No description available.

No description available.

This thesis extends the work of Crimmins (1995) on the control of glycolytic flux in yeast by the enzymes 6-phosphofructo-1-kinase and pyruvate kinase (Pyk1p). This study also examines the influence of Pf1kp and Pyk1p upon yeast resistance to the weak acid preservative, benzoic acid. In <I>Saccharomyces cerevisiae</I>, Pyk1p is encoded by <I>PYK1</I>, and the α and β subunits of Pf1kp are encoded by <I>PFK1</I> and <I>PFK2</I>, respectively. To test the influence of these genes upon glycolytic control, an isogenic set of <I>S. cerevisiae</I> mutants were utilised in which <I>PYK1, PFK1</I> and <I>PFK2</I> expression is dependent on the <I>PGK1</I> promoter. Increased Pf1k levels had little effect upon rates of glucose utilisation or ethanol production during fermentative growth. However, overexpressing Pyk1p resulted in an increased growth rate and an increase in glycolytic flux. This suggests that Pyk1p, but not Pf1kp, exerts some degree of control over the glycolytic flux under these conditions. The effects of reducing Pf1kp and Pyk1p levels were also studied by placing <I>PYK1, PFK1</I> and <I>PFK2</I> under the control of the weak <I>PGK1Δuas</I> promoter. The double Pf1kp mutant showed no significant changes in doubling time, ethanol production or glucose consumption. However, a mutant with a 3-fold reduction ion Pyk1p levels displayed slower growth rates and reduced glycolytic flux. In addition, there was an imbalance in the carbon flow in this mutant, with reductions in ethanol and glycerol production evident, along with increased TCA cycle activity. Hence, while Pf1kp levels did not affect cell physiology significantly under the conditions studied, reduced Pyk1p levels seemed to disturb glycolytic flux and carbon flow. Decreased Pf1kp levels caused an increase in the sensitivity of yeast cells to benzoate, whereas the Pyk1p mutant was not affected. This confirmed that benzoic acid specifically inhibits Pf1kp rather than glycolysis in general.

A selection system for yeast cells expressing mutated invertase (EC 3.2.1.26, SUC2) with altered fructotransferase activity was developed based on the survival of a fructose-intolerant strain in the presence of suitable acceptor substrates and sucrose. Cells of such a strain expressing a wild-type hydrolase activity will not grow due to the release of free fructose from sucrose. Cells expressing an inactive invertase mutant will not grow since they cannot cleave the sucrose, the sole carbon source. Only cells expressing sucrose fructosyl-transferase activity will thrive, growing on the released glucose, the fructosyl moiety not being released. A strain of Saccharomyces cerevisiae was engineered to be intolerant of the presence of fructose in its growth media. This was achieved by inducing a condition in yeast similar to liver cells of humans suffering from hereditary fructose intolerance (MIM 22960). This disorder results from a deficiency of aldolase B (EC 4.1.2.13), and phosphorylation of fructose by ketohexokinase (EC 2.7.1.3) results in an accumulation of fructose 1-phosphate, with a consequent depletion of cytoplasmic phosphate and ATP. Thus, cells in which ketohexokinase phosphorylates fructose, but which lack aldolase B, are intolerant of fructose. Yeast possess neither of these enzymes, and so expression of ketohexokinase in yeast would result in fructose-intolerance. A strain of yeast, for ketohexokinase expression, was initially bred to be unable to metabolize sucrose or fructose, yet remain capable of utilizing glucose, as well as lacking non-specific phosphatases, to prevent remobilization of sequestered fructose 1-phosphate. Rat liver ketohexokinase was purified to heterogeneity, and the partial amino acid sequence subsequently generated exploited to amplify a region of the ketohexokinase cDNA by PCR. This was used to probe a cDNA library, yielding clones encoding the entire ketohexokinase coding region. This was cloned into pMA91, and subsequent expression in yeast resulted in a strain intolerant of fructose in its growth medium, although still capable of growing on glucose. In order to produce a stable fructose-intolerant selection strain, a vector (pIADl) was constructed that allowed multiple integration of an expression cassette containing ketohexokinase cDNA into the rDNA locus of yeast chromosome XII. Expression of wild type invertase from the episomal plasmid pIAD3 in this strain resulted in sucrose-intolerance. A preliminary programme of mutagenesis of the SUC2 gene yielded eight libraries of about one hundred clones each. None of these contained any mutants showing solely sucrose fructosyl-transferase activity, although this system would clearly provide an ideal selection for such mutants from a much larger library.

No description available.

No description available.

No description available.

