Meiosis is a specialized form of cell division in which haploid gametes are produced from diploid progenitors. This reduction in ploidy results from proper meiotic chromosome segregation and is ensured by crossover recombination events. Given their importance, it is no surprise that crossover formation is regulated in most eukaryotes. Crossover assurance is a regulatory mechanism that helps to ensure that each pair of chromosomes gets at least one crossover during meiosis. We seek to better understand how crossover assurance works. To do so, we have developed a system in which crossover formation between a pair of chromosomes is restricted to a defined region. If crossover assurance functions in this context, then crossovers should frequently form in this defined region. Our experiments involve three yeast strains: Homolog: diploid Saccharomyces cerevisiae. Homeolog: Diploid S. cerevisiae, but with one copy of III derived from S. paradoxus and one from S. cerevisiae. Homo-meolog: The homeolog strain, but with the HIS4 region of the S. paradoxus III replaced with the corresponding S. cerevisiae sequence. S. cerevisiae and S. paradoxus are largely syntenic and have 80-90% sequence homology. This level of sequence divergence greatly reduces the incidence of meiotic crossing over. Thus, in the Homeolog strain chromosomes III will frequently fail to form crossovers. In the Homo-meolog strain, a defined region of homology surrounding HIS4 (a hotspot for meiotic recombination) exists in a chromosomal context of homeology. In the Homo-meolog strain, crossover assurance should result in a high incidence of crossover formation in the HIS4 region. By comparing the spectrum of meiotic recombination events in the HIS4 region in the three strains, we will gain insight into the means through which crossover assurance is enforced. These experiments are in the preliminary stage. Strain construction and data collection are ongoing, but our preliminary results demonstrate an elevated incidence of crossing over in the HIS4 region in the homo-meolog strain relative to both the homolog and homeolog strains. Spore viability patterns in the homo-meolog strain are not statistically distinguishable from that of the homolog strain, but are different from that of the homeolog strain. Taken together, these results suggest that the crossovers are targeted to the HIS4 region in the homo-meolog strain, possible through the action of a crossover assurance mechanism. Further analysis of the patterns of recombination in these strains may provide insight into the means through which this regulation is exerted.

Meiosis is a specialized cell division that leads to a reduction of ploidy in sexually reproducing organisms through segregation of homologous chromosomes at the first meiotic division. Improper segregation of chromosomes during meiosis results in anueploidy, which is usually fatal during embryonic development. The meiotic process is therefore tightly regulated. Typically, proper segregation of homologs at meiosis I requires pairing of homologous chromosomes, followed by crossover recombination between homologs. Crossovers enable proper chromosomal segregation during the first meiotic division in part by establishing tension in the meiotic spindle. However, in the absence of crossovers, some cells maintain the ability to direct homologous chromosomes to opposite spindle poles, through a poorly understood mechanism known as distributive segregation. We are using the common brewers yeast Saccharomyces cerevisiae to determine possible roles of two genes in distributive segregation. The genes of interest, Ndj1 and Tid1, have been previously demonstrated to play a role in crossover interference, but their roles in distributive segregation are not well understood. Ndj1 has been shown to function in the tethering of telomeres to the nuclear envelope and may aid in the homology search chromosomes undergo. Tid1 has been characterized as a recombination accessory factor and may stimulate crossovers by directing recombinases to double strand break sites early in meiosis. To assay distributive segregation, we use yeast in which crossing over between one chromosome pair is prevented (due to sequence divergence). Using this system, we can assay the ability of yeast to carry out distributive segregation. Our results indicate that mutations in Ndj1 impair the ability of yeast to carry out distributive segregation, while mutations in Tid1 do not affect distributive segregation. These results, in turn, suggest that Ndj1 may play a role in distributive segregation. This experiment is part of a larger question to determine whether crossover assurance and crossover interference are independent mechanisms.

Lipid droplet (LD) autophagy (lipophagy) is a recently discovered selective form of autophagy and is a pathway for LD catabolism through the lysosome or vacuole. Therefore, lipophagy has therapeutic potential in the treatment of a variety of lipid related diseases in which increased cellular LDs are associated with pathophysiologies, such as obesity or atherosclerosis. This ubiquitous process has been an ongoing area of research within the budding yeast, Saccharomyces cerevisiae. However, there remains a need to better understand the regulators of this process. I have developed and validated a lipophagy library in yeast for the assessment of novel genetic regulators of stationary phase induced lipophagy. Through the screening of my library for roles in lipophagy I have identified many genetic regulators of lipophagy which include CUE1, UBC7, LHS1, HSP31, PLN1, TFS1, LAM6, OSH3, OSH4 and OSH7, among others. My screen highlights the power of this library to identify lipophagy regulators in S. cerevisiae, which can be utilised in the future to further the understanding of lipophagy.

No description available.

No description available.

