No description available.

No description available.

A collection of salt-sensitive vacuolar (ssv) Saccharomyces cerevisiae mutants were selected for analysis in an attempt to reveal components of the osmosensing signal transduction pathway. A previous screen of these mutants had been used to select those with an impaired glycerol response to salt stress. In this study the glycerol-3-phosphate dehydrogenase activity was measured in all the strains which showed reduced glycerol accumulation to demonstrate a corresponding low enzyme activity which could be caused by lack of signalling through the high osmolarity glycerol (HOG) response pathway. However, enzyme activity was found to be impaired in only one of the strains tested. This demonstrated that the measurement of glycerol accumulation is not a particularly useful screening method for defects in the HOG pathway. The activity of the promoter of CTTl, another stress-responsive gene, was measured in selected ssv strains using a lacZ reporter gene attached to the CTTl promoter stress response element. This gave further information about the stress-responsiveness in the strains tested. CTTl promoter activity did not correlate with GPDH activity in all of the strains tested. As CTTl is subject to control by more than one type of stress the results imply that in at least one of the strains another stress response may be impaired. The VACl homologue (VACIH) on chromosome XIV was characterised as a candidate for one of the 55V genes, 55W7. Although it was demonstrated not to be 55W 7, a role for VACIH in vacuolar protein sorting was discovered. The Δvaclh strain also displayed class E vacuolar morphology. Sequence analysis and complementation experiments demonstrated that VACIH is identical to VPS27.

<p>Inositol phosphates (IPs) are eukaryotic signaling molecules that play important roles in a wide range of biological processes. IPs are required for embryonic development and patterning, insulin secretion, the regulation of telomere length, proper progression through the cell cycle, and the regulation of ion channels. This work uses the yeast Saccharomyces cerevisiae as a model system for investigating the functions of IPs and focuses on the transcriptional regulation of the gene encoding the secreted mating pheromone MF&alpha;2 by the IP kinase Ipk2 (also called Arg82, ArgR3, and IPMK). This work shows that Ipk2 has both kinase-dependent and kinase-independent functions in regulating the transcription of MF&alpha;2. Transcription of MF&alpha;2 is also dependent upon the integrity of an Mcm1-binding site in its promoter. This is the first description of a role for this binding site in the transcription of MF&alpha;2. </p><p><italic>In vivo</italic> and <italic>in vitro</italic> screening approaches to identify additional factors associated with MF&alpha;2 expression or with IP biology generally are also described. These unbiased approaches provide some valuable insight for further investigations.</p> / Dissertation

No description available.

The three-dimensional spatial architecture of chromosomes is integrally connected to chromatin function. Budding yeast telomeres cluster at the nuclear periphery, the ribosomal genes are localised to the nucleolus, tRNA genes may also tend to localise to the nucleolus or centromeres, while the later cluster near the spindle pole body. Recently, in the fission yeast Schizosaccharomyces pombe, a novel role has been revealed for the RNA polymerase III transcriptional apparatus, and TFIIIC in particular, in chromosome spatial organisation and boundary function. In this project, I investigate whether Saccharomyces cerevisiae Extra TFIIIC (ETC) sites, which bind the TFIIIC transcription factor but do not recruit RNA polymerase III, act to position chromosomal domains. I show that six of the eight known S. cerevisiae ETC sites localise predominantly at the nuclear periphery. An ETC site retains its tethering function when moved to a new chromosomal location. TFIIIC binding is necessary for peripheral localisation, since deleting the TFIIIC binding consensus ablates ETC site peripheral positioning. I find that any of the six TFIIIC subunits can drive peripheral tethering, suggesting that the TFIIIC complex is central to the positioning mechanism. Interestingly, anchoring of ETC sites to the nuclear periphery also requires Mps3, a Sad1-UNC-84 domain protein that spans the inner nuclear membrane. Moreover, I show that the mechanism of ETC site peripheral tethering requires chromatin remodelling proteins, and in particular Histone 3 - Lysine 56 (H3K56) acetylation. Finally, I investigate the biological function of ETC sites and examine the connection between this biological function and their ability to anchor at the nuclear periphery. In summary, TFIIIC and Mps3 together position a new class of genomic loci crucial for correct spatial organisation of S. cerevisiae chromosomes.

