Interchromosomal and intrachromosomal recombination in rad 18 mutants of Saccharomyces cerevisiae

Summary The frequency of intra- and interchromosomal recombination was determined in RAD18 and rad18 deletion and rad18-3 mutant strains. It was found that spontaneous interchromosomal recombination at trp5, his1, ade2, and MAT was elevated 10- to 70-fold in the rad18-3 and rad18 mutants as compared to the RAD ⁺ strains. On the other hand the frequencies of spontaneous intrachromosomal recombination for the his33, his35 and the his4C ^–, his4A ^– duplications and for heterothallic mating type switching were only marginally elevated in the rad18 deletion mutant, and recombination between ribosomal DNA repeats was only 2-fold elevated in the rad18-3 mutant. These differences may be due to a haploid versus diploid specific difference. However interchromosomal recombination was elevated 40-fold and intrachromosomal recombination was only marginally (1.5-fold) elevated in a diploid homozygous for rad18, arguing against a haploid versus diploid specific difference. Possible explanations for the difference in the elevated levels of intra- versus interchromosomal spontaneous recombination are discussed.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

A genome-wide screen in Saccharomyces cerevisiae reveals pathways affected by arsenic toxicity

We have used Saccharomyces cerevisiae to identify toxicologically important proteins and pathways involved in arsenic-induced toxicity and carcinogenicity in humans. We performed a systemic screen of the complete set of 4733 haploid S. cerevisiae single-gene-deletion mutants to identify those that have decreased or increased growth, relative to wild type, after exposure to sodium arsenite (NaAsO₂). IC₅₀ values for all mutants were determined to further validate our results. Ultimately we identified 248 mutants sensitive to arsenite and 5 mutants resistant to arsenite exposure. We analyzed the proteins corresponding to arsenite-sensitive mutants and determined that they belonged to functional categories that include protein binding, phosphate metabolism, vacuolar/lysosomal transport, protein targeting, sorting, and translocation, cell growth/morphogenesis, cell polarity and filament formation. Furthermore, these data were mapped onto a protein interactome to identify arsenite-toxicity-modulating networks. These networks are associated with the cytoskeleton, ubiquitination, histone acetylation and the MAPK signaling pathway. Our studies have potential implications for understanding toxicity and carcinogenesis in arsenic-induced human conditions, such as cancer and aging.     

Crabtree-negative characteristics of recombinant xylose-utilizing Saccharomyces cerevisiae

For recombinant xylose-utilizing Saccharomyces cerevisiae, ethanol yield and productivity is substantially lower on xylose than on glucose. In contrast to glucose, xylose is a novel substrate for S. cerevisiae and it is not known how this substrate is recognized on a molecular level. Failure to activate appropriate genes during xylose-utilization has the potential to result in sub-optimal metabolism and decreased substrate uptake. Certain differences in fermentative performance between the two substrates have thus been ascribed to variations in regulatory response. In this study differences in substrate utilization of glucose and xylose was analyzed in the recombinant S. cerevisiae strain TMB3400. Continuous cultures were performed with glucose and xylose under carbon- and nitrogen-limited conditions. Whereas biomass yield and substrate uptake rate were similar during carbon-limited conditions, the metabolic profile was highly substrate dependent under nitrogen-limited conditions. While glycerol production occurred in both cases, ethanol production was only observed for glucose cultures. Addition of acetate and 2-deoxyglucose pulses to a xylose-limited culture was able to stimulate transient overflow metabolism and ethanol production. Application of glucose pulses enhanced xylose uptake rate under restricted co-substrate concentrations. Results are discussed in relation to regulation of sugar metabolism in Crabtree-positive and -negative yeast.     

Isolation and characterization of temperature-sensitiveplc1 mutants of the yeastSaccharomyces cerevisiae

ThePLC1 gene of the yeastSaccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitiveplc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutantplc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature,plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests thatPLC1 is required at several or all stages of the cell cycle.     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

Characterization of the cyrl-2 UGA mutation in Saccharomyces cerevisiae

Summary cyrl-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyrl-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyrl-2 mutation. Construction of chimeric genes restricted the region containing the cyrl-2 mutation, and the cyrl-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyrl-2 mutation site result in null mutations. The complete open reading frame of the cyrl-2 gene expressed under the control of the GAL1 promoter complemented cyrl-dl in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyrl-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications.     

