Masters Degrees (Microbiology)

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Browsing Masters Degrees (Microbiology) by Author "Bihl, Georgia Gladys"

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    Regulation of the TPS1 gene expression in Saccharomyces Cerevisiae
    (Stellenbosch : Stellenbosch University, 1999-03) Bihl, Georgia Gladys; Van Vuuren, H. J. J.; Stellenbosch University. Faculty of Science. Dept. of Microbiolog.
    ENGLISH SUMMARY: For many years the disaccharide trehalose was thought to be simply a carbon and energy reserve in the yeast Saccharomyces cerevisiae. However, a positive correlation between the trehalose content and thermotolerance, freeze tolerance, desiccation resistance, osmotolerance and exposure to toxic chemicals was observed in yeast as well as in other organisms. Therefore, trehalose is now considered to be a stress protectant rather than an energy reserve. This property of trehalose as a possible stress protectant has been used by the baking and brewing industries to increase the freeze tolerance of yeast. However, there is still a need in both industries to further optimise the freeze tolerance of yeast and to use the protective function of trehalose to the fullest extent. Genes encoding enzymes involved in the trehalose metabolic pathway have been manipulated in order to increase trehalose levels in yeast cells. However, it has been shown that higher trehalose levels do not necessarily increase the stress resistance of yeast. It therefore seems that factors other than high trehalose levels are responsible for stress resistance in yeast and these factors could function either in combination with or independently from trehalose. The focus of this study was to unravel the regulation of the TPS1 gene that encodes the first enzyme of the trehalose biosynthesis pathway, namely trehalose- 6-phosphate synthase. A deletion of the TPS1 gene leads to various phenotypes, including defects in trehalose synthesis and defective growth on glucose. This implies additional functions for the TPS1 gene and prompted us to investigate the transcriptional regulation of the gene. Based on previous results, two control mechanisms were investigated: an autoregulatory mechanism and regulation by stress responsive elements. Autoregulation implies that the gene product regulates expression of its own gene by interacting with the DNA sequences either within the promoter or within the open reading frame (ORF). The latter hypothesis was investigated by scanning the ORF for possible DNA-protein interactions and by expression studies with different truncation constructs of the TPS1 gene under glucose shift conditions. Our results, however, showed no evidence for an autoregulatory mechanism. The correlation between trehalose content and stress conditions led us to investigate the possibility of stress-regulated expression of the TPS1 gene. Stress responsive elements (STRE) have been identified in several stress-related genes where they confer stress-induced expression. Six putative STRE elements were identified in the promoter of the TPS1 gene. Gel retardation experiments and competition studies were used to show that nuclear proteins bind specifically to the STRE elements in the TPS1 gene promoter. Mutation analysis of these elements further showed that the STRE element closest to the ATG (STRE1) played a prominent role in regulating expression of the gene. However, this does not explain the various phenotypes observed for the TPS1 mutant. Further research is required to elucidate the various aspects concerning the control mechanisms involved in expression of the TPS1 gene.