Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling

In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain 1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.     

Comparative assay of amyloid and prion contents in yeast cells

Amyloid contents were quantitatively assayed in crude yeast lysates treated with thioflavin T that specifically stained amyloid fibrils. We demonstrated that guanidine hydrochloride (GuHCl) treatment and overexpression of Hsp104p chaperone resulted in the elimination of the [PSI ⁺] factor and that the stable decline in amyloid contents followed from the reduced fluorescence intensity (IF) of thioflavin T. Overexpression of the SUP35 gene coding the protein prionizable to [PSI ⁺] results in the generation of [PSI ⁺] clones with increased thioflavin T IF. Transmission of [PSI ⁺] factor by cytoduction in crossings of recipients with low IF was also accompanied by stable IF enhancement in cytoductants, indicating enriched amyloid contents. Thus, in model experiments, modifying the quantity of [PSI ⁺] factor, a yeast prion amyloid, the change in thioflavin T IF corresponds to the expected shift in amyloid contents, the IF shift behaving as a cytoplasm hereditary determinant. It is concluded that thioflavin T IF allows for the quantitative estimation of amyloid contents in cells. The stable mitotic IF shift induced by agents affecting the prion composition permits the quantitative evaluation of prion contribution into amyloid pool. It is possible to assume that the monitoring of thiophlavin T IF shifts under the exposure of agents affecting prion pattern may be helpful to disclose previously unknown prions in yeast strains.     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg     

Alterations in the cell wall ofSaccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases inSaccharomyces cerevisiae after arrest of acdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it representedde novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and thecdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.Abbreviations GlcNAc N-acetyl glucosamine - UDPGIcNAc uridine-diphosphate-N-acetyl glucosamine - UDPGlc uridine-diphosphate glucose - TCA trichloroacetic acid - EDTA ethylene diamino tetraacetate - TAME tosyl-L-arginyl methyl ester - GTP guanosine triphosphate - WGA wheat germ agglutinin     

Spontaneous amplification of yeast CEN ARS plasmids

Summary Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-4 allele (as well as the URA3 and TRP1 markers) yielded His⁺ transformants at 0.1%–50% the frequency of Ura⁺ Trp⁺ transformants. Additional His⁺ derivatives arose on continuous growth of transformants originally scored as His^- Ura⁺ Trp⁺. In all cases, the His⁺ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His⁺ phenotype was lost more readily than the Ura⁺ Trp⁺ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura⁺ Trp⁺ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.     

Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast

Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.     

A catalytic subunit of the sugar beet protein kinase CK2 is induced by salt stress and increases NaCl tolerance in Saccharomyces cerevisiae

Salinity is an important limiting factor in plant growth and development. We have cloned a catalytic subunit of the sugar beet protein kinase CK2 (BvCKA2) by functional expression in yeast of a NaCl-induced cDNA library. BvCKA2 was able to increase the yeast tolerance to NaCl and to functionally complement the cka1 cka2 yeast double mutant upon over-expression. Southern blot analysis indicated that, in sugar beet, the BCKA2 gene is a member of a multigene family. The mRNA levels of BvCKA2 were up-regulated in response to NaCl stress which suggests that protein kinase CK2 may be involved in the plant response to salt stress.     

Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast

A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K ^– R ^– was subjected to the K₂ killer effect of Saccharomyces cerevisiae T206 K ⁺ R ⁺ in a liquid grape medium. The lethal effect of the K₂ mycoviral toxin was confirmed by methylene blue staining. Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface. Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.     

Influence of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains on wine total antioxidant capacity evaluated by photochemiluminescence

A preliminary investigation on 20 Aglianico del Vulture commercial wines from the Basilicata region proved the existence of a significant variability in total antioxidant capacity which can exert a potential impact on wine quality. Nineteen Saccharomyces cerevisiae strains were tested in Aglianico del Vulture on pilot scale fermentation and the experimental wines obtained were evaluated for the antioxidant capacity, ethanol and total polyphenols. At the ninth day of fermentation the experimental wines had an antioxidant capacity, measured by photochemiluminescence, between 2.88 and 6.25 mM of ascorbic acid equivalent, ethanol concentration, measured by GC, between 5.49% and 10.99% and total polyphenols, determined by Folin Ciocalteau reagent, from 1153 to 1867 mg catechins/l. After 12 days the total antioxidant capacity was increased in most wines but decreased in some wines. These results, statistically analysed by principal component analysis, revealed a significant influence of S. cerevisiae strain on total antioxidant capacity and total polyphenols content of wine.     

