ccz1p, in a complex with Mon1p, is a close partner of Ypt7p in the processes of fusion of endosomes to vacuoles and homotypic vacuole fusion. In this work, we exploited the Ca(2+)-sensitivity of the ccz1Delta mutant to identify genes specifically interacting with CCZ1, basing on functional multicopy suppression of calcium toxicity. The presented results indicate that Ccz1p functions in the cell either in association with Mon1p and Ypt7p in fusion at the vacuolar membrane, or--separately--with Arl1p at early steps of vacuolar transport. We also show that suppression of calcium toxicity by the calcium pumps Pmr1p and Pmc1p is restricted only to the subset of mutants defective in vacuole morphology. The mechanisms of Ca(2+)-pump-mediated suppression also differ from each other, since the action of Pmr1p, but not Pmc1p, appears to require Arl1p function.     

The intron of tRNA-TrpCCA is dispensable for growth and translation of Saccharomyces cerevisiae

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.     

Genetic improvement of Saccharomyces cerevisiae for xylose fermentation

There is considerable interest in recent years in the bioconversion of forestry and agricultural residues into ethanol and value-added chemicals. High ethanol yields from lignocellulosic residues are dependent on efficient use of all the available sugars including glucose and xylose. The well-known fermentative yeast Saccharomyces cerevisiae is the preferred microorganism for ethanol production, but unfortunately, this yeast is unable to ferment xylose. Over the last 15 years, this yeast has been the subject of various research efforts aimed at improving its ability to utilize xylose and ferment it to ethanol. This review examines the research on S. cerevisiae strains that have been genetically modified or adapted to ferment xylose to ethanol. The current state of these efforts and areas where further research is required are identified and discussed.     

Metabolic engineering of Saccharomyces cerevisiae for increased bioconversion of lignocellulose to ethanol

The absence of pentose-utilizing enzymes in Saccharomyces cerevisiae is an obstacle for efficiently converting lignocellulosic materials to ethanol. In the present study, the genes coding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) from Pichia stipitis were successfully engineered into S. cerevisae. As compared to the control transformant, engineering of XYL1 and XYL2 into yeasts significantly increased the microbial biomass (8.1 vs. 3.4 g/L), xylose consumption rate (0.15 vs. 0.02 g/h) and ethanol yield (6.8 vs. 3.5 g/L) after 72 h fermentation using a xylose-based medium. Interestingly, engineering of XYL1 and XYL2 into yeasts also elevated the ethanol yield from sugarcane bagasse hydrolysate (SUBH). This study not only provides an effective approach to increase the xylose utilization by yeasts, but the results also suggest that production of ethanol by this recombinant yeasts using unconventional nutrient sources, such as components in SUBH deserves further attention in the future.     

Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.     

Vesicular Transport of Extracellular Acid Phosphatases in Yeast Saccharomyces cerevisiae

A method for isolation of secretory vesicles from the yeast Saccharomyces cerevisiae based on the disintegration of protoplasts by osmotic shock followed by separation of the vesicles by centrifugation in a density gradient of Urografin was developed in this study. Two populations of the secretory vesicles that differ in density and shape were separated. Acid phosphatases (EC 3.1.3.2) were used as markers of the secretory vesicles. It was shown that the constitutive acid phosphatase (PHO3 gene product) is mainly transported to the cell surface by a lower density population of vesicles, while the repressible acid phosphatase (a heteromer encoded by PHO5, PHO10, and PHO11 genes) by a vesicle population of higher density. These data provide evidence that at least two pathways of transport of yeast secretory proteins from the place of their synthesis and maturation to the cell surface may exist. To reveal the probable reasons for transport of Pho3p and Pho5p/Pho10p/Pho11p enzymes by two different kinds of vesicles, we isolated vesicles from strains that synthesize the homomeric forms of the repressible acid phosphatase. It was demonstrated that glycoproteins encoded by the PHO10 and/or PHO11 genes could be responsible for the choice of one of the alternative transport pathways of the repressible acid phosphatase. A high correlation coefficient between bud formation and secretion of Pho5p phosphatase and the absence of correlation between bud formation and secretion of minor phosphatases Pho10p and Pho11p suggests different functional roles of the polypeptides that constitute the native repressible acid phosphatase.     

Novel Connections Between DNA Replication,Telomere Homeostasis,and the DNA Damage Response Revealed by a Genome-Wide Screen for TEL1/ATM Interactions in Saccharomyces cerevisiae

Tel1 is the budding yeast ortholog of the mammalian tumor suppressor and DNA damage response (DDR) kinase ATM. However, tel1-Δ cells, unlike ATM-deficient cells, do not exhibit sensitivity to DNA-damaging agents, but do display shortened (but stably maintained) telomere lengths. Neither the extent to which Tel1p functions in the DDR nor the mechanism by which Tel1 contributes to telomere metabolism is well understood. To address the first question, we present the results from a comprehensive genome-wide screen for genetic interactions with tel1-Δ that cause sensitivity to methyl methanesulfonate (MMS) and/or ionizing radiation, along with follow-up characterizations of the 13 interactions yielded by this screen. Surprisingly, many of the tel1-Δ interactions that confer DNA damage sensitivity also exacerbate the short telomere phenotype, suggesting a connection between these two phenomena. Restoration of normal telomere length in the tel1-Δ xxx-Δ mutants results in only minor suppression of the DNA damage sensitivity, demonstrating that the sensitivity of these mutants must also involve mechanisms independent of telomere length. In support of a model for increased replication stress in the tel1-Δ xxx-Δ mutants, we show that depletion of dNTP pools through pretreatment with hydroxyurea renders tel1-Δ cells (but not wild type) MMS-sensitive, demonstrating that, under certain conditions, Tel1p does indeed play a critical role in the DDR.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function

The wild-type yeast Saccharomyces cerevisiae (S. cerevisiae) is able to export less than 1 percent of the protein to be secreted. The reasons for retention of most of the secretory proteins on the cell surface of S. cerevisiae are unknown. Recently, temperature-sensitive (ts) mutants of S. cerevisiae showing an oversecretion phenotype were isolated. In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S. cerevisiae strain or their derivative ts protein-overexporting mutants. In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.     

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.