Dissertations / Theses on the topic 'Cerevisaie'

Il existe un intérêt croissant pour le développement de levures S. cerevisiae œnologiques à rendement abaissé de conversion des sucres en alcool. Nous proposons ici une approche originale basée sur la réorientation du flux carboné vers la voie des pentoses phosphate (VPP). Dans un premier temps, nous avons montré que le flux à travers la VPP est limité par le niveau de réoxydation du NADPH et par la capacité de la voie elle même. Nous avons ensuite mis en évidence le rôle crucial du facteur de transcription Stb5 dans le maintien d'un flux basal à travers la VPP. La surexpression de STB5, couplée à l'introduction d'un système de réoxydation du NADPH, est une stratégie intéressante pour amplifier le flux à travers la VPP. En parallèle, une stratégie d'évolution dirigée basée sur l'adaptation des souches sur gluconate, un hexose mal assimilé et incorporé au niveau de la VPP, a été développée. Des souches évoluées présentant une meilleure assimilation du gluconate ont été obtenues après 70, 180 et 240 générations. En fermentation, ces souches produisent la même quantité d'éthanol que la souche parentale mais présentent des phénotypes complètement nouveaux, en particulier des performances fermentaires accrues, de faibles besoins en azote, une production d'acétate réduite et une forte production de composés aromatiques. L'analyse 13C-flux et transcriptomique d'une souche évoluée ECA5 révèle une amplification de la VPP d'un facteur 1.5 par rapport à la souche parentale EC1118, en lien avec la surexpression de GND1 et TKL1. L'expression de nombreux gènes du métabolisme azoté et de la voie Ehrlich, de l'homéostasie des protons et de la glycolyse est augmentée chez ECA5, alors que les gènes de stress et de la respiration sont globalement réprimés, de façon cohérente avec les phénotypes observés. Outre le développement de nouvelles souches d'intérêt œnologique, ce travail apporte un éclairage nouveau sur le fonctionnement de la VPP et sur ses liens avec le métabolisme central et secondaire
There is an ever-growing interest in the development of S. cerevisiae wine yeast strains with reduced ethanol yield. We proposed a novel approach based on rerouting the carbon flux towards the pentose phosphate (PP) pathway. First, we showed that the flux through the PP pathway is limited both by the absence of a mechanism for reoxidation of NADPH and by the intrinsic capacity of the pathway. We also showed that the transcription factor Stb5 plays a key role in maintaining a basal flux through the PP pathway to meet the requirements for NADPH and biosynthetic precursors. Over-expression of STB5 is a potentially useful strategy for increasing the flux through the PP pathway, provided that an alternative system of reoxidation of NADPH is expressed. In parallel, we investigated an evolutionary engineering strategy based on long-term batch culture on gluconate, a substrate poorly assimilated by S. cerevisiae cells and metabolized by the PP pathway. We selected strains that had evolved a greater gluconate consumption capacity after 70, 180 and 240 generations. During wine fermentation, these evolved strains produced similar amounts of ethanol as the parental strain but displayed completely novel phenotypes, including higher fermentation rates, lower nitrogen requirements, lower levels of acetate production, and enhanced production of aroma compounds. 13C flux analysis and transcripomic analysis of one of these strains, ECA5, showed a greater flux through the PP pathway consistent with the observed increased expression of GND1 and TKL1. The expression of genes associated with nitrogen metabolism, the Ehrlich pathway, proton homeostasis and glycolysis was stronger than in the parental strain, whereas genes involved in stress response and respiration were down-regulated, in agreement with the phenotypes of ECA5. In addition to providing strains with considerable potential for wine making, this work sheds new light on the operation of PP pathway and its links with central and secondary metabolism

A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea.
The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.

Ubiquinol-cytochrome c reductase (complex III) is a central component of the respiratory chain of the inner mitochondrial membrane. It transfers electrons from reduced ubiquinone to ferricytochrome c. Correctly assembled and functional complex III is an essential prerequisite for oxidative energy metabolism. Complex III deficiency has been reported to be associated with several neurodegenerative diseases. Formation and assembly of complex III requires a multitude of specific nuclearly encoded proteins. For example, gene specific translational activators for cytochrome b synthesis as well as three non-subunit proteins, which are important for assembly and/or stability have been detected. The role of Bcs1p in assembly of Rieske FeS protein and Qcr10p into complex III has been clasified recently. The role of the two putative chaperones, Cbp3p and Cbp4p, is not known. In spite of the similar phenotype of cbp3D and cbp4D strains, that suggests the role of both proteins in the same step of complex III assembly, we were able for the first time to demonstrate differences on the molecular level between both deletion mutants. We show by BN-PAGE that cbp3D and cbp4D mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high molecular weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high level expression of Cbp3p can partially compensate for the lack of Cbp4p. Because lipids play an important role for complex III assembly and stability, we analysed the mitochondrial lipid composition of cbp3D and cbp4D mutants. Our data show that mitochondria of both mutants exhibit a wild type-like lipid composition, that favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism. By complementation studies we have shown that Cbp3 proteins of S. cerevisiae, S. pombe and human are (partially) functional homologues. A yeast model based on chimeric constructs of S. cerevisiae and human proteins was constructed, which allows to test the pathogenicity of human mutations. To define the role/s of Cbp3p and Cbp4p in the assembly pathway of complex III, interactions of selected subunits with both assembly factors were analysed by TAP- or co-immunoprecipitation. Based on the results of Cbp3p and Cbp4p topologies, BN-PAGE analysis of null mutant strains and interaction studies a model for complex III assembly and the roles of Cbp3p and Cbp4p in this process are proposed. I present a hypothesis, according to which Cbp3p and Cbp4p form a ?scaffold? for the assembly of all three putative sub-complexes, may act independently in the first steps of bc1 complex assembly (e. g. the formation of sub-complexes) and interact together to assist the final assembly of sub-complexes into a mature enzyme
Der Ubiquinol-Cytochrom c Reductase (Komplex III) ist eine zentrale Komponente der Atmungskette der inneren Mitochondrienmembran. Er transferiert Elektronen von reduziertem Ubiquinon auf Ferricytochrom c. Der korrekt assemblierte und funktionale Komplex III ist eine essenzielle Voraussetzung für den oxidativen Energiemetabolismus. Komplex III Defizienz ist assoziiert mit verschiedenen neurodegenerativen Krankheiten

