Thèses sur le sujet « Saccharomyce »

Orientadora: Cecilia Laluce
Banca: Kelly Johana Dussan Medina
Banca: Edwil Aparecida de Lucca Gattas
Resumo: A levedura mais utilizada nos processos fermentativos é a Saccharomyces cerevisiae, por apresentar uma grande eficiência de conversão dos açúcares em etanol, permitindo assim a produção de etanol combustível em larga escala, porem essas leveduras não predominam durante toda a safra, sendo substituídas por leveduras não - Saccharomyces. Durante o processo fermentativo fatores como estresse alcoólico, térmico, ácido, nutricional e osmótico causam prejuízo ao processo. Na busca por um microrganismo capaz de fermentar em condições de estresse a levedura híbrida, S. cerevisiae IQAr/45-1 (PI 0806141-6) construída no Laboratório de Unidades das Leveduras Industriais do Instituto de Química - UNESP, apresenta características de rápida fermentação e resistência ao estresse térmico. Sendo assim o objetivo principal do presente trabalho é testar a capacidade de fermentar da levedura IQAr/45-1 quando inoculada junto ao creme de levedura industrial, que contem leveduras Saccharomyces e não - Saccharomyces avaliando sua capacidade de melhorar a fermentação. Fermentações de 5 ciclos sucessivos com reuso de células foram conduzidas utilizando um fluxo de alimentação 0,39 mL/min, por 3 horas com melaço 20 % (ART) e foram realizadas a 35 °C e 40 °C, durante 10 horas, utilizando como inóculo creme de levedura industrial com adição da levedura IQAr/45-1 na proporção de 3:1. Durante a fermentação foi analisado a concentração celular, viabilidade e ART. Após as análises observou-se quando adiciona... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

En la superfície del raïm coexisteixen poblacions de llevats, fongs i bacteris. D’aquests llevats, coneguts també com a llevats no-Saccharomyces, n’hi ha algunes espècies que són d’interès enològic. En aquesta tesi, s’ha estudiat els efectes de diferents factors biòtics i abiòtics sobre les comunitats fúngiques i bacterianes durant la fermentació alcohòlica, posant especial èmfasi en els llevats no-Saccharomyces. Per una banda, s’ha avaluat tan l’estat sanitari del raïm com les concentracions de nitrogen i sucre del most sobre el procés fermentatiu i la microbiota. Els resultats mostren que l’estat del raïm defineix les comunitats microbianes al llarg de la fermentació alcohòlica. A més, el nitrogen ha demostrat ser el nutrient determinant en l’èxit de la fermentació. Per altra banda, s’han analitzat les fermentacions i els vins obtinguts amb Hanseniaspora vineae, un llevat no-Saccharomyces d’interès enològic, i Saccharomyces cerevisiae. Els vins obtinguts amb H. vineae presentaven un perfil més afruitat i floral gràcies a la producció de 2-fenil etil acetat. A més, s’ha observat la presència del mecanisme de repressió per catabòlit de nitrogen (NCR) en H. vineae gràcies al perfil d’expressió dels gens AGP1, GAP1, MEP2 i PUT2 i al consum de nitrogen. Per últim, s’ha analitzat la diversitat genotípica i fenotípica de Brettanomyces bruxellensis a Catalunya, un llevat no-Saccharomyces contaminant del vi. Els diferents aïllats de B. bruxellensis es distribuïen segons la zona d’aïllament i exhibien una tolerància a SO2 variable i una gran capacitat de producció de fenols volàtils.
En la superficie de la uva coexisten poblaciones de levaduras, hongos y bacterias. De estas levaduras, conocidas como levaduras no-Saccharomyces, hay algunas especies de interés enológico. En esta tesis, se han estudiado los efectos de diferentes factores bióticos y abióticos sobre las comunidades fúngicas y bacterianas durante la fermentación alcohólica, con especial énfasis en las no-Saccharomyces. Se ha evaluado tanto el estado sanitario de la uva como las concentraciones de nitrógeno y azúcar del mosto sobre el proceso fermentativo y la microbiota. Los resultados muestran como el estado de la uva define las comunidades microbianas durante la fermentación alcohólica. Además, el nitrógeno ha demostrado ser el nutriente decisivo para el éxito de la fermentación. Por otro lado, se han analizado las fermentaciones y vinos obtenidos con Hanseniaspora vineae, una no-Saccharomyces de interés enológico, y Saccharomyces cerevisiae. Los vinos obtenidos con H. vineae presentaban un perfil más frutado y floral gracias a la producción de 2-fenil etil acetato. También se ha observado la presencia del mecanismo de represión catabólica por nitrógeno (NCR) en H. vineae mediante la expresión de los genes AGP1, GAP1, MEP2 y PUT2 y el consumo de nitrógeno. Por último, se ha analizado la diversidad genotípica y fenotípica de Brettanomyces bruxellensis en Cataluña, una no-Saccharomyces contaminante del vino. Los aislados de B. bruxellensis se distribuían según la zona de aislamiento y exhibían una tolerancia a SO2 variable y una gran capacidad de producción de fenoles volátiles.
Populations of yeasts, fungi and bacteria coexist on grape berry surface. Some species belonging to these yeasts, also known as non-Saccharomyces, are of oenological interest. In this thesis, the effects of different biotic and abiotic factors on fungal and bacterial communities during alcoholic fermentation have been studied, emphasizing its effect on non-Saccharomyces yeasts. Therefore, the health status of the grape together with nitrogen and sugar concentrations of the must on the fermentation process and the microbiota has been evaluated. The results show that health status of the grape defines the microbial communities along the alcoholic fermentation. Moreover, nitrogen has demonstrated to be the decisive nutrient for fermentation success. On the other hand, the fermentations and wines obtained using Hanseniaspora vineae, a non-Saccharomyces yeast of oenological interest, and Saccharomyces cerevisiae have been analysed. H. vineae’s wines exhibited a more fruity and flowery aroma thanks to 2-phenetyl acetate production. Additionally, it has been observed the presence of nitrogen catabolite repression(NCR) mechanism in H. vineae considering the expression profile of AGP1, GAP1, MEP2 and PUT2 genes and the nitrogen consumption. Finally, genotypic and phenotypic diversity of Brettanomyces bruxellensis, a non-Saccharomyces spoiler wine yeast, from Catalonia has been evaluated. The different B. bruxellensis isolates distributed according to the isolation region and exhibited a variable SO2 tolerance and a great ability to produce volatile phenols.

The improvement of feed efficiency in dairy cattle has traditionally been approached through an increase in production levels. However, the effectiveness of the energy partitioning underlying the dilution of maintenance phenomenon decreases with each successive increment in production relative to body size, and therefore will lose importance in the next years. The selection of more efficient animals (i.e. animals that consume less feed to achieve a fixed production) is limited by the cost and inherent difficulty in recording individual feed intake data, which prove pivotal to improve the accuracy of the estimated breeding values for feed efficiency traits. First aim of my project was to record and collect individual phenotypic data on the dry matter intake of dairy cattle under different dietary conditions and at different production stages. The collected data will become part of a larger collaborative dataset aimed at increasing the prediction power of genomic selection plans including traits related to the feed efficiency. Furthermore, following a multimodal approach essential for an effective improvement of the dairy enterprise efficiency, the effect of feed additives and alternative feed ingredients on the production, health status and efficiency of dairy cattle has been investigated. In the first trial, the effect of camelina cake – a byproduct of camelina oil rich in proteins and unsaturated fatty acids – on milk fatty acid profile was investigated. Results from the study evidenced a higher concentration of n-3 PUFA (p = 0.02) and a lower n-6/n-3 ratio (p = 0.01) in the milk of cows fed camelina compared to control. The analysis of the ruminal content suggested a moderate effect of camelina on the ruminal environment and biochemistry. In the second trial, the effect of Saccharomyces cerevisiae (live yeast) on the ruminal environment and its interaction with the production and health status of dairy cows was investigated. Dietary treatment with S. cerevisiae did not affect the investigated ruminal parameters and had only mild effects on blood biochemistry. However, an effect on the ruminal pH was observed when cows were fed low-quality forages, with higher pH values in cows supplemented with S. cerevisiae compared to control (p < 0.05). In the last trial, the effect of camelina on the ruminal microbial populations and the cumulus-oocyte complexes of growing dairy heifers was evaluated. The inclusion of camelina in the diet was associated with higher expression of all selected molecular markers of oocyte quality (p < 0.05). Moreover, feeding camelina affected the ruminal microbiota, as shown by the significant reduction in the alpha diversity of the treatment group (p < 0.05).