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifiers. Like ubiquitin, most Ubls are covalently attached to a lysine residue on target proteins. The small ubiquitin-related modifiers (SUMO) play important roles in a number of critical biological processes, such as proliferation and regulation of the cell cycle, yet their specific cellular functions have remained poorly understood. Like ubiquitin, SUMO proteins can also form oligomeric “chains”, but the functions of these structures were even less well understood. To this end, I created the first spectral library for the identification of Ub/Ubl proteins and Ub/Ubl chain linkages in mass spectrometry experiments. This tool has dramatically improved our ability to use MS to analyze the contents of biological samples for Ub and Ubls, and to identify specific types of Ub and Ubl chains in model organisms. I also used MS to conduct the first comprehensive SUMO system protein-protein interactome in any organism. In total, 452 high confidence protein-protein interactions were detected for S. cerevisiae SUMO system proteins, encompassing a total of 321 interacting partners. Yeast SUMO system components were found to interact with proteins involved in a number of different biological processes, and my mapping effort increased the number of known SUMO system interacting partners >50-fold. This study revealed that a number of transcriptional co-repressors and chromatin remodelling proteins interact physically with specific SUMO system components, with a clear division of labour between SUMO system enzymes. Finally, I conducted the first global analysis of SUMO chain function, using a combination of genetic, high-content microscopy, and high-density transcriptomics screens. Consistent with my interactomics work, this study demonstrated that inhibition of SUMO chain synthesis leads to severe chromatin condensation defects, which in-turn leads to chromosome missegregation, unscheduled transcription of stress-and nutrient-regulated genes, and aberrant intragenic transcription. Together, my work thus revealed a major role for the SUMO system in the maintenance of higher order chromatin structure and transcriptional repression.

Global quantitative analysis of genetic interactions provides a powerful approach for deciphering the roles of genes and mapping functional relationships amongst path-ways. Using colony size as a proxy for fitness, I developed a method for measuring ge-netic interactions from high-density arrays of yeast double mutants generated by synthet-ic genetic array (SGA) technology. I identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate fitness measurements. I used this scoring method to map quantitative genetic interactions among 5.4 million yeast double mutants and generated the first functionally unbiased genetic interaction map of a eukaryotic cell. My map produced an unprecedented view of the cell in which genes of similar biological processes cluster together in coherent subsets and functionally interconnected bioprocesses map next to each other. We discovered several physiological and evolutionary gene features that are characteristic of genetic interaction hubs, and explored the relationship between genetic and protein-protein interaction networks. In particular, by comparing quantitative single and double mutant phenotypes, we identified specific cases of positive genetic interactions, termed genetic suppression, and constructed a global network of suppression interactions among protein complexes. I also demonstrated that an extensive and unbiased mapping of genetic interactions provides a key for interpreting chemical-genetic interactions and identifying drug targets. In addition, I used genome-wide SGA data to map profiles of genetic linkage along all sixteen yeast chromosomes. These linkage profiles recapitulated previously identified recombination patterns and uncovered an unexpected correlation between chromosome length and the extent of centromere-related recombination repression. These findings suggest a chromosome size-dependent mechanism for ensuring proper chromosome segregation and highlight the SGA methodology as a unique approach for systematic analysis of yeast meiotic recombination.

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifiers. Like ubiquitin, most Ubls are covalently attached to a lysine residue on target proteins. The small ubiquitin-related modifiers (SUMO) play important roles in a number of critical biological processes, such as proliferation and regulation of the cell cycle, yet their specific cellular functions have remained poorly understood. Like ubiquitin, SUMO proteins can also form oligomeric “chains”, but the functions of these structures were even less well understood. To this end, I created the first spectral library for the identification of Ub/Ubl proteins and Ub/Ubl chain linkages in mass spectrometry experiments. This tool has dramatically improved our ability to use MS to analyze the contents of biological samples for Ub and Ubls, and to identify specific types of Ub and Ubl chains in model organisms. I also used MS to conduct the first comprehensive SUMO system protein-protein interactome in any organism. In total, 452 high confidence protein-protein interactions were detected for S. cerevisiae SUMO system proteins, encompassing a total of 321 interacting partners. Yeast SUMO system components were found to interact with proteins involved in a number of different biological processes, and my mapping effort increased the number of known SUMO system interacting partners >50-fold. This study revealed that a number of transcriptional co-repressors and chromatin remodelling proteins interact physically with specific SUMO system components, with a clear division of labour between SUMO system enzymes. Finally, I conducted the first global analysis of SUMO chain function, using a combination of genetic, high-content microscopy, and high-density transcriptomics screens. Consistent with my interactomics work, this study demonstrated that inhibition of SUMO chain synthesis leads to severe chromatin condensation defects, which in-turn leads to chromosome missegregation, unscheduled transcription of stress-and nutrient-regulated genes, and aberrant intragenic transcription. Together, my work thus revealed a major role for the SUMO system in the maintenance of higher order chromatin structure and transcriptional repression.