Dissertations / Theses on the topic 'Yeast propagation'

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2008.
Committee Chair: Chernoff,Yury; Committee Member: Cairney,John; Committee Member: Choi,Jung; Committee Member: Doyle,Donald; Committee Member: Lobachev,Kirill. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Analysis of how prions are propagated and transmitted in the yeast Saccharomyces cerevisiae has begun to reveal how an amyloid-forming protein can act as an epigenetic determinant of cell phenotype. Through the ability of prions to self propagate, genetic traits encoded by prions are inherently dominant, yet the underlying mechanism is only just beginning to emerge. One approach to elucidating the mechanism of prion propagation is to establish why certain mutations can impact negatively on inheritance of a prion-based trait. This thesis reports on a combined in vivo and structural analysis of one such class of mutant - PNM2-1 - a dominant negative mutation that inhibits propagation of [PS/+], the prion form of the translation termination factor Sup35p. The original PNM2-1 allele, a G58D mutation lies in one of a series of five oligopeptide repeats in the amino-terminal prion domain of the Sup35p (eRF3) and cells expressing this allele cannot efficiently propagate the [PS/+] prion. To establish the mechanism by which the PNM2-1 allele mediates this effect, a series of PNM2-1G58/G59/Y60 mutants of Sup35p was constructed. By combining genetic crossing, phenotypic analysis and solution NMR structural studies, a clear correlation between the conformational changes in the oligopeptide repeat caused by these mutations and the relative impact of these mutations on the in vivo propagation of [PS/+] was demonstrated. The conformational constraints associated with these mutations were also shown to the affect the ability of the protein to form and/or incorporate different of prion variants. These findings provide a molecular explanation of the dominant negative effects of PNM2-1 mutation on the maintenance of the [PS/+] prion and provide important new insights into the importance of conformation in prion propagation.

Includes bibliographical references.
The traditional batch brewing process is characterised by serial yeast propagation to build sufficient yeast for pitching. This results in cyclic variations in yeast environment, leading to a slow brewing process. In high gravity brewing the carbohydrate utilisation is inefficient as a result of the Crabtree effect that occurs in the presence of high sugar concentration. When optimising the brewing process the characteristics of conventional batch brewing should be maintained. Fed-batch propagation of yeast is used to improve carbohydrate utilisation and the yeast biomass formation by controlling nutrient supply.

[PSI+] is the prion form of the Sup35 protein in Saccharomyces cerevisiae. The molecular Hsp 104 chaperone is involved in the propagation of the [PSI+] by dissolving Sup35p prion fibres into the small seeds (propagons) needed to generate a new round propagation. Overexpression of the HSP104 gene results in elimination of the [PSI+] prion, but the mechanism by which [PSI+] loss is triggered remains undetermined. To gain insight into the mechanism of such induced [PSr] elimination, the cellular factors with a known functional connection with Hsp104 and that may necessary for Hsp 104 overexpression-induced prion loss, were investigated. The Hsp90 cochaperones Cpr7p and Stilp that also bind to the C-terminus of Hspl04 are required for [PSI+] elimination by overexpression of both wild type Hsp104 and two different ATPase-defective mutants of Hspl04 but are not essential for [PSI+] prion propagation. This indicated that [PSr] elimination by elevated Hsp104 may occur as a consequence of activities of Hsp104 in addition to its remodelling activity.

Within the dissertation thesis “Study of Biological Material Attributes by Using Image Analysis Methods”, attention is focused on monitoring of the application of image analysis methods, mostly a fractal analysis, in studying the properties of various yeast species. The thesis includes determining the number of yeast cells and vegetative propagation of yeast using fractal parameters – fractal measure D and fractal dimension K. Attention is also paid not only to the application of the existing image analysis methods, but also to their renovation. The obtained images were evaluated using the box counting method specified by implementation of wavelet transformation. To monitor yeast cells for a longer time, it was first necessary to prepare a suitable microscopic preparation. To distinguish live and dead cells, the following fluorescent dyes were used: acridine orange, fluorescein diacetate, FUN-1, and Calcofluor White M2R. The images of yeast cells were recorded using a still camera or a CCD camera and microscope. Clips of the same size were obtained from the acquired digital photographs and processed by the HarFA program developed at the Faculty of Chemistry, Brno University of Technology. On the results it is possible to see a change in the fractal dimension depending on time, i.e. on the change of a budding cell structure, or to determine the number and radius of yeast cells upon predefined conditions.