La melatonina (N-acetil-5 metoxytryptamine) que es sintetitza a partir de triptòfan, es forma durant la fermentació alcohòlica, no obstant el seu paper en el llevat és desconegut. Aquest estudi va utilitzar espècies de Saccharomyces i no Saccharomyces per avaluar els possibles efectes antioxidants de melatonina. Es va avaluar la resistència al H2O2, la producció d’espècies reactives d'oxigen, la peroxidació lipídica, l'activitat catalasa i a la composició lipídica (àcids grassos, fosfolípids i esterols) tant en llevats de Saccharomyces com no-Saccharomyces. A més, a S. cerevisiae es va avaluar el contingut de glutatió reduït i oxidat, es va quantificar la melatonina endògena i es va realitzar un assaig transcriptòmic. Els resultats van mostrar que els llevats que contenen àcids grassos insaturats com els àcids linoleic o linolènic són més tolerants a l'estrès oxidatiu. Per altra banda, la suplementació amb melatonina va facilitar que les cèl·lules fessin front a possibles estressos futurs. Tanmateix, quan les cèl·lules van ser sotmeses a estrès oxidatiu induït per H2O2, la melatonina va poder mitigar parcialment el dany cel·lular reduint la producció de ROS, la peroxidació de lípids i el glutatió oxidat a la vegada que augmentava el glutatió reduït i la viabilitat cel·lular. L`anàlisi de transcriptòmica va demostrar que la melatonina és capaç de modular la resposta a l'estrès oxidatiu a nivell transcripcional. Els resultats demostren que la melatonina pot actuar com antioxidant tant en llevats Saccharomyces com en no-Saccharomyces.
La melatonina (N-acetil-5 metoxytryptamine) que se sintetiza a partir del triptófano, se forma durante la fermentación alcohólica, no obstante su papel en la levadura es desconocido. Este estudio utilizó especies de Saccharomyces y no Saccharomyces para evaluar los posibles efectos antioxidantes de la melatonina. Se evaluó la resistencia al H2O2, la producción de especies reactivas de oxígeno, la peroxidación lipídica, la actividad catalasa y la composición lipídica (ácidos grasos, fosfolípidos y esteroles) tanto en levaduras de Saccharomyces como no-Saccharomyces. Además, en S. cerevisiae se evaluó el contenido de glutatión reducido y oxidado, se cuantificó la melatonina endógena y se realizó un ensayo transcriptómico. Los resultados mostraron que las levaduras que contienen ácidos grasos insaturados como los ácidos linoleico o linolénico son más tolerantes al estrés oxidativo. Por otra parte, la suplementación con melatonina facilitó que las células hicieran frente a posibles estreses futuros. Sin embargo, cuando las células fueron sometidas a estrés oxidativo inducido por H2O2, la melatonina pudo mitigar parcialmente el daño celular reduciendo la producción de ROS, la peroxidación de lípidos y el glutatión oxidado a la vez que aumentaba el glutatión reducido y la viabilidad celular. El analisis de transcriptómica demostró que la melatonina es capaz de modular la respuesta al estrés oxidativo a nivel transcripcional. Los resultados demuestran que la melatonina puede actuar como antioxidante tanto en levaduras Saccharomyces como no-Saccharomyces.
Melatonin (N-acetyl-5 methoxytryptamine) which is synthesized from tryptophan, is formed during alcoholic fermentation, though its role in yeast is unknown. This study employed Saccharomyces and non-Saccharomyces species to evaluate the possible antioxidant effects of melatonin. Resistance to H2O2, reactive oxygen species, lipid peroxidation, catalase activity and lipid composition (fatty acids, phospholipids and sterols) were evaluated in both Saccharomyces and non-Saccharomyces yeasts. Furthermore, cell viability, reduced and oxidized glutathione levels, endogenous melatonin levels as well as transcriptomics study were assessed in S. cerevisiae. Results showed that non-Saccharomyces yeast containing unsaturated fatty acids such as linoleic or linolenic acids are more tolerant to oxidative stress. Melatonin supplementation enables cells to resist better further stresses. However, when cells were subjected to oxidative stress induced by H2O2, melatonin was able to partially mitigate cell damage by decreasing ROS production, lipid peroxidation and oxidized glutathione and increasing reduced glutathione and viability. Transcriptomics assays showed that melatonin is able to modulate the oxidative stress response at transcriptional level. The findings demonstrate that melatonin can act as antioxidant in both Saccharomyces and non-Saccharomyces yeasts.

La maîtrise des procédés fermentaires a largement contribué à l'essor de la pratique du levurage en vinification. Toutefois, dans un souci de respect et de préservation des spécificités de chaque région viticole, de nouvelles souches de levures sont sélectionnées. Ainsi nous nous intéressons dans ce travail à l'étude de souches hybrides S. Cerevisiae x S. Uvarum et plus particulièrement dans l'optique de leur production industrielle sous forme de levures sèches actives. Tout d'abord, les caractéristiques physiologiques de la souche parentale S. Uvarum nécessaires pour la conduite d'une production de biomasse ont été identifiées. Par ailleurs, une perte de viabilité assez atypique chez une levure a été décelée sous certaines conditions opératoires. Puis après une étude comparative des souches hybrides et parentales, notamment en ce qui concerne leurs potentialités fermentaires, les modalités pour la production optimale d'un hybride ont été abordées. La détermination de deux profils d'apport du substrat, dont la validité dépend de la concentration en sucre dans l'alimentation et des souches employées, ainsi que l'accumulation de tréhalose intracellulaire en constituent les paramètres établis. Ces parmètres clé du procédé ont ensuite été confirmés par des productions de biomasse sous forme de levures sèches actives à une échelle pilote.

