No / We have used the budding yeast Saccharomyces cerevisiae to identify genes that may confer sensitivity in vivo to the antimalarial and cytotoxic agent cryptolepine. Five S. cerevisiae strains, with different genetic backgrounds in cell permeability and DNA damage repair mechanisms, were exposed to several concentrations of cryptolepine. Cryptolepine showed a relatively mild toxicity for wild-type strains, which was augmented by either increasing cell permeability ( erg6 or ISE2 strains) or disrupting DNA damage repair ( rad52 strains). These results are compatible with the ability of cryptolepine to intercalate into DNA and thus promote DNA lesions. The effects of low concentrations of cryptolepine (20% and 40% inhibitory concentrations [IC20 and IC40]) were analyzed by comparing the gene expression profiles of treated and untreated erg6 yeast cells. Significant changes in expression levels were observed for 349 genes (117 upregulated and 232 downregulated). General stress-related genes constituted the only recognizable functional cluster whose expression was increased upon cryptolepine treatment, making up about 20% of upregulated genes. In contrast, analysis of the characteristics of downregulated genes revealed a specific effect of cryptolepine on genes related to iron transport or acid phosphatases, as well as a significant proportion of genes related to cell wall components. The effects of cryptolepine on the transcription of iron transport-related genes were consistent with a loss of function of the iron sensor Aft1p, indicating a possible disruption of iron metabolism in S. cerevisiae. Since the interference of cryptolepine with iron metabolism is considered one of its putative antimalarial targets, this finding supports the utility of S. cerevisiae in drug-developing schemes.

No

Yes / We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division. / Biotechnology and Biological Sciences Research Council (BB/K009346/1)

The goal of this research is to determine the effects of added soybean lipoxygenase (LOX) on bread dough rheological properties and physical properties of bread loaves compared to controls, and to determine sensory attributes of bread loaves using quantitative descriptive analysis (QDA). Protein fractions were obtained through the use of isoelectric precipitation. The pH 4.8 precipitate was found to yield the greatest LOX activity when compared with other fractions (p<0.05). The addition of pH 4.8 precipitate improved rheological properties of bread dough, examined in a farinograph, when compared to the all-purpose control (p<0.05). Addition of soy flour also increased the gluten strength of all-purpose flour (p<0.05). The addition of pH 4.8 precipitate to all-purpose flour did not improve bread loaf volume or texture. Sensory panelists described pH 4.8 supplemented bread as having firmer crumb when compared with controls (p<0.05). There were slight color differences among the loaves. The crust and crumb of bread flour loaves was lighter in color than any other sample. It was concluded that the addition of pH 4.8 precipitate to all-purpose flour greatly improved the rheological properties when compared with all-purpose flour alone. / Master of Science

The effect of soy as a natural antioxidant against flaxseed rancidity in yeast breads was tested. Variables included: control (100% bread flour); yeast bread with 15% flax meal in place of part of the total bread flour; yeast bread with 15% flax meal and 5% soy; and yeast bread with 15% flax meal and 10% soy. Objective and sensory tests were used to evaluate breads. Peroxide values indicated that the hydroperoxides in breads increased during the first four weeks of the study, and then decreased, as would be expected as breads are exposed to more elements with time. Moisture content was not significantly different between the breads. Breads containing flax were significantly firmer (p < 0.02) in texture. Breads containing flax were also significantly lower in volume (p < 0.005) and significantly darker in crumb color (p < 0.01). The level of 10% soy contributed to a significantly darker crust color (p < 0.04). Quantitative descriptive analysis (QDA) found the level of 10% soy also contributed to an increased stale taste and aftertaste, firmer texture, coarser crumb, and drier loaf (p < 0.05). Musty aroma was not significantly different among breads and all breads containing flax had an increased grainy taste (p < 0.0001). Soy was found to have no significant antioxidant effect on the prevention of flaxseed rancidity in yeast breads. / Master of Science

Thesis (PhD(Agric) Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / The main goal of this project was to elucidate the underlying genetic factors responsible for the different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic profiling during alcoholic fermentation in simulated wine-making conditions. The aim was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions. The five strains were also characterized in terms of their adhesion/flocculation phenotypes, tolerance to various stresses and survival under conditions of nutrient starvation. Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). Analysis of changes in gene expression profiles during the course of fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to changing conditions during fermentation. Comparison of differentially expressed transcripts between strains also enabled the identification of genetic factors responsible for differences in the metabolism of these strains, and paved the way for genetic engineering of strains with directed modifications in key areas. In particular, the integration of exo-metabolite profiles and gene expression data for the strains enabled the construction of statistical models with a strong predictive capability which was validated experimentally. Proteomic analysis enabled correlations to be made between relative transcript abundance and protein levels for approximately 450 gene and protein pairs per analysis. The alignment of transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain comparisons, there was almost no correlation between trends in protein and transcript levels, except in certain functional categories such as metabolism. The data also provide interesting insights into molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains. Overall, the systems biology approach to the study of yeast metabolism during alcoholic fermentation opened up new avenues for hypothesis-driven research and targeted engineering strategies for the genetic enhancement/ modification of wine yeast for commercial applications.

