The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae

MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Käfer, 1988). However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with γ-valerolactone and propionitrile. In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M. An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and γ-valerolactone in yeast strain D7.     

Expression of cancer related BRCA1 missense variants decreases MMS-induced recombination in Saccharomyces cerevisiae without altering its nuclear localization

BRCA1 tumor suppressor gene is found mutated in familial breast and ovarian cancer. Most cancer related mutations were found located at the RING (Really Interesting New Gene) and at the BRCT (BRca1 C-Terminal) domain. However, 20 y after its identification, the biological role of BRCA1 and which domains are more relevant for tumor suppression are still being elucidated. We previously reported that expression of BRCA1 cancer related variants in the RING and BRCT domain increases spontaneous homologous recombination in yeast indicating that BRCA1 may interact with yeast DNA repair/recombination. To finally demonstrate whether BRCA1 interacts with yeast DNA repair, we exposed yeast cells expressing BRCA1wt, the cancer-related variants C-61G and M1775R to different doses of the alkylating agent methyl methane-sulfonate (MMS) and then evaluated the effect on survival and homologous recombination. Cells expressing BRCA1 cancer variants were more sensitive to MMS and less inducible to recombination as compared to cell expressing BRCA1wt. Moreover, BRCA1-C61G and -M1775R did not change their nuclear localization form as compared to the BRCA1wt or the neutral variant R1751Q indicating a difference in the DNA damage processing. We propose a model where BRCA1 cancer variants interact with the DNA double strand break repair pathways producing DNA recombination intermediates, that maybe less repairable and decrease MMS-induced recombination and survival. Again, this study strengthens the use of yeast as model system to characterize the mechanisms leading to cancer in humans carrying the BRCA1 missense variant.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Effects of camptothecin or TOP1 overexpression on genetic stability in Saccharomyces cerevisiae

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation.     

The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae

Summary The induction of mitotic gene conversion and crossing-over inSaccharomyces cerevisiae diploid cells homozygous for thepso4-1 mutation was examined in comparison to the corresponding wild-type strain. Thepso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth. Concerning the lethal effect, diploids homozygous for thepso4-1 mutation are more sensitive to all agents tested in any growth phase. However, this effect is more pronounced in the G2 phase of the cell cycle. These results imply that the ploidy effect and the resistance of budding cells are under the control of thePSO4 gene. On the other hand, thepso4-1 mutant is mutationally defective for all agents used. Therefore, thepso4-1 mutant has a generalized block in both recombination and mutation ability. This indicates that thePSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between thepso4-1 mutation and theRecA orLexA mutation ofEscherichia coli.     

Different life strategies in genetic backgrounds of the Saccharomyces cerevisiae yeast cells

Changes in the natural environment require an organism to make constant adaptations enabling efficient use of environmental resources and ensuring its success in competition with other organisms. Such adaptations are expressed through various life strategies, largely determined by the rate of consumption and use of available resources, affecting the life-history traits and the related trade-offs. Allocation of available resources must take into consideration the costs of cell maintenance as well as reproduction. Given that carbon metabolism plays a crucial role in resource allocation, yeast living in different ecological niches show various life-history traits. There are a lot of data about life-history strategies in yeast living in various ecological niches; however, the question is whether different life strategies will be noted for yeast strains growing under strictly controlled conditions. Our studies based on three laboratory yeast strains representing different genetic backgrounds show that each of these strains has specified life strategies which are mainly determined by the glucose uptake rate and its intracellular usage. These results suggest that specific life strategies and related differences in the physiological and metabolic parameters of the cell are the key aspects that may explain various features of cells from different yeast strains, either industrial or laboratory.     

The effect of a-factor on hybrid formation by protoplast fusion in Saccharomyces cerevisiae

Abstract a_˜-Factor, unlike α-factor, does not significantly enhance hybrid formation by protoplast fusion in the yeast Saccharomyces cerevisiae . When Mat α cells are treated with a-factor prior to being proto-plasted and fused, the frequency of hybrid formation is only slightly increased over unarrested controls.     