Mestrado em Bioquímica - Bioquímica Alimentar
A indústria cervejeira usa diferentes espécies de leveduras Saccharomyces para a produção de cerveja. As leveduras são normalmente reutilizadas em 3-7 ciclos fermentativos e em seguida, são descartadas, sendo designado como levedura excedentária da cerveja (BSY). A BSY é um dos maiores subprodutos resultantes da indústria cervejeira e é fonte de polissacarídeos, nomeadamente glucanas, manoproteínas e quitina, provenientes da parede celular. No presente trabalho foram analisados os polissacarídeos das leveduras S. cerevisiae com 1 e 3 ciclos fermentativos e S. pastorianus com 2 e 6 ciclos fermentativos. Os polissacarídeos da parede celular das leveduras foram extraídos sequencialmente recorrendo à extração com água a 100 ºC e à extração aquosa assistida por micro-ondas (MWE) a 180 ºC. A extração com água quente permitiu extrair os polissacarídeos da superfície da parede celular que, no caso da S. cerevisiae, são constituídos essencialmente por resíduos de glucose em ligação (1→4) e, no caso da S. pastorianus são constituídos por resíduos de manose em ligação terminal, (1→2)-Man e (1→2,6)-Man. Os extratos solúveis da MWE, de S. cerevisiae são ricos em (1→4)-Glc e os da S. pastorianus são ricos em (1→2)-Man e (1→2,6)-Man. O resíduo insolúvel é composto por (1→4) e (1→3) glucanas. Os resíduos de glucose em ligação (1→4) foram sensíveis à hidrólise com α-amilase e com celulase, permitindo inferir a presença de resíduos com configuração anomérica α e β. Por microscopia eletrónica de varrimento verificou-se que a estrutura tridimensional das leveduras se mantém no resíduo após extração aquosa dos polissacarídeos. Uma potencial valorização deste resíduo poderá ser como microcápsula para a incorporação de compostos bioativos na área alimentar ou clínica. A levedura excedentária da cerveja apresenta grande variabilidade dependendo da estirpe/espécie da levedura e ainda do número de ciclos fermentativos a que está sujeita. Os extratos solúveis de MWE de S. cerevisiae são fonte de glucose em ligação (1→3), quando provenientes de um baixo número de reutilizações, e/ou ligação (1→4), se provenientes de um elevado número de reutilizações. As S. pastorianus são fonte de manoproteínas.
Beer industry uses different Saccharomyces yeast species, which are reused during 3-7 fermentative cycles. When discarded, they are named brewer’s spent yeast (BSY). BSY is one of the major by-products resultant of brewery industry and it is a source of glucan, mannoprotein and chitin components of yeast cell wall polysaccharides. In the present work, the cell wall polysaccharides of S. cerevisiae with 1 and 3 fermentative cycles and S. pastorianus with 2 and 6 fermentative cycles were analyzed. Cell wall polysaccharides were sequentially extracted with water at 100 ° C and with microwave assisted water extraction (MWE) at 180 ° C. The hot water extraction allowed to obtain the cell wall surface polysaccharides. Extracted S. cerevisiae polysaccharides were mainly constituted by (1→4) linked glucose and S. pastorianus ones were constituted by terminally-linked mannose, (1→2)-Man and (1→2,6)-Man. S. cerevisiae MWE extracts were enriched in (1→4)-Glc while MWE extracts of S. pastorianus were rich in (1→2)-Man e (1→2,6)-Man. The insoluble residue was composed mainly of (1→ 4) and (1→ 3) glucan. The (1→4) linked glucose was hydrolysed by amylase and cellulase, allowing to infer the presence of α and β anomeric configurations. The residue that remain after the extraction of the polysaccharides was found by scanning electron microscopy, to maintain the three dimensional structure of the yeast. This residue can be valued as a microcapsule for the incorporation of bioactive compounds in food or clinical applications. Depending on the number of yeast reutilizations, MWE extracts of S. cerevisiae are a source of (1→3)-glucans or (1→4)-glucans, while MWE extracts of S. pastorianus are a source of mannoproteins. As BSY showed a high variability depending on the yeasts strain/ species and reutilization, able to be recovered by MWE.

L'effet de plusieurs pesticides envers Saccharomyces cerevisiae et Metschnikowia pulcherrima a été évalué. Une étude de la cytotoxicité et la génotoxicité du penconazole en fonction des conditions de culture et du stade métabolique a été effectuée. Le penconazole inhibe les cinétiques de croissance et de fermentation des deux levures en cas d'exposition dès l'inoculation aux concentrations résiduelles faibles (0,2-2 ppm), M. pulcherrima est plus sensible ; des adduits à l'ADN ont été détectés dans les cultures contaminées durant la phase exponentielle. Ces résultats ont été validés en testant notre système expérimental levurien vis-à-vis du benzo(a)pyrène et de l'aflatoxine B1 (0,2 et 2 ppm), deux génotoxiques de référence. Des adduits à l'ADN ont été obtenus dans toutes les cultures exposées au benzo(a)pyrène, et l'aflatoxine B1 a induit la formation d'adduits uniquement lors de son addition durant la phase exponentielle ; alors que la croissance n'a pas été altérée par aucun des deux.

Il existe des millions d'espèces différentes dans le monde qui ont évolué grâce à des interactions complexes avec leur environnement. La biologie évolutive contemporaine connaît une révolution grâce au séquençage de génomes ainsi qu’au criblage et la manipulation génétique, mais l'objectif reste le même qu'il y a 160 ans: comprendre les mécanismes sous-jacents impliqués dans la spéciation. Cela peut être réalisé en étudiant les mécanismes génétiques impliqués dans l'adaptation locale et la spécialisation écologique lors des premiers événements de spéciation. L'objectif principal de cette thèse est d'étudier les mécanismes moléculaires qui sous-tendent l'adaptation et la différenciation des populations dans un complexe de jeunes espèces de la levure Saccharomyces paradoxus, naturellement présentes dans les forêts de feuillus d'Amérique du Nord. En utilisant diverses approches, telles que la génomique des populations, la biologie expérimentale, la transcriptomique et le phénotypage à haut débit, nous (1) disséquons les bases génétiques de la spécialisation écologique et (2) étudions les effets de l’hybridation sur la divergence rapide et la spéciation. Nous documentons d’abord que la spécialisation écologique à différentes températures (un phénotype reconnu pour jouer un rôle important dans la divergence de deux principales lignées de S. paradoxus) est en partie causée par une sélection assouplie avec des compromis. Les travaux portant sur deux événements d'hybridation inter-espèces démontrent, quant à eux, un croisement entre une espèce hybride et son espèce parentale, ce qui indique que l'hybridation est probablement plus fréquente dans l'évolution des espèces qu'on ne le pensait auparavant. Nos travaux soulignent l’importance de la différenciation écologique par une sélection relaxée plutôt que par une divergence adaptative de la fixation de mutations bénéfiques. En outre, nos travaux montrent que l'hybridation dans la nature joue probablement un rôle important dans la création d'une nouvelle diversité par le biais de la ségrégation transgressive et que cela peut se répéter par des croisements incluant des espèces hybrides. Des études à venir sur des espèces jeunes et des complexes hybrides permettront de comprendre davantage les bases génétiques de la différenciation des populations, les conséquences de l'hybridation inter-espèces et de sa récurrence dans l'origine des espèces.
Millions of different species inhabiting the world have evolved through complex interactions with their environment. Contemporary evolutionary biology is experiencing a revolution in genome sequencing, screening and genetic manipulation technologies. Its aim, however, remains the same as 160 years ago when pioneers like Darwin and Wallace published the first articles about the evolutionary theory: to understand the underlying mechanisms involved in speciation, because such knowledge is key to shed light into species diversification. This can be achieved by studying the genetic mechanisms involved in local adaptation and ecological specialization during early speciation events. The main objective of this work is to investigate the molecular mechanisms underlying adaptation and population differentiation in a young species complex of the budding yeast Saccharomyces paradoxus, naturally found in the North American deciduous forests. Using different approaches, such as population genomics, experimental biology, transcriptomics and high-throughput phenotyping we (1) dissect the genetic bases for ecological specialization and (2) investigate the effect of hybridization in facilitating rapid divergence and speciation. First, we document that the ecological specialization to different temperatures, a phenotype that has been previously shown to play an important role in the divergence of two main S. paradoxus lineages, is partially driven by relaxed selection with trade-offs. Second, with the work on two inter-species hybridization events, we document a back-cross between a hybrid taxa and its parental species, which highlights that hybridization is likely more common in the evolution of species than previously thought. Our work underlines the importance of ecological differentiation through relaxed selection, rather than adaptive divergence from the fixation of beneficial mutations. Additionally, our findings show that hybridization in nature likely plays an important role in creating new diversity through transgressive segregation, and that this can reiterate through crosses that include hybrid species. Studies on young species and hybrid complexes will enable to further understand the genetic bases of population differentiation and the consequences of inter-species hybridization and its recurrence in the origin of species.

Thesis (Ph. D.)--University of Oregon, 2003.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 94-104). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph.D.)-- University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1999.
Errata slip inserted on back end-paper. Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1999. Bibliography: leaves 106-125.

The theoretical part discusses the yeasts and their taxonomic classification using traditional methods and using modern methods. Detail the work is concerned with descriptions of modern molecular-biology methods. The practical part was analyzed DNA by PCR-fingerprinting (rep-PCR) type of yeasts, which we received from the CCY and subsequent analysis of yeast samples obtained from grape musts. One of the grape must was obtained in 2009 (white grape variety) and the second in 2010 (red grape variety). Both grape musts come as integrated vineyards and organic. Grape musts samples were obtained from the winery Holánek from Ivaň. The cross-comparison of images PCR-fingerprint type yeasts and yeasts PCR-fingerprint samples using BioNumerics was to evaluate the results and conclude that the diversity of yeast flora in grape must.

Mestrado em Biologia Molecular e Celular
The genetic code is defined as a series of biochemical reactions that establish the cellular rules that translate DNA into protein information. It was established more than 3.5 billion years ago and it is one of the most conserved features of life. Over the years, several alterations to the standard genetic code and codon ambiguities have been discovered in both prokaryotes and eukaryotes, suggesting that the genetic code is flexible. However, the molecular mechanisms of evolution of the standard genetic code and the cellular role(s) of codon ambiguity are not understood. In this thesis we have engineered codon ambiguity in the eukaryotic model Sacharomyces cerevisiae to clarify its cellular consequences. As expected, such ambiguity had a strong negative impact on growth rate, viability and protein aggregation, indicating that it affects fitness negatively. However, it also created important selective advantages in certain environmental conditions, suggesting that it has the capacity to increase adaptation potential under environmental variable conditions. The overall negative impact of genetic code ambiguity on protein aggregation and cell viability, suggest that codon ambiguity may have catastrophic consequences in multicellular organisms. In particular in tissues with low cell turnover rate, namely in the brain. This hypothesis is supported by the recent discovery of a mutation in the mouse alanyl-tRNA synthetase which creates ambiguity at alanine codons and results in rapid loss of Purking neurons, neurodegeneration and premature death. Therefore, genetic code ambiguity can have both, negative or positive outcomes, depending on cell type and environmental conditions.
O código genético pode ser definido como uma série de reacções bioquímicas que estabelecem as regras pelas quais as sequências nucleotídicas do material genético são traduzidas em proteínas. Apresenta um elevado grau de conservação e estima-se que tenha tido a sua origem há mais de 3.5 mil milhões de anos. Ao longo dos últimos anos foram identificadas várias alterações ao código genético em procariotas e eucariotas e foram identificados codões ambíguos, sugerindo que o código genético é flexível. Contudo, os mecanismos de evolução das alterações ao código genético são mal conhecidos e a função da ambiguidade de codões é totalmente desconhecida. Nesta tese criámos codões ambíguos no organismo modelo Saccharomyces cerevisiae e estudámos os fenótipos resultantes de tal ambiguidade. Os resultados mostram que, tal como seria expectável, a ambiguidade do código genético afecta negativamente o crescimento, viabilidade celular e induz a produção de agregados proteicos em S. cerevisiae. Contudo, tal ambiguidade também resultou em variabilidade fenótipica, sendo alguns dos fenótipos vantajosos em determinados condições ambientais. Ou seja, os nossos dados mostram que a ambiguidade do código genético afecta negativamente a capacidade competitiva de S. cerevisiae em meio rico em nutrientes, mas aumenta a sua capacidade adaptativa em condições ambientais variáveis. Os efeitos negativos da ambiguidade do código genético, nomeadamente a agregação de proteínas, sugerem que tal ambiguidade poderá ser catastrófica em organismos multicelulares em que a taxa de renovação celular é baixa. Esta hipótese é suportada pela recente descoberta de uma mutação na alaniltRNA sintetase do ratinho que induz ambiguidade em codões de alanina e resulta numa forte perda de neurónios de Purkinge, neurodegeneração e morte prematura. Ou seja, a ambiguidade do código genético pode ter consequências negativas ou positivas dependendo do tipo de células e das condições ambientais.

This thesis examines genome-wide polymorphisms amongst 20 strains of Saccharomyces paradoxus, a yeast strain which has recently emerged as a model organism for population genetic studies. Three major studies are included in this thesis. The first study attempts to quantify the life cycle of yeast undergoing different modes of reproduction in nature. Measures of mutational and recombinational diversity are used to make two independent estimates of the population size. In an obligatory sexual population these estimates should be approximately equal. Instead, there is a discrepancy of about three orders of magnitude, indicating that S. paradoxus goes through one sexual cycle once in every ~1,000 asexual generations. This study illustrates the utility of population genomic data in quantifying the life cycles of organisms undergoing different modes of reproduction. Second, a map showing the distribution of rates of population recombination along chromosome III of S. paradoxus is presented. Several regions of very high recombination (hotspots) are identified in chromosome III. Comparison of hotspot regions between S. paradoxus and S. cerevisiae shows evidence of conservation of recombination hotspot regions. I argue that these observations reflect the weak impact of recombination due to the reduced frequency of sex of yeasts in nature. Recombination rates correlate with GC content, consistent with various studies in yeasts and humans, but there is no correlation with diversity or divergence. In addition, regions of extremely high recombination (hotspots) show an increased rate of GC→AT than rest of the chromosome. The reason for this is not clear at present ii Finally, a catalogue of polymorphisms within each population, and divergences between the two populations of S. paradoxus is presented. Tests of selection on the chromosomal sequence of S. paradoxus suggest a predominant mode of purifying selection. At least a third of mutations in synonymous sites and ~90% of mutations in replacement sites are removed by purifying selection. We estimate that around 12-31% of replacement mutations are deleterious in S. paradoxus, and that the average selection strength acting on these mutations is 1%. I also present a summary of data and subsequent analyses from the Saccharomyces Genome Resequencing Project (SGRP). Population genetic measures are applied to data using different basecalling quality cutoffs. From the results I recommend that at least a quality score of 40 is necessary to achieve the confidence required in data to be used in population genomic analyses.

mRNA decay is an important step in the control of gene expression. To study mRNA degradation I have exploited the genetic, biochemical, and molecular tools available in Saccharomyces cerevisiae. These studies provided insight into the signals within individual transcripts which specify their half-lives, the various mechanisms by which mRNAs are degraded, and the trans-acting factors which both perform and control nucleolytic events. I identified a 65 nucleotide segment from the coding region of the unstable MATɑl mRNA which was capable of targeting both the MATɑl and stable PGKI transcripts for rapid degradation. This "instability element" was divided into two parts, one located in the first 33, and the second in the latter 32, nucleotides. The first part could be functionally replaced by different mRNA sequences containing rare codons, and while unable to promote mRNA decay by itself, enhanced degradation mediated by the second part. I determined that the MATɑl Instability Element (MIE) targets mRNAs for rapid degradation by increasing the rates of two nucleolytic steps in a pathway of mRNA decay common to several stable and unstable yeast transcripts. The initial step in this pathway is shortening of the poly(A) tail of an mRNA. Subsequently, mRNAs are decapped, after which the transcript body is degraded in a 5' to 3' exonucleolytic manner. The MIE promotes decay of the MATɑl mRNA through an increase in its decapping rate. In contrast, PGKI mRNA decay was stimulated through an increase in its rate of deadenylation. The observation that the poly(A) tail must be removed prior to mRNA decapping suggests that the poly(A) tail inhibits decapping. I determined that the major poly(A)binding protein (Pablp) is required for the inhibition of decapping mediated by the poly(A) tail. Pablp is also required for normal deadenylation rates. Pablp therefore affects mRNA decapping and deadenylation, the two rate determining steps in a common pathway of mRNA decay. Determining how Pablp, and additional trans-acting factors, exert influence over both decapping and deadenylation will provide a greater understanding of the basis of differential rates of mRNA degradation.

The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5' to 3' exonucleolytic decay of the transcript body. The products of the DCP1 and DCP2 genes are required for mRNA decapping. DCP1 encodes a conserved mRNA decapping enzyme. Dcp2p is a highly conserved protein that is required for the activation of Dcp1p. The Dcp2p contains a functional Muff motif that is required for its decapping function, suggesting that Dcp2p encodes a pyrophosphatase. These results suggest that Dcp2p hydrolyzes a specific pyrophosphate bond that either directly activates Dcp1p or removes a specific inhibitor of Dcp1p. In addition to Dcp2p, several additional proteins were identified that influence mRNA decapping. Edc1p and Edc2p are related proteins whose overexpression suppressed conditional mutations in dcp1 and dcp2, respectively. The Edc1 protein interacts in vivo with Dcp1p and Dcp2p. Based on similar genetic data for EDC1 and EDC2, the Edc2p also likely interacts directly with the mRNA decapping machinery. Edc1p and Edc2p may function to activate transitions in the decapping complex that lead to the Dcp2p-dependent activation of Dcp1p. The SBP1 protein was identified as an overexpression suppressor of a conditional dcp2 allele, termed dcp2-7. SBP1 overexpression also suppressed a conditional allele of the decapping enzyme (dcp1-2). In addition, the sbp1Delta was found to partially suppress the decapping defect of the dcp2-7 allele. This suggests that SBP1, which is a highly conserved RNA binding protein related to nucleolin, may influence the assembly or organization of the mRNP. Lastly, loss of function mutations in the previously uncharacterized IDC1 gene were shown to stimulate decapping in the presence of the dcp2-7 mutation. This suggests that the wild-type Idc1p inhibits mRNA decapping. Interestingly, the idc1 mutations described here represent the only known loss of function mRNA decapping suppressors that are not known to influence the rate of translation initiation, suggesting a more direct role for Idc1p in the inhibition of Dcp2p function. Combined, these results indicate that mRNA decapping is a highly controlled process involving the intricate and coordinated function of multiple proteins, in addition to the Dcp1p decapping enzyme.

Baker's yeast, Saccharomyces cerevisiae, is a well-studied model system in genetics and molecular biology. It is also a promising system for experimental ecology, evolution, and epidemiology, and is very important in the fermentation industry. The large amount of information generated by studies using this organism cannot be fully exploited until sufficient ecological data is gathered. Only when the natural environment of S. cerevisiae is well characterized can research using this yeast as a model system be put into context. The lack of information about the natural environment of S. cerevisiae is what prompted this work. First, I review the current available data on the ecology and evolution of S. cerevisiae and its sister species (the sensu stricto species complex). I then report results from fieldwork in an old growth forest. Finally, I report a community ecology experiment carried out using three naturally coexisting yeasts from this forest.

L’objectif principal de cette étude était d’identifier les déterminants génétiques causant une différenciation phénotypique entre les levures Saccharomyces cerevisiae et Saccharomyces paradoxus. Ces espèces peuvent s’hybrider malgré des différences écologiques importantes, dont la capacité de croître à haute température. S. cerevisiae et l’hybride sont capables de croître à 37 °C tandis que S. paradoxus ne peut pas. L’approche qui a été utilisée est un test de complémentation. D’abord, les chromosomes de S. cerevisiae ont été enlevés un à un du génome de l’hybride, puis les gènes afin d’augmenter la résolution de l’approche. Ainsi, les chromosomes et les gènes de S. paradoxus qui ne peuvent pas complémenter ceux de S. cerevisiae, empêchant l’hybride de croître à 37 °C, ont pu être identifiés. Les résultats indiquent que la différence entre les taux de croissance de S. cerevisiae et S. paradoxus est causée par de multiples locus ayant des effets mineurs sur le phénotype.

Les levures non-Saccharomyces, naturellement présentes dans les moûts, peuvent impacter positivement ou négativement la qualité des vins. Depuis quelques années, l’utilisation de cultures mixtes comme starters, associant une souche de Saccharomyces cerevisiae et une souche d’une autre espèce est proposée aux œnologues. C’est le cas du couple S. cerevisiae/Torulaspora delbrueckii. L’étude des interactions entre la souche T. delbrueckii Zymaflore Alpha et S. cerevisiae Zymaflore X5, de la société Laffort, a été réalisée. Les fermentations alcooliques ont été effectuées dans un réacteur à double compartiment permettant la séparation physique des levures tout en conservant l’homogénéité du milieu de culture. Les résultats ont mis en évidence que la séparation impacte la croissance des deux souches suggérant l’existence d’interactions de type cell-cell contact entre ces deux souches. Si une grande majorité de praticiens utilise désormais les levures sélectionnées, certains ont fait le choix de favoriser les populations autochtones de levures S .cerevisiae et de levures non-Saccharomyces. L’incidence de deux facteurs de l’environnement a été étudié sur un mélange de cinq espèces de non-Saccharomyces (T. delbrueckii, Metschnikowia spp., Candida zemplinina, Hanseniaspora uvarum, Pichia kluyveri) et de deux souches de S. cerevisiae (une à phase de latence courte, une à phase de latence longue) en cultures pures et en mélange. L’inoculation de la souche de S. cerevisiae à phase de latence longue dans un moût saturé en CO2 permet de stimuler les levures non-Saccharomyces d’intérêt (T. delbrueckii/P. kluyveri) tout en inhibant les espèces indésirables (H. uvarum, C. zemplinina)
Non-Saccharomyces yeasts, naturally found in grape must, can impact wine quality positively or negatively. In recent years, the use of mixed cultures as starters (association of S. cerevisiae species and other species) such as the couple Saccharomyces cerevisiae/Torulaspora delbrueckii is proposed to winemakers. Interactions between these two species have been studied with two commercial strains, T. delbrueckii Zymaflore Alpha and S. cerevisiae Zymaflore X5 (Laffort). Alcoholic fermentations were carried out in a fermentor with double compartment allowing a physical separation of yeasts and preserving the homogeneity culture medium. The results highlighted that the physical separation impacts the growth of both strains, suggesting interactions of type cell-cell contact between these two strains. If a large majority of winemakers use selected yeasts strains, some of them chose to favor native yeasts, S. cerevisiae species and non- Saccharomyces species. The impact of two environmental factors was investigated on five non-Saccharomyces species (T. delbrueckii, Metschnikowia spp., Candida zemplinina, Hanseniaspora uvarum, Pichia kluyveri) and two strains of S. cerevisiae (one with short fermentation lag phase, one with long fermentation lag phase), in pure and mixed cultures. The inoculation with S. cerevisiae with a long fermentation lag phase in a must saturated with CO2 allowed to stimulate some of non-Saccharomyces which present an interest in winemaking (T. delbrueckii/P. kluyveri) and inhibit the undesirable ones (H. uvarum, C. zemplinina)

Dissertation (PhD)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Cells of the yeast Saccharomyces cerevisiae are able to change their morphological appearance in response to a variety of extracellular and intracellular signals. The processes involved in morphogenesis are well characterised in this organism, but the exact mechanism by which information emanating from the environment is integrated into the regulation of the actin cytoskeleton and the yeast cell cycle, is still not clearly understood. Considerable progress has, however, been made. The processes are investigated on various levels including: (i) the nature of the signals required to elicit a morphological adaptation, (ii) the mechanism by which these signals are perceived and transmitted to the nucleus for gene transcription regulation (signal transduction pathways), (iii) the role of the cytoskeleton, particularly actin, in morphogenesis, and (iv) the relationship between cell cycle regulators and factors required for alterations in cellular shape. The focus of this study was on elements involved in the regulation of one of these morphological processes, pseudohyphal formation, in S. cerevisiae. During pseudohyphal differentiation normal oval yeast cells become elongated and mother and daughter cells stay attached after cytokinesis to give rise to filaments. These filaments are able to penetrate the growth substrate, a phenomenon referred to as invasive growth. Actin remodelling is a prerequisite for the formation of elongated cells during pseudohyphal development and invasive growth. Its main contribution to this event is the directing of vesicles, containing cell wall constituents and enzymes, to specific sites of cell wall growth at the cell periphery. In order to fulfil this cellular function, actin is regulated on several levels. Signal transduction pathways that are activated in response to external nutritional signals play important roles in the regulation of the actin cytoskeleton during pseudohyphal differentiation. For this reason a literature review was compiled to introduce various aspects of actin-structure, the regulation of this structure and the functions actin performs during morphogenesis. The connection between signal transduction elements involved in morphological processes and actin remodelling is also reviewed. This study entailed the genetic analysis of numerous factors involved in the regulation of pseudohyphal differentiation, invasive growth and starch metabolism. Several transcriptional regulators playing a role in these phenomena were investigated. Apart from the transcription factors, which include Mss11p, Msn1p, Ste12p, F108p,Phd1p and Tec1p, additional elements ranging from transporters to G-proteins, were also investigated. Mutant strains deleted for one or more of these factors were constructed and tested to assess their abilities to form filaments that penetrate the growth substrate, and to utilise starch as a carbon source. Complex genetic relationships were observed for various combinations of these factors. Specifically, F108p,Msn1p and Ste12p were shown to act independently in controlling invasive growth and starch metabolism, suggesting that these factors are regulated by different signal transduction pathways. Mss11p, on the other hand, was found to play an indispensable role and seems to act as a downstream factor of Msn1 p, Fl08p, Ste12p and Tec1 p. The exception to this is Phd1 p, since multiple copies of PHD1 partially suppress the effect of a MSS11 deletion. The data suggests that Mss11 p functions at the confluence of several signalling pathways controlling the transcriptional regulation of genes required for invasive growth and starch degradation. Different nutritional signals were also found to differentially regulate specific signalling elements during the invasive growth response. For example, Tec1 p requires Msn1 p activity in response to growth on media containing a limited nitrogen source. This dependency, however, was absent when invasive growth was tested on glucose and starch media. Evidence was also obtained that confirmed the transcriptional co-regulation of MUC1 and STA2. MUC1 encodes a mucin-like protein that is required for invasive growth and pseudohyphal differentiation, whereas STA2 encodes a glucoamylase required for starch degradation. Unpublished results indicated that several transcriptional regulators of invasive growth also exert an effect on starch metabolism. The data generated during this study complemented and confirmed published results. It also contributed to the compilation of a more detailed model, integrating the numerous factors involved in these signalling processes.
AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae gisselle beskik oor die vermoë om hul morfologiese voorkoms in responstot 'n verskeidenheid van ekstrasellulêre en intrasellulêre seine te verander. Die prosesse betrokke by morfogenese is goed gekarakteriseerd in hierdie organisme, maar die presiese meganisme waardeur inligting vanuit die omgewing geïntegreer word in die reguleringvan die aktien-sitoskelet en die gisselsiklus, word nog nie ten volle verstaan nie. Aansienlike vordering in die verband is egter gemaak. Die prosesse word op verskeie vlakke ondersoek, insluitende: (i) die aard van die seine wat benodig word om 'n morfologiese aanpassing te inisïeer; (ii) die meganisme waardeur hierdie seine waargeneem en herlei word na die selkern vir die regulering van geen-transkripsie (seintransduksie paaie); (iii) die rol van die sitoskelet, spesifiek aktien, in morfogenese en (iv) die verhouding tussen selsiklusreguleerders en faktore wat benodig word vir verandering in selvorm. Hierdie navorsing fokus op elemente betrokke by die regulering van een van hierdie morfologiese prosesse in S. cerevisiae, naamlik pseudohife-vorming. Gedurende pseudohife-differensiëring neem tipiese ovaalvormige selle 'n verlengde voorkoms aan wat tot die vorming van filamente lei. Hierdie filamente is in staat om die groeisubstraat te penetreer, 'n verskynsel bekend as penetrasie-groei. Aktienherrangskikking is 'n voorvereiste vir die vorming van verlengde selle tydens pseudohife-ontwikkeling. Die hoofbydrae van aktien tot hierdie verskynsel is die oriëntering van uitskeidingsvesikels, wat selwandkomponente en ensieme bevat, na spesifieke areas van selwandgroei op die seloppervlak. Aktien word op verskeie vlakke gereguleer om hierdie sellulêre funksie te vervul. Seintransduksiepaaie wat geaktiveer word in respons tot ekstrasellulêre voedingsseine speel 'n belangrike rol in die regulering van die aktien-sitoskelet tydens pseudohife-differensiëring. Op grond hiervan is 'n literatuuroorsig saamgestel vir die bekendstelling van verskeie aspekte van aktienstruktuur, die regulering van hierdie strukture en die funksies wat deur aktien gedurende morfogenese vervul word. Die verband tussen seintransduksie-elemente betrokke by morfologiese prosesse en aktien herrangskikkingword ook behandel. Hierdie studie het die genetiese analisering van verskeie faktore betrokke by pseudohife-differensiëring, penetrasie-groei en styselmetabolisme, behels. Verskeie transkripsionele reguleerders wat In rol speel in hierdie prosesse was bestudeer. Buiten die transkripsiefaktore Mss11p, Msn1p, Ste12p, F108p,Phd1P en Tec1p, was addisionele faktore, wat gewissel het van transporters tot G-proteïene, ook ondersoek. Mutante-rasse met geendelesies vir een of meer van hierdie faktore is gekonstrueer en getoets om vas te stel hoe dit hul vermoë raak om penetrerende filamente te vorm, asook om te bepaal of stysel as koolstofbron gebruik kan word. Komplekse genetiese interaksies vir verskeie kombinasies van hierdie faktore is waargeneem. Dit was waargeneem dat F108p,Msn1p en Ste12p onafhanklik funksioneer tydens die regulering van penetrasie-groei en styselmetabolisme, wat impliseer dat hierdie faktore deur verskillende seintransduksiepaaie gereguleer word. Mss11 p word beskou as In onmisbare rolspeler in hierdie prosesse en dit kom voor asof hierdie protein as 'n stroom-af faktor is en vereis word vir die funksionering van Msn1p, F108p, Ste12p en Tec1p. Phd1p is egter 'n uitsondering, aangesien veelvuldige kopieë van PHD1 die effek van 'n MSS11-delesie gedeeltelik oorkom. Die data impliseer dat Mss11 p by die samevloei van verskeie seintransduksiepaaie, benodig vir die transkripsionele regulering van gene betrokke by penetrasie-groei en styselmetabolisme, funksioneer. Dit was ook waargeneem dat verskillende voedingsseine die faktore betrokke by die penetrasie-groeirespons differensieel reguleer. Tec1 p byvoorbeeld benodig Msn1paktiwitieit in respons tot groei op media met 'n beperkte stikstofbron. Hierdie afhanklike interaksie is egter afwesig wanneer penetrasie-groei bestudeer word op glukose- en styselmedia. Resultate wat die gesamentlike transkripsionele regulering van MUC1 en STA2 bevestig, is ook verkry. MUC1 kodeer vir 'n mukienagtige proteïen wat benodig word vir pseudohife-vorming en penetrasie-groei, terwyl STA2 kodeer vir 'n glukoamilase essensieël vir styselafbraak. Ongepubliseerde resultate dui daarop dat verskeie transkripsionele reguleerders van penetrasie-groei ook In effek uitoefen op styselmetabolisme. Die data wat gegenereer is tydens hierdie studie komplementeer en bevestig reeds gepubliseerde resultate. Dit het ook bygedra tot die samestelling van 'n gedetaileerde model wat die verskillende faktore, betrokke by hierdie seintransduksieprosesse, integreer.

Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 117 pages. Includes Vita. Includes bibliographical references.

Thesis (PhD)--University of Stellenbosch, 2002
ENGLISH ABSTRACT: Upon nutrient limitation, normal cells of the budding yeast, Saccharomyces cerevisiae, undergo a transition from ovoid cells that bud in an axial (haploid) or bipolar (diploid) fashion to elongated cells that bud in a unipolar fashion. The daughter cells stay attached to the mother cells, resulting in chains of cells referred to as pseudohyphae. These filaments can grow invasively into the growth substrate (haploid), or away from the colony (diploid), and are hypothesised to be an adaptation of yeast cells that enables them to search for nutrientrich substrates. This filamentous growth response to nutrient limitation was shown to be dependent on the expression of, amongst others, the MUC1 gene. MUC1 (also known as FL011) encodes a large, cell wall-associated, GPI-anchored threonine/serine-rich protein that bears structural resemblance to mammalian mucins and to the yeast flocculins. Deletion and overexpression studies demonstrated that it is critical for pseudohyphal differentiation and invasive growth, and that overexpression of the gene also results in strongly flocculating yeast strains. The upstream regulatory region of MUC1 comprises the largest yeast promoter identified to date and areas as far as 2.4 kb upstream of the translational start site have been shown to confer regulation on MUC1 expression. The large promoter region is not unique to MUC1, however, since it is almost identical to that of the functionally unrelated STA2 gene. The STA2 gene, as well as the identical STA1 and STA3 genes, encodes extracellular glucoamylase isozymes that enable the yeast cell to utilise starch as a carbon source. Glucoamylases liberate glucose residues from the non-reducing end of the starch molecule, thereby making it accessible to yeast cells. The high identity between the promoters of MUC1 and STA1-3 suggests that the two genes are co-regulated. In addition, several transcription factors that regulate the transcriptional levels of both MUC1 and STA2 have been identified and include Msn1p and the previously uncharacterised Mss11p. Overexpression of either Msn1p or Mss11p results in elevated levels of MUC1 and STA2 transcription and a dramatic increase in flocculation, invasive growth, pseudohyphal differentiation and the ability to utilise starch, suggesting that the two genes are indeed co-regulated. The main objective of this study was to characterise Mss11p and its role in the co-regulation of MUC1 and STA2 (as a representative member of the STA gene family). A detailed expression analysis, using Northern blots and Lacl reporter gene expression studies in different media, confirmed that these genes are indeed co-regulated to a large extent. MUC1 and STA2 are also regulated by the same transcriptional regulators, which include not only Msn1pand Mss11p, but also Ste12p, the transcription factor of the mating pheromone/filamentous growth signalling cascade, and Flo8p, a transcriptional activator of the flocculation genes. Overexpression of the genes encoding these factors results in elevated expression levels of both MUC1 and STA2 in most nutritional conditions and enhances the filamentous growth phenotypes of the strain, as well as the ability to degrade starch. On the other hand, the deletion thereof results in severe reductions in the transcription levels of MUC1 and STA2, with equally severe reductions in filamentous growth and the ability to hydrolyse starch. These expression studies also showed that the repressive effect of STA10, a previously uncharacterised negative regulator of STA2, is actually a phenotype conferred by a FLOB mutation in some laboratory strains of S. cerevisiae. The upstream regulatory regions of MUC1 and STA2 are the largest promoters in the yeast genome. By sequencing the upstream areas of STA2 and STA3 and comparing them to the sequence of MUC 1, it was shown that these upstream areas are 99.7%identical over more than 3 900 base pairs (bp) upstream of the translational start. With the exception of a few minor substitutions, the only significant difference between the MUC1 and STA2 promoters is the presence of a 20-bp and a 64-bp sequence found in the MUC1 promoter, but not in the promoters of any of the STA1-3 genes. Through a promoter-deletion analysis, it was shown that Mss11p, Msn1pand Flo8p exert their control over the transcription of MUC1 and STA2 from an 90-bp sequence located at -1 160 to -1 070 in the STA2 and -1 210 to -1 130 in the MUC1 promoters. This sequence also mediates the effect of carbon catabolite repression on the transcription of STA2 and MUC1. Despite the similarities in the expression patterns of MUC1 and STA2, some discrepancies also exist. The most significant difference is that, in wild-type cells and under all nutritional conditions tested, MUC1 transcription is reduced significantly if compared to the transcription levels of STA2. This was attributed to the presence of the 20- and 64-bp sequences, that are present in the promoter region of MUC1, but absent from that of STA2. To place the transcriptional regulators of MUC1 and STA2 in the context of known signal transduction pathways, an epistasis analysis was conducted between MSN1, MSS11 and components of the mating pheromone/filamentous response MAPkinase cascade and cAMPPKA pathway that were shown to be required for the filamentous growth response. This analysis revealed that Msn1p functions in a third, as yet uncharacterised, signal transduction pathway, also downstream of Ras2p,but independent of the two identified pathways, i.e. the cAMP-PKA and pheromone response/filamentous growth response MAP kinase pathways. However, Mss11p seems to function downstream of all three the identified pathways. This suggestsa critical and central role for Mss11p in determining the transcription levels of MUC1 and STA2. To further characterise Mss11p and its role in the transcriptional regulation of MUC1 and STA2, it was also subjected to a detailed deletion and mutation analysis. Mss11p was shown to harbour two distinct activation domains required for the activation of MUC1 and STA2, but also able to activate a reporter gene expressed from under the GALl promoter. The more prominent of the activation domains of Mss11p was shown to be one of the domains with homology to Flo8p, designated H2. The H2 domain has significant homology to a number of proteins of unknown function from a range of different organisms. A multi-sequence alignment allowed the identification of conserved amino acids in this domain. Mutations in two of the four conserved amino acid pairs in the H2 domain completely eliminated the activation function of Mss11p. The poly-glutamine and poly-asparagine domains of Mss11p are not required for its activation function. The deletion of these domains has no impact on the ability of Mss11p to activate MUC1 or STA2 or of the Gal4p-Mss11p fusion to activate the lacl reporter gene expressed from under the GAL7 promoter. Gal4p fusions of either of these domains were also unable to trans-activate the PGAL7-lacl reporter gene. As such, it was concluded that neither of these domains performs a function in the role of Mss11p as a transcriptional activator. We also demonstrated that the putative ATP/GTP-binding domain (P-loop) is not required for the transcriptional activation function of Mss11p. In an attempt to identify other target genes of Mss11p, the use of micro-arrays was employed to assessthe impact of the overexpression and deletion of MSS11 on the total yeast transcriptome. These results showed that MUC1 and STA2 are the only two genes in the ISP15 genetic background that are significantly (more than 15-fold) enhanced by the overexpression of MSS11. Mss11p therefore seemsto playa very specific or dedicated role in MUC1 and STA2 transcription. This analysis also identified several genes (DBP2, ROM2, YPLOBOC, YGR053C, YNL179C, YGR066C) that are repressed by overexpression of MSS11 and activated when MSS11 is deleted. To integrate all the results, three possible models for the activation of MUC1 and STA2 transcription by Mss11p are proposed: (i) Mss11p performs the role of a transcriptional mediator, possibly in a protein complex, to convey information from upstream regulatory elements to the transcription machinery assembledat the core promoters of MUC1 and STA2; (ii) Mss11p plays a more direct role in transcriptional activation, possibly as a transcription factor itself; and (iii) Mss11p facilitates transcription of the MUC1 and STA2 promoters as part of a larger complex that removes or releases the chromatin barrier over the MUC1 and STA2 promoters in responseto specific nutritional signals.