Thesis (MSc)--Stellenbosch University, 2003. / Full text to be digitised and attached to bibliographic record. / ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape juice by yeast that originate from the grapes and winery equipment. Research has shown that the population composition and dynamics of these yeasts and other microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora, dominate the yeast population found on the surfaces of grapes, although prevailing Saccharomyces cerevisiae strains complete the fermentation process. The yeast S. cerevisiae is an important factor contributing to the quality of wines and, therefore, the improvement of wine yeasts receives considerable attention worldwide. Apart from classical yeast breeding studies, genetic engineering and recombinant DNA techniques are increasingly being used in strain development research programmes. These techniques might enable the wine yeasts to produce heterologous enzymes that degrade polysaccharides, convert malic acid to lactic acid, increase glycerol production, release roam and flavour compounds, secrete antimicrobial peptides, etc. The release of recombinant yeast strains (genetically modified organisms, GMOs) is subject to statutory approval. Therefore, it is important to answer several questions prior to the use of such genetically improved yeast in the commercial production of wine. For example, will recombinant yeast strains be able to multiply and spread in nature, and will this GMO be able to out-compete the natural microflora because of its newly acquired genetic traits. Since existing commercial wine yeasts are used in the abovementioned strain development research, it is essential to determine already at this early stage to what extent these wine yeast strains survive and spread in nature and to what extent they influence the fermentations of the following vintages. This study is divided into two sections. The aim of the first section is to sample a representative number of yeast strains from various vineyards in different climatological areas, mainly in the Western Cape, South Africa. These yeast strains were identified mainly by electrophoretic karyotyping (contour-clamped homogenous electric field electrophoresis; CHEF). The second part of the study summarises the results obtained when Fourier transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine yeast strains. Sets of data, containing the spectra of the mostly used commercial wine yeast strains, were constructed and used as a reference library. The spectra of the isolated yeast strains were then compared to the reference dataset with specific FT-NIR computer software using mathematical calculations. In conclusion, the two methods used in conjunction with one another proved that the commercial wine yeast strains do not easily disperse from the cellar into the vineyard. The commercial wine yeast strains are also more likely to be found near the cellar and the places where the grape skins are dumped. Therefore, should a recombinant yeast strain be used in winemaking, it would not be dispersed into the vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial population in the environment / AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die samestelling en dinamika van die gispopulasie en ander mikro-organismes baie kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik voltooi. Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag. Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens. Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle wynfermentasies van die daaropvolgende jare beïnvloed. Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om 'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte homogene elektriese veld; CHEF) geïdentifiseer. Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR) spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die kommersiële wyngiste te onderskei. As die twee metodes saam gebruik word vir identifikasie, kan die afleiding gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.

The yeast Saccharomyces cerevisiae is used in many industrial applications including beer brewing, bread making, and winemaking. Winemaking yeast strains have the ability to convert grape sugars into alcohol and other metabolites consistent with good wine. An exploratory comparative approach was undertaken to identify the genes and corresponding proteins that give wine yeast strains of S. cerevisiae their distinctive phenotype, with a focus on studying genes that provide tolerance to ethanol. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.

Yeast prions e.g. [PSI+], [PIN+] and [URE3] are similar to mammalian amyloids that cause neurodegenerative diseases. [PSI+] is the aggregated self-perpetuating (prion) isoform of Sup35, a translation termination factor. The molecular chaperone Hsp104 plays a crucial role in the maintenance and propagation of [PSI+]. Deletion of the GET2 gene has been shown to cause a [PSI+] curing defect by excess Hsp104 and [PSI+] instability on synthetic medium (S. Muller, J. Patterson and Y. Chernoff, unpublished data; and J. Patterson Honors Thesis). Get2 is a membrane protein working in a complex with Get1 and Get3 proteins. This complex, called GET (Golgi-to-ER Traffic), is known to retrieve resident ER proteins from Golgi. In this particular study we provide further evidence for the connection between the GET pathway and yeast prions. The get2 deletion also leads to a detectable loss of [PIN+] prion on synthetic medium. The role of the other two members of the Get complex in prion propagation is also explored. The levels and the activity of Hsp104 in the get2 mutants is analyzed. The size of [PSI+] aggregates in the get2Δ strain is compared to that found in wild type. Finally, other possible mechanisms for the effect of get2 on prion maintenance and propagation are addressed.

Thesis (Ph.D.)--Indiana University, 2010. / Title from screen (viewed on July 21, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Ronald C. Wek, Howard J. Edenberg, Peter J. Roach, Martin Bard. Includes vitae. Includes bibliographical references (leaves 125-132).