Mapping candidate hotspots of meiotic recombination in segments of human DNA cloned in the yeast<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The hotspots of meiotic recombination in the human genome can be localized by genetic techniques. The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci. We thought it would be interesting to localize these sites with higher resolution. Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA. A number of potential recombination hotspots could be identified in the clones studied. Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.Communicated by G. P. Georgiev     

Influence of magnesium ions on heat shock and ethanol stress responses of Saccharomyces cerevisiae

This study has highlighted the role of magnesium ions in the amelioration of the detrimental effects of ethanol toxicity and temperature shock in a winemaking strain of Saccharomyces cerevisiae. Specifically, results based on measurements of cellular viability and heat shock protein synthesis together with scanning electron microscopy have shown that, by increasing the bioavailability of magnesium ions, physiological protection is conferred on yeast cells. Elevating magnesium levels in the growth medium from 2 to 20 mM results in repression of certain heat shock proteins following a typical heat shock regime (30–42°C shift). Seed inocula cultures prepropagated in elevated levels of magnesium (i.e. ‘preconditioned’) also conferred thermotolerance on cells and repressed the biosynthesis of heat shock proteins. Similar results were observed in response to ethanol stress. Extra- and intracellular magnesium may both act in the physiological stress protection of yeast cells and this approach offers potential benefits in alcoholic fermentation processes. The working hypothesis based on our findings is that magnesium protects yeast cells by preventing increases in cell membrane permeability elicited by ethanol and temperature-induced stress.     

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.     

Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants

Abstract The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42–46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristics of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna 1 = cdc 22, dna 6 = cdc 34, dna 19 = cdc 36, and dna 39 = dbf 3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhausive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.     

The intramitochondrial location of cytochrome c peroxidase in wild-type and petite Saccharomyces cerevisiae

Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.     

Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes

Yeast artificial chromosome (YAC) cloning systems have advanced the analysis of complex genomes considerably. They permit the cloning of larger fragments than do bacterial artificial chromosome systems, and the cloned material is more easily modified. We recently developed a novel YAC cloning system called transformation-associated recombination (TAR) cloning. Using in vivo recombination in yeast, TAR cloning selectively isolates, as circular YACs, desired chromosome segments or entire genes from complex genomes. The ability to do that without constructing a representative genomic library of random clones greatly facilitates analysis of gene function and its role in disease. In this review, we summarize how recombinational cloning techniques have advanced the study of complex genome organization, gene expression, and comparative genomics.     

非随机方法构建酿酒酵母基因工程菌

酿酒酵母是基因工程产品研究和生产的一个重要表达系统,表达载体和宿主细胞是构成表达系统的两大要素,虽然外源基因表达的方式、强度主要由表达载体控制．但宿主细胞的选择对最终获取产品的质量和数量也具有十分关键的作用。酿酒酵母基因工程宿主菌除要求具有高的DNA转化效率、细胞生长密度和稳定性、低的内源蛋白水解酶活性外,还必须具备与表达载体相对应的营养缺陷筛选标记,用传统随机诱变方法得到的营养缺陷变异株,因含有本底和隐性突变,在细胞生长密度和稳定性方面往往不能满足基因工程产品研究和生产的要求,甚至不能有效地表达外源基因。本文报道用重组技术,通过非随机方法构建了酿酒酵母基因工程宿主茁。研究表明用该方法得到的宿主菌在细胞生长密度、稳定性和表达外源基因方面优于用传统随机诱变方法得到的宿主菌。     

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.     

酿酒酵母渗透敏感新基因的定位及其作用机理的研究

本文通过四分子分析将酿酒酵母中另一控制渗透敏感的基因osm3进行了定位。遗传分析表明,osm3是位于染色体Ⅱ上的1个新基因,与gal1座位相距大约45厘摩。其第二次分裂分离频率为51.01%,它与着丝粒的图距为25.51厘摩,属着丝粒连锁基因。回复突变的研究结果表明,渗透敏感性状很可能是因为osm3座位上发生了错义或无义突变所致。 根据渗透敏感菌株和正常菌株对高渗透压反应试验,证明了高的胞内甘油含量是酵母在高渗条件下生长所必需的。osm3菌株不能耐高渗的主要原因是由于甘油转运失调所致,OSM3基因产物可能与甘油的转运过程有关。本文还就高渗对酿酒酵母发酵力的影响进行了讨论。     

Formaldehyde, glyoxal, urethane, methyl carbamate, 2,3-butanedione, 2,3-hexanedione, ethyl acrylate, dibromoacetonitrile and 2-hydroxypropionitrile induce chromosome loss in Saccharomyces cerevisiae.

Induction of mitotic chromosome loss could be demonstrated for the dialdehyde glyoxal, the diketones 2,3-butanedione and 2,3-hexanedione, ethyl and methyl carbamate, ethyl acrylate, dibromoacetonitrile, 2-hydroxypropionitrile and formaldehyde, but only when they were combined with subacute concentrations of propionitrile, which is a strong inducer of chromosomal malsegregation. The same chemicals did not induced mitotic chromosome loss when applied in pure form. However, glyoxal, ethyl acrylate, dibromoacetonitrile and formaldehyde when applied in pure form also induced mitotic recombination. Respiratory deficiency was induced, in the absence of propionitrile, by these recombinogenic agents and also by 2,3-hexanedione and 2-hydroxypropionitrile which are not recombinogenic.