Diaminotoluenes induce intrachromosomal recombination and free radicals in Saccharomyces cerevisiae

The carcinogenicity of aniline-based aromatic amines is poorly reflected by their activity in short-term mutagenicity assays such as the Salmonella typhimurium reverse mutation (Ames) assay. More information about the mechanism of action of such carcinogens is needed. Here we report the effects on DEL recombination in Saccharomyces cerevisiae of the carcinogen 2,4-diaminotoluene and its structural isomer 2,6-diaminotoluene, which is reported to be non-carcinogenic. Both compounds are detected as equally mutagenic in the Salmonella assay. In the absence of any external metabolizing system both compounds were recombinagenic in the DEL assay, with the carcinogen being a more potent inducer of deletions than the non-carcinogen. In the presence of Aroclor-induced rat liver S9, however, the carcinogen 2,4-diaminotoluene became a 2-fold more potent inducer of deletions, and the non-carcinogen 2,6-diaminotoluene was rendered less toxic and no induced recombination was observed. 2,4-Diaminotoluene is distinguished from its non-carcinogen analog in the DEL assay, therefore, on the basis of a preferential activation of the carcinogen in the presence of a rat liver microsomal metabolizing system. Free radical species are produced by several carcinogens and have been implicated in carcinogenesis. We further investigated whether exposure of yeast to either 2,4-diaminotoluene or 2,6-diaminotoluene resulted in a rise in intracellular free radical species. The effects of the free radical scavenger N-acetylcysteine on toxicity and recombination induced by the two compounds and intracellular oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate were studied. Both 2,4- and 2,6-diaminotoluene produced free radical species in yeast, indicating that the reason for the differential activity of the compounds for induced deletions is not reflected in any difference in the production of free radical species.     

Effects of beta-amyloid on proliferation and morphology of yeast Saccharomyces cerevisiae

Deposition of beta-amyloid peptide (1–42) (AP) in the brain is an early event linked with pathogenesis of cell injury and death in Alzheimer disease. Previous studies have demonstrated that AP induces cytotoxicity in several types of human cells. Surprisingly, the peptide was found not only to be non toxic for yeast cells, but to stimulate growth of yeast culture. The results are consistent with AP binding to yeast cell as illustrated by binding isotherms with theapparent dissociation constant of 8 × 10^-7 M and B_max of 4.7 × 10⁴ molecules/cell.     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

Isolation of the DLD gene of Saccharomyces cerevisiae encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase

In Saccharomyces cerevisiae the utilization of lactate occurs via specific oxidation of l- and d-lactate to pyruvate catalysed by l-lactate ferricytochrome c oxidoreductase (L-LCR) (EC 1.1.2.3) encoded by the CYB2 gene, and d-lactate ferricytochrome c oxidoreductase (D-LCR) (EC 1.1.2.4), respectively. We selected several lactate^– pyruvate⁺ mutants in a cyb2 genetic background. Two of them were devoid of D -LCR activity (dld mutants, belonging to the same complementation group). The mutation mapped in the structural gene. This was demonstrated by a gene dosage effect and by the thermosensitivity of the enzyme activity of thermosensitive revertants. The DLD gene was cloned by complementation for growth on d-, l-lactate in the strain WWF18-3D, carrying both a CYB2 disruption and the dld mutation. The minimal complete complementing sequence was localized by subcloning experiments. From the sequence analysis an open reading frame (ORF) was identified that could encode a polypeptide of 576 amino-acids, corresponding to a calculated molecular weight of 64000 Da. The deduced protein sequence showed significant homology with the previously described microsomal flavoprotein l-gulono--lactone oxidase isolated from Rattus norvegicus, which catalyses the terminal step of l-ascorbic acid biosynthesis. These results are discussed together with the role of L-LCR and D-LCR in lactate metabolism of S. cerevisiae.     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.     

Plasmid multimerization is dependent on RAD52 activity in Saccharomyces cerevisiae

Summary A mutant plasmid, pX, derived from the 1453 base pair small plasmid, YARp1 (or TRP1 RI circle), consists of 849 base pairs of DNA bearing the TRP1 gene and the ARS1 sequence of Saccharomyces cerevisiae and, unlike YARp1 and other commonly used yeast plasmids, highly multimerizes in a S. cerevisiae host. The multimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S. cerevisiae. Based upon this observation, a regulated system of multimerization of pX with GAL1 promoter-driven RAD52 has been developed. We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids.