The objective of the experiments was to compare qualitatively the monosaccharides in the hydrolysis products of the extracellular polysaccharides of several yeasts and yeast-like fungi. Specifically, the study was aimed at finding similarities and differences that might be useful in suggesting and supporting taxonomic relationships. Gas chromatography and paper chromatography were used as methods of analyses in an effort to find out what method is sufficient at the qualitative level for distinguishing some genera of yeasts and yeast-like fungi; and what method would be best at the quantitative level for distinguishing amongst some species of the same genus. From the analytical results it was found that paper chromatography using the solvents ethyl acetate: pyridine: water, (8:2:2) was sufficient for qualitative determination of the monosaccharides in the extracellular polysaccharide hydrolysis products. However, indications were that quantitative analyses by gas chromatography, using the trimethylsilyl derivatives of the monosaccharides would have been successful in distinguishing among species of the same genus. Two groups were formed on the bases of the qualitative results. Group I contained two subgroups. Subgroup I encompassed those yeasts and yeast-like fungi with the monosaccharides galactose, glucose, mannose, xylose present in the hydrolysis products of their extracellular polysaccharides. Included in this Subgroup I are: Cryptococcus laurentii, Tremella mesenterica, Bullera alba, Sporobolomyces odorus, Sporobolomyces singularis, and Rhodotorula glutinis. Subgroup II is Ustilago hordei only, with the monosaccharides galactose, glucose, mannose, and lacking xylose. Group II contains Taphrina populina only, with glucose and mannose present and both galactose and xylose absent. The two groups formed support some of the taxonomic relationships that have already been suggested. The Tremella - Cryptococcus taxonomic relationship that had previously been postulated on the basis of similarities in extracellular polysaccharide hydrolysis products, morphology, carbon assimilation patterns, enzymatic xylosylation reaction, and starch formation was supported. Secondly, the Cryptococcus-Bullera relationship that had been suggested on the basis of inositol assimilation, lack of pseudomycelium, and similarities in starch synthesis, was supported by the qualitative analysis of the monosaccharides present in the extracellular polysaccharide hydrolysis products. The monosaccharides found in both Cryptococcus laurentii and Bullera alba extracellular polysaccharides were the same qualitatively. Duality amongst species of Sporobolomyces might be supported with further work using quantitative gas chromatographic analyses. This duality had been postulated on account of the duality shown in antigenic analyses and percent G+C base analyses of DNA. Taphrina populina can be distinguished from Rhodotorula glutinis and Cryptococcus laurentii. Cryptococcus laurentii produces starch and assimilates inositol: Rhodotorula glutinis assimilates inositol but does not produce starch; and Taphrina populina produces starch but does not assimilate inositol. Two monosaccharides present in the extracellular polysaccharide hydrolysis products of both Cryptococcus laurentii and Rhodotorula glutinis are galactose and xylose whereas Taphrina populina lacks these two monosaccharides. Results obtained from the qualitative analyses of the extracellular polysaccharides produced by fungi may be important taxonomically. This is because the qualitative information may be used when deciding on Perfect-Imperfect fungal relationships. However, this information should be considered along with data from other fields such as morphology, cytology, and genetics before hypothesizing on a taxonomic relationship. / Science, Faculty of / Botany, Department of / Graduate

Saccharomyces cerevisiae, the best-known representative of the Saccharomycotina subphylum, is an intron-poor organism with introns in only 5 % of its protein-coding genes. The most popular model of intron evolution suggests that intron-poor eukaryotes, such as S. cerevisiae, have undergone extensive intron loss throughout their evolutionary history. Against this background of intron loss, the retention of specific introns in the S. cerevisiae genome might be attributable to an evolutionary advantage that they provide. Introns have been shown to exhibit ‘function’ in various ways: through recognition of their sequence by RNA binding proteins, the adoption of secondary structures after transcription, the mechanism of splicing itself, and noncoding RNA genes embedded within them. In order to understand how RNA structures contribute to intron function, we first performed a computational screen using 306 alignments of S. cerevisiae intron orthologs. We identified conserved RNA structures in 19 introns that act either in trans as independent intron-encoded ncRNA genes or in cis within the pre-mRNA. Our results showed that introns with conserved secondary structures are conserved in yeast and experimental validation revealed they are frequently maintained in the cells after splicing. Our results suggest that the intron in GLC7 contains a novel ncRNA that regulates expression of its host transcript under stress conditions. Secondly, we focused on the HAC1 intron, which is known to be spliced upon the unfolded protein response by an endoribonuclease IRE1. We showed that the conservation of known intron-defining RNA hairpins in HAC1 extends to Fungi and Metazoa. Concurrently, we identified with high confidence those species that have lost the mechanism of this unconventional splicing. Thirdly, we investigated rates and mechanisms of intron loss within the whole Saccharomycetaceae family in order to develop our findings on the conservation of introns with RNA structures within the context of yeast evolution on both the species and clade level. Computational intron prediction supplemented by RNAseq data from four yeast species demonstrated that both intron loss and conservation of intronic ncRNAs were prevalent in yeast species, and that these patterns have been shaped by whole genome duplication. Lastly, we hypothesise that intron loss in recent yeast evolutionary history has been promoted by double strand break repair machinery.