The present study was initiated with the purpose of determining and comparing the effect of gamma radiation on the capacity of yeast cells to grow and sporulate. Using a new technique by which irradiated and non-irradiated yeast cells could be scored directly, it was found that sporulating yeast cells were more sensitive to radiation than growing cells, and that the inactivation of the capacity of an irradiated yeast cell to sporulate did not affect its ability to grow. Observations on irradiated sporulating cells indicated that spore-formation and reduction division of the nucleus, are closely allied phenomena. A short discussion of a probable mechanism of action of gamma radiation on sporulating yeast is included, together with suggestions for future research. / Thesis / Master of Science (MS)

The cell cycle of the budding yeast, Saccharomyces cerevisiae, is regulated by a complex network of chemical reactions controlling the activity of the cyclin-dependent kinases (CDKs), a family of protein kinases that drive the major events of the cell cycle. A previous mathematical model by Chen et al. (2000) described a molecular mechanism for the Start transition (passage from G1 phase to S/M phase) in budding yeast. In this thesis, my main goal is to extend Chen's model to include new information about the mechanism controlling Finish (passage from S/M phase to G1 phase). Using laws of biochemical kinetics, I transcribed the hypothetical molecular mechanism into a set of differential equations. Simulations of the wild-type cell cycle and the phenotypes of more than 60 mutants provide a thorough understanding of how budding yeast cells exit mitosis. / Master of Science

Yeast mannoproteins released during the process of aging wine on the yeast lees have been reported to make important contributions to wine quality. However, few mannoproteins have been identified in wine and their lifespan during aging is unknown. As a first step towards better understanding the contributions of yeast mannoproteins to wine quality, a model system was used to follow changes in yeast protein release, and to identify the released proteins over a 9-month time course following completion of fermentation. Model musts were fermented in duplicate by a number of commercial yeast strains, including BM45 and RC212, and were stored on the yeast lees post-fermentation with monthly stirring at 15°C. Wine samples were taken during and after fermentation, and following removal of suspended solids, total protein and total mannoproteins were measured, and individual proteins were identified--but not quantified--by HPLC-MS of tryptic fragments. The total number of identified proteins in all samples increased from between 3-15 following inoculation, to between 70-80 after one month on the lees, and decreased to about 20 after 6 months on the lees. Over 50% of the identified proteins were shared among all yeast samples. For most strains, protein and mannoprotein concentrations increased during, but decreased by the end of fermentation. Both protein and mannoprotein concentrations were found to increase again post-fermentation, reaching values about 2- and 6-fold higher than values measured at 2 days, respectively. Consistent with the increase in mannoprotein concentration, cell wall mannoproteins were the predominant proteins identified after 6 months of aging on the lees. Most cytosolic proteins found during and soon after fermentation were not found after 6 months of aging on the lees. / Graduation date: 2009

Through a full factorial design experiment, the effects of time between worts, wort aeration and yeast dosage in production of a pale lager beer were examined in the beer process at Spendrups Bryggeri AB. The aim was to learn how different parameters may affect the yeast fermentation performance during beer production. Response variables used were the concentrations of ethyl acetate and isoamyl acetate, free amino nitrogen (FAN) degradation and change in extract. A statistical analysis showed that the concentration of ethyl acetate is dependent on yeast dosage and the interaction between time between worts and aeration while the isoamyl acetate concentration is dependent on yeast dosage and time between worts. No parameters are statistically significant for FAN degradation while the change in extract is dependent on the yeast dosage. Due to botched runs, mostly because of aeration problems, it was not possible to verify theoretical parameter values and responses. Since the aeration was not properly performed, the management of the aeration control should be further investigated. Ester analysis and analysis of FAN were performed as worts entered and exited horizontal fermentation tanks. An additional analysis of ester content was also performed as the early stage beer was transferred into lagering tanks. Cell viability as well as extract, pH and tank temperature was measured daily to verify the state of fermentation. Statistical calculations showed that when using NucleoCounter YC-100, there is no significant difference between analysis made of samples homogenized by a magnetic stirrer and samples shaken by hand.

This thesis presents a systematic investigation ofyeasts associated with wine grapes cultivated in several Australian vineyards during the 2001-2003 vintages. Using a combination of cultural and molecular methods, yeast populations of red (Cabernet sauvignon, Merlot, Tyrian) and white (Sauvignon blanc, Semilion) grape varieties were examined throughout grape cultivation. The yeast-like fungus, Aureobasidium pullulans, was the most prevalent species found on grapes. Various species of Cryptococcus, Rhodotorula and Sporobolomyces were frequently isolated throughout grape maturation. Ripe grapes showed an increased incidence of Hanseniaspora and Metschnikowia species for the 2001-2002 seasons, but not for the drought affected, 2002-2003 seasons. Atypical, hot and dry conditions may account for this difference in yeast flora and have limited comparisons of data to determine the influences of vineyard location, grape variety and pesticide applications on the yeast ecology. More systematic and controlled studies of these variables are required. Damaged grape berries harboured higher yeast populations and species diversity than intact healthy berries. PCR-DGGE analysis was less sensitive than plate culture for describing the diversity of yeast species on grapes; it detected prevalent species, but subdominant populations below 103 CFU/g were not detected. In some cases, PCR-DGGE revealed the presence ofyeasts (Candida galli, C. zemplinina) not isolated by culture. Fermentative wine species (Kluyveromyces, Torulaspora, Saccharomyces) were rarely isolated, and only detected by enrichment cultures. Significant morphological and genetic variability were detected among A. pullulans and other black yeasts isolates from grapes. Taxonomic characterization of 61 strains by ITS-RFLP and rDNA sequencing revealed that they belonged to several distinct species within the generic groupings ofAureobasidium, Hormonema and Kabatiella. Isolates were strong producers of extracellular enzymes and polysaccharides that could have oenological significance, and, using a plate assay, some were antagonistic towards Bacillus thuringiensis, several wine yeasts, and some spoilage and mycotoxigenic fungi found on grapes. Growth of Saccharomyces cerevisiae was not inhibited by these organisms in grape juice. A species-specific probe was developed for the identification of the wine spoilage yeast, Zygosaccharomyces bailii in a microtitre plate hybridization assay. The probe detected 102 cells/ml in wine, reliably differentiating Z. bailii from other Zygosaccharomyces and other wine-related yeasts.