Two Cases of Vaginitis Caused by Itraconazole-Resistant Saccharomyces cerevisiae and a Review of Recently Published Studies

Genitourinary infections caused by non-Candida yeasts are uncommon, and especially due to Saccharomyces cerevisiae. We describe the cases of two adult females with vulvovaginal infections caused by itraconazole-resistant S. cerevisiae who made a full recovery after oral fluconazole therapy. We also provide a concise review of recently published studies on this topic.     

Choreography of recombination proteins during the DNA damage response

Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.     

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.     

Loss of heterozygosity and DNA damage repair in Saccharomyces cerevisiae

Loss of heterozygosity (LOH) of tumor suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Understanding how LOH events arise may provide an opportunity for the prevention or early intervention of cancer development. In an effort to investigate the source of LOH events, we constructed MATalphacan1Delta::LEU2 and MATa CAN1 haploid yeast strains and examined canavanine-resistance mutations in a MATa CAN1/MATalphacan1Delta::LEU2 heterozygote formed by mating UV-irradiated and nonirradiated haploids. An increase in LOH was observed when the irradiated CAN1 haploid was mated with nonirradiated can1Delta::LEU2, while reversed irradiation only marginally increased LOH. In the rad51Delta background, allelic crossover type LOH increased following UV irradiation but not gene conversion. In the rad52Delta background, neither type of LOH increased. The chromosome structure following LOH and the requirement for Rad51 and Rad52 proteins indicated the involvement of gene conversion, allelic crossover and break-induced replication. We argued that LOH events could have occurred during the repair of double-strand breaks on a functional (damaged) but not nonfunctional (undamaged) chromosome through recombination.     

利用酿酒酵母转座子文库筛选线粒体镁代谢相关基因

镁离子对于维持细胞正常功能十分重要,糖尿病、高血压、慢性呼吸道疾病、骨质疏松、心律失常等多种疾病都与镁代谢失衡有关．MRS2 基因编码线粒体镁离子转运蛋白,MRS2缺失会导致酵母线粒体镁离子浓度下降、线粒体内Ⅱ型内含子剪接缺陷和非发酵碳源培养基上的生长缺陷．为了增进对线粒体镁离子代谢调控基因的了解,利用酿酒酵母mTn-lacZ/LEU2转座子文库筛选MRS2的抑制基因,发现线粒体载体家族成员YMR166C基因的缺失可以挽救MRS2基因缺失突变体的生长缺陷、Ⅱ型内含子剪接缺陷,并可以调节线粒体镁离子浓度,首次发现YMR166C是线粒体镁代谢相关基因.     

Selection in yeast I: Assessing genetic stability and relative fitness of commercial yeasts

Summary The potential for changes in allele frequencies in yeast populations by selection was examined. Cells from the wine yeastSaccharomyces cerevisiae (strain Montrachet) were grown over a large number of generations using two different culturing techniques, each with two variations: serial transfers on WLN agar plates with and without UV irradiation, and continuous culture in autoclaved and in filter-sterilized grape must. A low frequency of variant isozyme patterns was found in samples taken at the end of the experiment. Growth rates in must and on agar plates were also examined, and it was found that all samples were faster-growing than the original strain, to varying degrees. Applications for the selection system developed are discussed.     

DNA damage induced by copper deficiency in cattle assessed by the Comet assay

Cattle hypocuprosis is a well-known endemic disease in several parts of the world. In a previous paper, the clastogenic effect of copper deficiency in cattle has been described although the occurrence of DNA damage was not directly tested. For this reason, the relation between DNA damage assessed by the Comet assay and Cu plasma concentration was studied in Aberdeen Angus cattle.Blood samples were obtained in heparinized Vacutainer^® tubes from 28 female Aberdeen Angus cows during pregnancy or immediately after to give birth. Each sample was divided into two aliquots for Comet assay and Cu plasma determination, respectively. From the 28 cattle sampled, 17 were normocupremic and 11 were hypocupremic.Results obtained showed that whereas the average plasma Cu level in normocupremic cattle was 67.6 μg/dl, in hypocupremic cattle it was 32.1 μg/dl. The increase of DNA damage was mostly evidenced by the decrease of comet degree 1 cells and an increase of comet degree 2 cells. Correlation analysis comparing plasma Cu levels and degree 1 cells showed a correlation coefficient 0.72 (P<0.01). The comparison between plasma Cu levels and comet degree 2 cells was −0.65 (P<0.01). The comparison between plasma Cu levels and the comet length-head diameter medians determined in 23 out of 28 animals showed a correlation coefficient of −0.54 (P<0.01).The induction of DNA damage was clearly supported by the fact that the decrease of plasma Cu levels was correlated with the increase of comet length-head diameter. These findings could be considered as a contribution to the hypothesis that DNA and chromosome damage are a consequence of the higher oxidative stress suffered by hypocupremic animals.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.