This research project is concerned with the use of an amylolytic yeast, Endomycopsis sp., for simultaneous production of yeast protein and crude amylase preparations from natural starch materials. The Endomycopsis yeasts were cultivated alone and in combination with other yeasts which are unable to attack starch directly. The propagations were carried out in the presence of urea and phosphate, under aerobic conditions, with vigorous agitation, at pH 5.0 and 28°C. At daily intervals, the cultures were analyzed for protein yield, cell density, and amylase activity. The cell crop harvested after propagation of Endomycops is yeasts on 6.0% potato media contained 19% protein and the culture filtrate obtained after biomass separation had an activity of 1.5 units. Variations in activity and protein content were observed, depending on the starch substrate used, the concentration of urea added, and apparently, the amount of oxygen supplied. Mixed preparations using Candida utilis as ancillary yeast, gave higher protein yields and amylase activities compared to single propagations of Endomycopsis sp. and mixed propagations with Saccharomyces cerevisiae. Purple yam and cassava tubers were examined for protein enrichment and amylase production. It was observed that the protein content of the cell crops obtained from these substrates could be increased about ten-fold but that the amylase activities of the culture filtrates were very low . / Land and Food Systems, Faculty of / Graduate

No description available.

The Silent Information Regulator (SIR) complex is responsible for the formation of silent chromatin domains in Saccharomyces cerevisiae, and consists of the NAD-dependent histone deacetylase Sir2, and histone binding proteins Sir3 and Sir4. The current model of silent chromatin assembly proposes that histone deacetylation by Sir2 is required to promote recruitment of Sir3 and Sir4, and assembly of full SIR complexes on chromatin. However, recent work has suggested unique roles for the histone binding proteins Sir3 and Sir4 in this process. Here we present data suggesting that Sir3 is primarily responsible for mediating the spreading of silent chromatin from sites of nucleation, while regulation of Sir4 abundance controls the rate of silencing establishment. We have also investigated a potential novel dimerization domain in Sir3, which may represent a conserved function in vertebrates. Investigations into the regulation of silent chromatin assembly in budding yeast will facilitate our understanding of the mechanisms that control heterochromatin-mediated gene repression in higher organisms.

Cell division and the maintenance of cellular integrity are key features of life itself and some vital aspects of these processes have been studied in this thesis, using the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis as model eukaryotes. In the first part of the thesis, three putative negative regulators of the cell wall integrity (CWI) signal transduction pathway were investigated, which have been isolated in previous genetic screens. Whereas the FIG4 gene seems to encode a protein which could be distantly related to CWI signaling, NTA1 and SET4 gene products were not found to have a major influence, as judged from phenotypes of deletion mutants, overproducers, and epistasis analyses with different CWI pathway mutants. In general, the data indicated rather indirect connections of all three protein functions with the maintenance of cellular integrity. Therefore, this line of research was discontinued, in order to investigate more closely the regulation of cytokinesis in the dairy yeast K. lactis. In this second and major part of the thesis the homologue of a recently found cytokinesis regulator in S. cerevisiae (INN1, accordingly designated as KlINN1), was cloned and characterized. It could be shown, that the gene is essential and that the encoded protein is species-specific, i.e. KlINN1 does not complement the lethality of a Scinn1 deletion mutant and vice versa. Analyses of hybrid proteins demonstrated that this specificity is most likely mediated by the C2-domain of the protein, which is thought to interact with membrane lipids. In S. cerevisiae, Inn1 interacts through its proline-rich motifs located in the C-terminal half of the protein with the cytokinesis regulators Hof1 and Cyk3. They both carry a SH3-domain which has been shown to mediate the interaction with Inn1. Consequently, the two encoding genes, KlHOF1 and KlCYK3, were also characterized in K. lactis. In contrast to S. cerevisiae, where the homologues seem to exert somewhat redundant functions and only a double deletion is lethal, each of the genes is essential in K. lactis. The exact nature of their roles in cytokinesis of this yeast remains to be determined. Unexpectedly, attempts to confirm an interaction between the proline-rich motifs of KlInn1 and the SH3-domains of KlHof1 and KlCyk3 in a yeast two-hybrid assay failed so far, but this line of research will be followed up in future experiments. In order to compare the timing of cytokinesis and the localization of the above mentioned regulators between S. cerevisiae and K. lactis, another homologue of a protein involved in cytokinesis in S. cerevisiae was investigated in K. lactis. Preliminary evidence from deletion of the KlMYO1 gene, which encodes a likely component of the contractile actomyosin ring (CAR) in K. lactis, indicates that this yeast may predominantly engage in a CAR-independent pathway of cytokinesis. Nevertheless, similar to S. cerevisiae GFP-fusions of KlInn1, KlHof1, KlCyk3, and KlMyo1 all were shown here to localize to the bud neck during cytokinesis in K. lactis. In summary, components identified to play a crucial role in yeast cytokinesis in S. cerevisiae display a similar localization in K. lactis, but may differ considerably in their detailed functions in vivo. This thesis represents the first detailed investigation of the molecular processes underlying cytokinesis in K. lactis and provides the basis for elaborate future studies.