AFRIKAANSE OPSOMMING: Wanneer voedingstowwe beperkend raak, ondergaan selle van die botselvormende gis, Saccharomyces cerevisiae, fn transformasie vanaf ronde selle, wat in fn aksiale (haploïede) of bipolêre (diploïede) patroon bot, tot verlengde selle, wat slegs op een punt bot. Die dogterselle blyaan die moederselle geheg, sodat kettings van selle, wat as pseudohifes bekend staan, gevorm word. Hierdie filamente kan fn groeisubstraat binnedring (haploïede) of vanaf die kolonie weggroei (diptoïede), en is moontlik fn aanpassing van die gisselle wat hulle in staat stelom na meer voedingstofryke substrate te groei. Die vermoë om filamente in respons tot voedingstoftekorte te vorm, is onderhewig aan die uitdrukking van, onder meer, die MUC1-geen. MUC1 (ook bekend as FL011) kodeer vir fn selwand-geassosieerde treonien/serien-ryke proteten met fn GPI-anker wat strukturele verwantskappe met die mukiene van soogdiere en die flokkuliene van giste toon. Delesie- en ooruitdrukkingstudies het bewys dat dit krities is vir die ontwikkeling van pseudohifes en penetrerende groei, terwyl die ooruitdrukking daarvan ook tot sterk flokkulerende gisrasse lei. Die stroom-op regulatoriese area van MUC1 vorm die grootste promotor wat tot dusver in gis geïdentifiseer is, en daar is bewys dat areas so ver as 2.4 kb stroom-op van die translasie-inisiëringsetel die regulering van MUC1 beïnvloed. Hierdie groot promotor is egter nie uniek tot MUC1 nie, aangesien fn amper identiese promotor die regulering van die funksioneelonverwante STA2-geen beheer. Die STA2-geen, asook die identiese STA1- en STA3-gene, kodeer vir ekstrasellulêre glukoamilase isosieme wat die gis in staat stelom stysel as koolstofbron te benut. Dit bevry glukosemolekules vanaf die nie-reduserende punt van die styselmolekuul en stel dit sodoende aan gisselle beskikbaar. Die hoë vlak van eendersheid tussen dié twee promotors veronderstel dat die twee gene op soortgelyke wyse gereguleer word. Verskeie transkripsiefaktore wat die transkripsievlakke van beide MUC1 en STA2 beheer, is ook geïdentifiseer, Dit sluit Msn1p en die tot dusver ongekarakteriseerde Mss11p in. Ooruitdrukking van Msn1p of Mss11p lei tot verhoogde vlakke van MUC1 en STA2 se transkripsie en fn dramatiese toename in flokkulasie, asook die vermoë om penetrerend te groei, pseudohifes te vorm en stysel te benut. Dit bevestig dat die twee gene wel tot fn groot mate op dieselfde wyse gereguleer word. Die hoofdoel van hierdie studie was om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 te karakteriseer. Gedetailleerde uitdrukkingsanalises met behulp van die Northern-kladtegniek en facZverklikkergeeneksperimente in verskillende media het bevestig dat die gene wel tot fn groot mate op dieselfde wyse gereguleer word. Transkripsie van MUC1 en STA2 word ook deur dieselfde transkripsionele reguleerders beheer, wat nie net Msn1pen Mss11p insluit nie, maar ook Ste12p, die transkripsiefaktor van die paringsferomoon/filamentagtige groei seintransduksiekaskade, en Fl08p, fn transkripsionele aktiveerder van die flokkulasiegene. Ooruitdrukking van die gene wat vir hierdie faktore kodeer, veroorsaak verhoogde uitdrukkingsvlakke van beide MUC1 en STA2 onder die meeste groeitoestande en verbeter die vermoë van die gisras om filamentagtig te groei en om stysel te benut. Andersyds veroorsaak delesies van die gene 'n dramatiese afname in die transkripsievlakke van MUC1 en STA2, met vergelykbare afnames in die vermoë van die gisras om filamentagtig te groei en om stysel te benut. Hierdie uitdrukkingstudies het ook bewys dat die onderdrukkingseffek van STA10, 'n tot dusver ongekarakteriseerde, negatiewe reguleerder van STA2, aan 'n mutasie in FLOB in sekere laboratoriumrasse van S. cerevisiae toegeskryf kan word. Die stroom-op regulatoriese areas van MUC1 en STA2 is die grootste promotors in die gis se genoom. Deur die nukleotiedvolgordes van die ver stroom-op areas van STA2 en STA3 te bepaal en hulle met dié van MUC1 te vergelyk, is daar vasgestel dat die stroom-op areas van die gene 99.7% identies is oor meer as 3 900 basispare (bp) stroom-op van die beginsetel van translasie. Met die uitsondering van enkele basispaarverskille, is die enigste noemenswaardige verskil tussen die promotors van MUC1 en STA2 die teenwoordigheid van 'n 20 bp- en 'n 64 hp-fragment wat in die MUC1-promotor aangetref word, maar nie in die promotors van die STA1-3 gene nie. Deur 'n promotordelesie-analise kon daar bewys word dat Mss11p, Msn1p en Flo8p beheer uitoefen oor die transkripsie van MUC1 en STA2 vanaf 'n 90-bp-fragment, wat by posisie -1 160 tot -1 070 in die STA2-promotor en posisie -1 210 tot -1 130 in die MUC1-promotor aangetref word. Koolstofkatabolietonderdrukking van MUC1 en STA2 se transkripsie geskied ook deur middel van hierdie fragment. Ten spyte van die ooreenkomste in die uitdrukkingspatrone van MUC1 en STA2, kom daar tog ook verskille voor. Die mees opvallende verskil is dat, in wilde-tipe selle en onder alle toestande tot dusver getoets, die transkripsievlakke van MUC1 aansienlik laer is as dié van STA2. Dit word toegeskryf aan die teenwoordigheid van die 20 bp- en 64 bp-fragmente, wat in die promotor van MUC1 teenwoordig is, maar in die promotor van STA2 afwesig is. Om die transkripsionele reguleerders van MUC1 en STA2 in die konteks van bekende seintransduksieweë te plaas, is 'n epistase-analise gedoen tussen MSN1, MSS11 en komponente van die paringsferomoon/filamentagtige groei MAP-kinasekaskade en die cAMPPKA- weg wat uitgewys het dat dit 'n rol in die filamentagtige groeirespons speel. Hierdie analise het onthul dat Msn1p in 'n derde, tot dusver onbeskryfde, seintransduksieweg funksioneer, wat ook stroom-af van Ras2p is, maar wat onafhanklik funksioneer van die twee bekende weë, die cAMP-PKA-weg en die paringsferomoon/filamentagtige groei MAPkinasekaskade. Mss11p blyk egter stroom-af van al drie dié weë te funksioneer. Dit wys dat Mss11p 'n kritiese en sentrale rol in die bepaling van MUC1 en STA2 se transkripsievlakke speel. Om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 se transkripsie verder te karakteriseer, is dit aan 'n volledige delesie- en mutasie-analise onderwerp. Dit het gewys dat Mss11p twee verskillende aktiveringsdomeine bevat wat vir die transkripsionele aktivering van STA2 en MUC1 benodig word, maar wat ook 'n verklikkergeen kon aktiveer wat onder die GAL7-promotor uitgedruk word. Die prominentste van die twee aktiveringsdomeine van Mss11p is een van die domeine wat homologie toon met 'n soortgelyke domein van Flo8p, die sogenaamde H2-domein. Die H2-domein toon hornologie met 'n verskeidenheid van organismesse proteïene, waarvan die funksie onbekend is. 'n Vergelyking van al die relevante aminosuurvolgordes uit dié proteïene het gehelp om 'n aantal gekonserveerde aminosure te identifiseer. Mutasies van twee van die vier gekonserveerde aminosuurpare het die vermoë van Mss11p om transkripsie te aktiveer, heeltemal geëlimineer. Die poliglutamien- en poliasparagiendomeine van Mss11p word nie vir die aktiveringsfunksie benodig nie. Die delesie van die domeine het geen impak gehad op die vermoë van Mss11p om die transkripsie van MUC1 en STA2 te aktiveer nie, of op die vermoë van die Gal4p-Mss11p fusie om die lacZ-verklikkergeen onder regulering van die GAL7-promotor te aktiveer nie. Gal4p-fusies met enige van die domeine was ook nie in staat om die PGAL7-lacZverklikkergeen te aktiveer nie. Daar kan dus afgelei word dat nie een van die twee domeine 'n funksie in die rol van Mss11p as transkripsionele aktiveerder het nie. Soortgelyke eksperimente het bewys dat die moontlike ATP/GTP-bindingsdomein (P-lus) nie vir die transkripsionele aktiveringsfunksie van Mss11p benodig word nie. In 'n poging om ander teikengene van Mss11p te identifiseer, is mikro-ekspressieroosters gebruik om die impak van die ooruitdrukking en delesie van MSS11 op die totale transkriptoom van die gis te bepaal. Dié resultate het gewys dat MUC1 en STA2 die enigste twee gene in die ISP15genetiese agtergrond is waarvan transkripsie noemenswaardig (meer as 15-voudig) deur die ooruitdrukking van MSS11 verhoog word. Dit wil dus voorkom asof Mss11p 'n baie spesifieke rol in die transkripsie van MUC1 en STA2 speel. Hierdie analise het ook verskeie gene (DBP2, ROM2, YPLOBOC,YGR053C, YNL179C, YGR066C) geïdentifiseer wat deur die ooruitdrukking van MSS11 onderdruk word en deur die delesie van MSS11 geaktiveer word. Ten einde al die resultate te integreer, word drie moontlike modelle vir die aktivering van MUC1- en STA2-transkripsie deur Mss11p voorgestel: (i) Mss11p vervul die rol van 'n transkripsionele tussenganger, moontlik as deel van 'n proteïenkompleks, om die inligting van die stroom-op regulatoriese elemente aan die transkripsiemasjinerie wat oor die kernpromotor van MUC1 en STA2 gebind is, oor te dra; (ii) Mss11p speel 'n meer direkte rol in transkripsionele aktivering, moontlik as 'n transkripsiefaktor self; en (iii) Mss11p maak die transkripsie van MUC1 en STA2 moontlik as deel van 'n groter kompleks wat die chromatienblokkade oor die promotors van STA2 en MUC1 in respons tot spesifieke seine verslap of verwyder.