Une protéine prion peut adopter deux conformations distinctes, l’une cellulaire et l’autre prion. La conformation prion est le résultat de son agrégation en fibre amyloïde. Cette fibre est le support de l’information prion à partir duquel les isoformes cellulaires sont convertis en forme prion de façon autocatalytique. La transmission de l’information prion repose donc sur la transmission de cette fibre au cours des divisions cellulaires, qui est réalisée par de petits polymères. Ceux-ci sont le résultat d’un équilibre entre la fragmentation et la polymérisation de la fibre. Une perturbation de cet équilibre provoque une agrégation massive de la protéine prion, menant à la perte de l’information prion.L’objectif de ma thèse était de comprendre ce qui définit in vivo la transmission du prion. Mon modèle d’étude est la protéine Ure2p propageant le prion [URE3] dans la levure S. cerevisiae. J’ai montré que la concentration cellulaire d’Ure2p détermine la vitesse d’agrégation de la protéine prion et donc son efficacité de transmission. En effet, de trop fortes concentrations cellulaires sont incompatibles avec la propagation du prion. La concentration cellulaire d’Ure2p définit également la diversité des souches prions. Un crible génétique m’a permit de mettre en évidence que la présence de séquences centromériques surnuméraires dans la cellule interfère avec la transmission du prion [URE3]. Le même phénomène est observé avec une augmentation du niveau de ploïdie de la cellule. Dans les deux cas, la surexpression du chaperon Hsp104 restaure une propagation normale du prion
A prion protein can adopt two distinct conformations, one cellular and one prion. Prion conformation is the result of its aggregation into amyloid fibers. This fiber is the support of the prion information from which the cellular isoforms are converted into prion form by autocatalytic manner. The prion information transmission is therefore based on the transmission of this fiber during cell division, which is done by small polymers. These are the result of a balance between fragmentation and polymerization of the fiber. A disturbance of this balance causes a massive aggregation of the prion protein, leading to the prion information loss.The objective of my thesis was to understand what defined in vivo the prion transmission. My studying model was the Ure2p protein propagating the [URE3] prion in S. cerevisiae yeast. I showed that the Ure2p cellular concentration determined the aggregation speed of the prion protein and thus its transmission efficiency. Indeed, too high cellular concentrations are incompatible with the prion propagation. The cellular concentration of Ure2p also defines the prion strains diversity. A genetic screen allowed me to highlight that the presence of centrometric supernumerary sequences in the cell interferes with the [URE3] prion transmission. The same phenomenon is observed with an increase in the cell ploidy. In both cases, overexpression of the Hsp104 chaperone restores normal prion propagation

Les prions sont des protéines qui suite à des changements de conformation acquièrent un caractère infectieux. Ils sont à l’origine de traits dominants, héritables de façon non-Mendélienne, chez les mammifères, les champignons filamenteux et les levures. Le mauvais repliement et l’agrégation des protéines sont à l’origine de plus de 40 maladies, parmi lesquelles on retrouve des maladies neurodégénératives telles que les maladies d’Alzheimer, de Parkinson et de Huntington. Il a été montré que les formes agrégées des protéines supposées responsables de ces maladies (i.e. peptide amyloïde-β, tau, α-synucléine, huntingtine) se propagent de cellule en cellule à la manière des prions. La levure Saccharomyces cerevisiae possède plusieurs prions qui sont autant d’excellents modèles biologiques pour la compréhension des mécanismes de formation et de propagation des prions.[PSI+] et [URE3], issus respectivement de la conversion sous forme prion du terminateur de la traduction Sup35p et d’un régulateur du métabolisme azoté Ure2p, sont à ce jour les deux prions les mieux documentés chez la levure. Les chaperons moléculaires et leurs co-chaperons modulent la formation, la réplication et la propagation des prions chez la levure. Cependant, l’élimination ou la dégradation de ces prions sont encore mal connus. Notre laboratoire a montré que le protéasome 26S est capable de dégrader les formes soluble et fibrillaire de Sup35p. Dans la première partie de ma thèse, nous avons étudié le rôle du protéasome 26S dans la dégradation des formes soluble et fibrillaire d’Ure2p. Nous avons montré que, comme pour Sup35p, le protéasome 26S dégrade Ure2p soluble en générant des peptides amyloïdes issus du domaine prion N-terminal ainsi qu’un fragment C-terminal résistant à la protéolyse. Nous avons montré que le domaine prion déstructuré est nécessaire pour la reconnaissance et la dégradation par le protéasome. Contrairement à ce qui avait été observé pour Sup35p, Ure2p sous sa forme fibrillaire est totalement résistante à la dégradation protéasomale. Nous suggérons que la variabilité structurale aux seins des particules de prions dans un contexte cellulaire dicte leurs interactions avec les machineries protéolytiques, et plus particulièrement avec le protéasome.Les prions de levure ont principalement été étudiés dans un contexte de cellules en division active. Cependant, dans la nature, la plupart des cellules sont retrouvées dans un état quiescent post-mitotique. Nous n’avons que très peu d’informations sur le devenir des particules de prions lorsque les cellules entrent dans un état quiescent. De même les conséquences physiologiques des prions sur la survie à long terme des levures sont très peu documentées. Dans la seconde partie de ma thèse, nous avons utilisé le prion [PSI+] comme modèle pour répondre à ces questions. Différentes conformations des agrégats de Sup35p conduisent à des souches phénotypiquement distinctes du prion [PSI+]. Nous avons constaté que les agrégats de Sup35p subissent des changements ultra-structuraux et fonctionnels au cours des différentes phases de croissance cellulaire. Ainsi, nous avons observés des changements importants dans la distribution de taille et dans l’infectiosité des polymères de Sup35p résistants au SDS formant les briques élémentaires du prion [PSI+]. Ces changements interviennent sans affecter les informations structurales spécifiques à chaque souche de prion [PSI+]. De façon remarquable, bien que [PSI+] n’affecte pas le taux de croissance des levures, ce prion semble prolonger significativement la durée de vie des levures. Cet effet bénéfique semble pouvoir se fixer de façon efficace et permanente dans les cellules et persister même après élimination de [PSI+]. La fixation génétique de caractéristiques épigénétiques induites par [PSI+] ont été déjà observées et l’ensemble de ces résultats suggère que [PSI+] (et éventuellement d’autres prions) peut jouer le rôle de capaciteurs évolutifs transitoires
“Proteinaceous infectious particles”, or prions, are self-perpetuating alternate conformations of proteins that are responsible for heritable non-Mendelian traits in mammals, filamentous fungi and yeast. On a more general note, protein misfolding and aggregation is at the origin of over forty protein folding disorders including devastating neurodegenerative diseases such as Alzheimer’s, Parkinson’s or Huntington’s diseases. The aggregated proteins responsible for these diseases (i.e. amyloid-β peptide/tau, α-synuclein and huntingtin) were shown to propagate from cell to cell in a prion-like manner. The yeast Saccharomyces cerevisiae hosts many prion or prion-like proteins, unrelated in sequence and function, which proved to be excellent models for understanding the dynamics of prion aggregation and distribution upon cell division.Sup35p and Ure2p which cause the [PSI+] and [URE3] heritable traits, respectively, stand out as the most studied and best characterized yeast prions to date. A plethora of cellular factors, mostly belonging to various molecular chaperone families, were shown to affect yeast prion formation and propagation. Clearance of protein aggregates and prion particles is however poorly understood and documented. Our laboratory showed that the 26S proteasome degrades both the soluble and prion-associated fibrillar forms of Sup35p. In the first part of my thesis, we investigated the role of the 26S proteasome in the degradation of the soluble and fibrillar forms of Ure2p. We found that, as with Sup35p, the 26S proteasome is able to degrade the soluble native Ure2p, generating an array of amyloidogenic N-terminal peptides and a C-terminal fragment which is resistant to proteolysis. The N-terminal prion domain was shown to act as a degron required for proteasomal engagement and degradation. In contrast to Sup35p, fibrillar Ure2p resisted proteasomal degradation. We expect the structural variability within prion assemblies in a cellular context to dictate their interaction with proteolytic machineries in general and the proteasome in particular.The biology of yeast prions has been mostly explored in the context of logarithmically dividing cells. In nature however, most cells are generally in a post-mitotic non-dividing quiescent state. Yet little is known about the fate and properties of prion particles upon yeast cells entry into the stationary or quiescent states and the physiological consequences of harboring these prions throughout the lifespan of yeast cells. In the second part of my thesis, we addressed this issue using the [PSI+] prion as a model. Structurally different conformers of Sup35p aggregates can lead to distinct [PSI+] strains with different prion phenotypes. We found that Sup35p prion particles undergo growth phase-dependent ultrastructural and functional changes. Indeed, the size distributions of SDS-resistant core-prion particles significantly change during growth without affecting the structural information specific to each prion strain. The infectious properties of Sup35p prion particles undergo dramatic growth phase-dependent changes. Importantly, we found that while [PSI+] has little to no effects on the growth rates of yeasts, it robustly prolongs their chronological lifespan. Furthermore, this beneficial effect can then be permanently and efficiently fixed in the cells even when [PSI+] is subsequently lost. Similar genetic fixation of [PSI+]-induced epigenetic characteristics were previously observed and suggested [PSI+] (and possibly other prions) can act as transient evolutionary capacitators

碩士
大同大學
生物工程研究所碩士在職專班
91
This study investigated the influence of the temperature and aeration on yeast propagation in a large beer brewery. The fermentation temperature （9,10,11℃）and aeration time interval were the control variables to study the optimum conditions for the subsequent yeast propagation tanks YPT-2 and YPT-3. Apparent plato, alcohol, and yeast concentration in the fermentation wort were measured for each experiment. The results indicated that the higher of incubation temperature the lower of apparent plato, and higher alcohol and yeast concentration of the transferred wort at fixed aeration. When the temperature was controlled at constant, more aeration resulted higher apparent plato and lower alcohol, however, the yeast concentration was almost the same. It was better to transfer the yeast in the exponential phase for YPT-2 tank, and the yeast concentration had better more than 7.50×107 cells/ml. Moreover, alcohol concentration should not be too high, apparent plato should be in the range of 5~7o P. The optimum conditions for propagation of yeast in the YPT-2 tank were 1 min. aeration in 10 min. interval, and the temperature should be 10℃ or 11℃. It was better to transfer the wort of YPT-3 in high yeast concentration (more than 7.50×107 cells/ml) to the following fermentation tank. The yeast could ferment wort in anaerobic condition (alcohol fermentation), the apparent plato should be in the range of 4 ~6o P. The optimum conditions for propagation of yeast in the YPT-3 tank were 4 min. aeration in 20 min. interval, and the temperature should be 10℃ or 11℃.

The baker's yeast Saccharomyces cerevisiae is potentially a very useful host for the production of pharmaceutical proteins by recombinant DNA technology. One requirement for efficient overproduction of a foreign protein in yeast is a stable recombinant DNA vector which is maintained at a high number of copies per cell. The rational design of such vectors requires knowledge concerning their propagation in a cell population. The purpose of this work is to develop mathematical and experimental tools for the study of multicopy plasmid propagation, and to apply these tools to the investigation of a particular type of yeast vector: a conditional centromere plasmid. A method for measuring the distribution of plasmid copy number in yeast populations was developed, using [beta]-galactosidase activity as a marker for plasmid copy number. Enzyme activity is assayed at the single-cell level using a fluorogenic substrate and flow cytometry. The relationship between single-cell fluorescence and enzyme activity is described by a mathematical reaction-diffusion model. A segregated mathematical modeling framework was established to link measured copy number distributions with probabilistic models of single-cell plasmid replication and partitioning. Simplifications of the general integral-partial differential population balance equations were obtained for a discrete state variable, resulting in a linear system of ordinary differential equations. Flow cytometry and segregated modeling were applied to the study of a conditional centromere plasmid. This type of plasmid can be amplified to high copy number by unequal partitioning, but the amplified copy number state is unstable in the absence of selection pressure. A segregated model of this plasmid's propagation was shown to be consistent with experimental observations. The conceptual model of plasmid instability suggests changes in the attributes of the host cell and plasmid construction to improve stability at high copy number. A segregated mathematical model of this type is necessary for the design of bioreactor operating conditions that optimize productivity

The 2 micron plasmid of Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number of 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-encoded partitioning system and an amplification system to ensure its stable, high-copy propagation. The partitioning system consists of the plasmid encoded proteins, Rep1 and Rep2 and a cis-acting partitioning locus STB. The Rep proteins, together with several host factors, assembled at STB couple plasmid segregation to chromosome segregation. A plasmid lacking an active partitioning system is subject to a ‘diffusion barrier’, which causes it to be retained in the mother cell with a strong bias (mother bias). Currently available evidence favors the hitchhiking model for plasmid segregation, in which the tethering of plasmids to chromosome provides the basis for faithful plasmid partitioning. However, direct evidence to support this hypothesis has been difficult to obtain because of the small size of the budding yeast nucleus and the poor resolution of chromosomes in live cells or in chromosome spreads. In this study, we have attempted to verify the hitchhiking model using single copy derivatives of the 2 micron plasmid as reporters. We demonstrate, using two single copy reporters present in the same nucleus, that plasmid association with chromosome spreads is authentic, and is dependent on the partitioning system. By using a strategy that forces all chromosomes to stay in either the mother or the daughter compartment, we show that plasmid segregation can be uncoupled from nuclear envelope segregation. However, plasmid segregation cannot be uncoupled from chromosome segregation under this condition. This tight coupling between plasmid and chromosome segregation is consistent with the hitchhiking model for plasmid segregation. The plasmid partitioning complex is assembled de novo at STB during each cell cycle during the G1-S window. Plasmid replication or cell cycle cues that signal cellular DNA replication appear to trigger this assembly. Furthermore, there is an apparent temporal hierarchy in the association and dissociation of protein factors at STB. When DNA replication is delayed or blocked, the dissociation of factors from STB from the previous portioning cycle and the association of factors for the new partitioning cycle are delayed or blocked, respectively. The precise role of replication in plasmid segregation has not been elucidated. We have addressed this question by blocking either plasmid replication or all cellular DNA replication. We find that replication is not required for plasmid to overcome mother bias. However, replication is critical for the equal segregation of sister plasmid copies. These results provide a refinement of the hitchhiking model by suggesting that sister plasmids tether to sister chromatids in a replication-dependent manner and hitchhike on them during chromosome segregation. Finally, we have attempted to reconstitute the 2 micron plasmid partitioning system in mammalian cells with the goal of exploiting their larger nuclear size and the considerably higher chromosome resolution they provide. In experiments completed so far, we show that Rep2 expressed in COS7 cells localizes to chromosomes, and Rep1 does so in the presence of Rep2. Furthermore, they show co-localization on sister chromatids in a symmetric fashion, implying that plasmids associated with them are likely to follow suit. These observations suggest, by extrapolation, the Rep1-Rep2 assisted association of sister plasmids with sister chromatids in yeast as well, and are consistent with the refined hitchhiking model for plasmid segregation.
text

Dissertação de mestrado integrado em Engenharia Biológica (área de especialização em Tecnologia Química e Alimentar)
Esta dissertação propunha-se projetar um laboratório de microbiologia nas instalações da cervejeira Sovina com a capacidade para armazenar culturas puras de estirpes de levedura, propagar leveduras desde a escala laboratorial até à escala de produção, armazenar leveduras de fermentações prévias para a reutilização e realizar análises microbiológicas. Foi também solicitada a elaboração de um Manual de Procedimentos para todas as atividades propostas. Determinaram-se as tarefas e respetivos procedimentos a realizar no laboratório, para que estes cumprissem com todos os objetivos propostos. O cumprimento dos objetivos propostos é, em geral, possível mediante realização de tarefas de fácil execução e aplicação. Elaborou-se um plano de construção e montagem do laboratório em que se incluem as plantas de construção, instalação de utilidades e instalação de equipamentos AVAC e as listas de equipamentos e materiais laboratoriais necessários. De forma a averiguar a viabilidade económica de implementação do projeto determinaram-se os custos de investimento inicial e manutenção e os benefícios gerados. A partir destes, calcularam-se os fluxos financeiros, o VAL, o TIR, a AE e o TR do projeto. A avaliação destes fatores determinou que o projeto é viável, embora seja de retorno financeiro a longo prazo.
The purpose of this thesis was to design a microbiology laboratory in the facilities of the brewery Sovina with the ability to store pure cultures of yeast strains, propagating yeast from the laboratory scale to the production scale, store yeast from previous fermentations for reuse and perform microbiological analyses. It was also requested that a Manual of Procedures for all proposed activities should be made. The tasks and the respective procedures to be performed in the laboratory were listed in order to comply with all the goals. Compliance with the proposed objectives is, generally, possible by performing tasks of easy implementation and enforcement. A lab construction and assembly plan was performed which includes the layouts for the construction, utilities installation and installation of HVAC equipment. Lists of necessary laboratory equipment and materials were also included. In order to ascertain the economic viability of the project’s undertaking the initial investment and maintenance costs and the benefits generated were measured. From these, the financial flows, NPV, IRR, EA and the project PT were calculated. The evaluation of these financial indicators determined that the project is viable, although financial returns are set to the long run.