An investigation of the factors influencing the inhibitory and toxic effects of ethanol and higher alcohols, byproducts of alcoholic fermentation, on yeast, is presented. The relative potency of alcohols was found to correlate strongly with the carbon chain-length or molecular size and the lipid solubility of the respective alcohols. Higher alcohols act synergistically with each other and with ethanol in causing cell death of suspensions of non-growing Saccharomyces cerevisiae. The presence of higher alcohols in fermented broth, even at low concentrations, and other by-products of alcoholic fermentation, could explain the higher potency of ethanol produced during fermentation compared to added ethanol. The kinetics of uptake of labelled ethanol supplied at different concentrations gave no evidence of enzymic involvement in the ethanol uptake process. The rate of release of labelled ethanol by cells fed labelled glucose paralled the rate of p14sC-C0b2s release. This does not support the view that ethanol accumulates within the cells to higher concentrations than occur in the medium. Supplementation of a basal synthetic medium with various nutrients did not confer additional survival capacity on yeast against the adverse effects of alcohol. Osmotic pressure did not influence alcohol toxicity below 10% (w/v) sorbitol equivalent of osmotic pressure. Alcohol toxicity is not influenced by hydrogen ion concentration (pH) over a range of pH 5.3 to 3.5.

Oleaginous yeasts are able to produce a high percentage of their weight as lipids, which can be used as the starting material for biodiesel production, producing a fuel with many of the same properties as petroleum-based diesel. The objective of this research was to compare three oleaginous yeast species, Rhodosporidium babjevae, Rhodosporidium diobovatum, and Yarrowia lipolytica to determine which species would be the best candidate for larger-scale production. Following the comparison work, it was determined that R. diobovatum was the best candidate for scale-up. Subsequent experiments used batch cultures in bioreactors at a volume of 3.5 L, followed by a 25x fold increase to 90 L production. The results of this scale-up showed that the high levels of production and growth continued in a reactor system. As such, R. diobovatum could be a possible organism to use in the production of lipids from waste glycerol for biodiesel production. / October 2015

This thesis describes attempts to investigate the regulation of the Candida albicans hyphal-specific gene HYR1 by a functional dissection of the HYR1 promoter, protein localisation studies and analysis of HYR1 expression in C. albicans morphological mutants. Sequencing of the HYR1 promoter revealed several putative cis-acting elements within 700 bp of the determined HYR1 transcriptional start site. The possibility of using the LAC4 gene from Kluyveromyces lactis as a reporter for dissection of the HYR1 promoter in C. albicans was investigated. Expression of LAC4 in S. cerevisiae and C. albicans was driven by the C. albicans ADH1 promoter. LAC4 expression was carbon-source-dependent in Saccharomyces cerevisiae as shown by a plate assay and -galactosidase assay, and was confirmed by northern analyses which showed high levels of LAC4 mRNA. However, -galactosidase activity was not detectable in C. albicans transformants using the plate assay or the enzyme assay, and this lack of LAC4 expression was confirmed by northern analysis of the LAC4 mRNA. Preliminary Southern analysis revealed that the LAC4 sequences in S. cerevisae and C. albicans are maintained at approximately equal copy numbers between transformants. Hence LAC4 was not sufficiently sensitive to act as reporter of HYR1 expression and therefore the recently developed yEGFP gene was used for a preliminary HYR1 promoter dissection. However, the HYR1 promoter-yEGFP fusions failed to confirm a role for these elements in the regulation of HYR1 expression . Nevertheless, the hyphal-specific nature of HYR1 expression was confirmed by analysis of the HYR1-yEGFP mRNA by northern analysis. The yEGFP reporter also proved to be too insensitive for use as a reporter of HYR1 expression in C. albicans. To investigate the proposed localisation of the Hyr1p, an in-frame Hyr1-yEGFP fusion was created and expressed in C. albicans and S. cerevisiae. However, this work was inconclusive and the status of Hyr1p as a component of the hyphal cell wall remains to be confirmed.

The aims of this work were to investigate the signal transduction pathways controlling the yeast-hyphal morphological transition of Candida albicans, and to gain a clearer understanding of the importance of hyphal formation of the virulence of this organism. The work can be divided into two areas. Firstly, the recently discovered species Candida dubliniensis, the only species in addition to C. albicans capable of forming true non-constricted hyphae, was examined in comparison to C. albicans to compare their virulence capability in vitro and in vivo. The two species were compared with respect to hypha formation, adherence, possession of SAP genes and virulence in the mouse model of systemic candidosis. C. dubliniensis possessed at least a homologue to each of the nine known C. albicans SAP genes, adhered to human cells to a greater degree on exposure in glucose, formed hyphae slightly less efficiently than C. albicans and was less virulent in mice. C. dubliniensis has been isolated particularly from the mouths of HIV positive and AIDS patients. The results of the virulence assessment could be interpreted as reflecting its epidemiological occurrence, - increased adherence on exposure to glucose may be a response to dietary sugar and the reduced virulence would explain in part its association with immunocompromised hosts. Secondly, the role of phosphoinositide signalling in control of the yeast-hyphal transition was investigated by the cloning and characterisation of two putative phosphatidylinositol 4-kinase genes from C. albicans, named CaPIK1 and CaPIK2. Both genes were cloned through their homology to the S. cerevisiae PIK1 gene. Disruption of the CaPIK1 gene in C. albicans indicated that it had no obvious role in the control of hypha formation.