L’ingénierie métabolique est une discipline qui vise à modifier artificiellement le métabolisme d’un organisme afin de lui faire produire un composé chimique d’intérêt. L’une des problématiques fréquemment rencontrées pour la production de nouvelles molécules est l’impossible diffusion du produit formé dans le cytoplasme vers l’extérieur de la cellule. Ce phénomène engendre une accumulation de ce dernier à l’intérieur du micro-organisme qui limite sa capacité de production pour des raisons de cinétiques chimiques et de toxicité pour l’hôte. Enfin, cela complique fortement la récupération et la purification de la molécule d’intérêt, ce qui peut réduire à néant le rendement économique du procédé industriel.Durant cette thèse, nous avons exploré la possibilité de réaliser les dernières étapes de synthèse de composés imperméables pour la membrane à l’extérieur de la cellule en utilisant des enzymes accrochées à la surface de la levure par la technique de “yeast surface display”.Dans une première partie, nous avons regardé l’intérêt industriel de la bioéconomie, puis nous nous sommes intéressés aux problématiques liées à la perméabilité des membranes plasmiques. Dans un second temps, nous avons évalué les méthodes de mesures de la perméabilité des membranes biologiques et exploré la possibilité de développer une méthode prédictive en utilisant une technique de relation structure-propriété. Dans un troisième temps, nous avons évalué les systèmes de “yeast surface display” disponibles et cherché à en découvrir de nouveaux, adaptés à nos problématiques. Dans l'objectif de construire ces bibliothèques nous avons aussi réalisé un outils informatique, permettant de calculer de grands nombres de primers. Enfin, nous avons réalisé différents circuits de production de molécules modèles pour évaluer la pertinence de l’approche pour la production de composés imperméables. / Sustainable chemical production is one of the endeavour of the post-oil era. Amongst the possible techniques, metabolic engineering which aims at producing novel compounds through genetic engineering of micro-organism is seen as one of the most promising techniques. One of the problem met by metabolic engineers is often the absence of diffusion or pumping mechanism expelling the compound of interest produced in the cell cytoplasm towards the outer environment, which reduces the process efficiency because of kinetic and toxicity concerns.During this PhD, we explored the possibility of producing impermeable compounds on the surface of a cell by anchoring the last reaction enzyme using « Yeast surface display » techniques.As PhD disputation we first looked at the industrial interest of metabolic engineering in the whole bioeconomy framework. We then looked at the membrane permeability issues met for the production of some compounds. We evaluated the different membrane permeability techniques and explored the possibility realizing a predictive technique using quantitative structure-property relationship (QSAR). We evaluated the different yeast-display systems available and paved the way for the discovery of new systems more suitable for metabolic engineenering. We developped a dedicated program tool for large PCR fragment library design. Finally we built several toy metabolic pathways in yeast in order to evaluate the interest of the technique.

The reconstituted steroid transcription unit in Saccharomyces cerevisae was used to screen for steroids in cell extracts prepared from 24 species of plants representing 16 families. Extracts from 7 species representing 5 families were able to activate both estrogen and progesterone receptors expressed in yeast.

No description available.

No description available.