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.

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.

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.

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 (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.

Saccharomyces cerevisiae est l’acteur principal de la fermentation du moût de raisin, mais l’influence de facteurs sur sa distribution dans les vignobles est peu connue. La région bordelaise, par son histoire et ses appellations, est une région d’intérêt pour étudier la diversité de S.cerevisiae. Au total, 2422 isolats de S.cerevisiae provenant de prélèvements de raisins et de cuves en fermentation spontanées sur deux années consécutives ont été analysés par 15 à 17 marqueurs microsatellites. Une très grande diversité génétique est mise en évidence, supérieure en mode de conduite conventionnel par rapport au mode biologique. Le mode de conduite influence faiblement la structure de la population de S.cerevisiae au vignoble. L’appellation et le domaine impactent significativement la structure de population, sans que des gradients de diversité n‘apparaissent, mais nos analyses révèlent des connections importantes dans le sens Pessac-Léognan vers les autres appellations du Bordelais, en particulier le Médoc. Des flux importants bidirectionnels sont mis en évidence entre les compartiments vigne et chai, illustrés par la présence de 25% de souches apparentées à des levures commerciales au vignoble, retour des souches du chai au vignoble jusqu’alors sous-estimé, alors qu’un flux d’importance similaire est observé entre le vignoble et le chai. La présence de populations ancestrales communes dans des prélèvements anciens (plus de 20 ans) et récents révèle la stabilité des populations sur le long terme à l’échelle d’une appellation. Une succession temporelle des populations du chai pourrait être favorisée par la mise en œuvre de pied de cuve avec repiquages successifs
Saccharomyces cerevisiae is the main actor of wine fermentation but still little is known about the factors impacting its distribution in the vineyards. Bordeaux region, by its history and its appellations, is one of the regions of interest to study S. cerevisiae diversity. A total of 2422 isolates of S.cerevisiae sampled from grapes and spontaneous fermentation tanks during two consecutive years were analyzed by 15 to 17 microsatellite markers. A very high genetic diversity is demonstrated, greater in conventional farming system than in organic one. The type of farming system weakly influences the population structure of S.cerevisiae in the vineyard. The appellation and the wine estate significantly impact the population structure, without appearance of diversity gradients, but our analyses reveal important connections from the Pessac-Léognan to other Bordeaux appellations, especially to the Medoc. Bidirectional strong flows are highlight between the vineyard and the cellar compartments as illustrated by the presence of 25% of commercial related strains in the vineyard, due to the unexpected return of strains through cellar to the vineyard, while a flow of similar magnitude is observed between the vineyard and the cellar. The presence of common ancestral populations in old (over 20 years) and recent samples showed population stability over the long-term at an appellation scale. A temporal succession of cellar populations was highlighted that could be link with the implementation of the Pied de Cuve method through successive inoculations

Membranes are hydrophobic barriers that define the outer boundaries and internal compartments of living cells. Membrane proteins are the gates in these barriers, and they perform vital functions in the highly regulated transport of matter and information across membranes. Membrane proteins destined for the endoplasmic reticulum are targeted either co- or post-translationally to the Sec61 translocon, the major translocation machinery in eukaryotic cells, which allows for lateral partitioning of hydrophobic segments into the lipid bilayer. This thesis aims to acquire insights into the mechanism of membrane protein insertion and the role of different translocon components in targeting, insertion and topogenesis, using the yeast Saccharomyces cerevisiae as a model organism. By measuring the insertion efficiency of a set of model proteins, we studied the sequence requirements for Sec61-mediated insertion of an α-helical transmembrane segment and established a ‘biological hydrophobicity scale’ in yeast, which describes the individual contributions of the 20 amino acids to insertion. Systematic mutagenesis and photo-crosslinking of the Sec61 translocon revealed key residues in the lateral gate that modulate the threshold hydrophobicity for membrane insertion and transmembrane segment orientation. Further, my studies demonstrate that the translocon-associated Sec62 is important not only for post-translational targeting, but also for the insertion and topogenesis of moderately hydrophobic signal anchor proteins and the C-terminal translocation of multi-spanning membrane proteins. Finally, nuclearly encoded mitochondrial membrane proteins were found to evade mis-targeting to the endoplasmic reticulum by containing short C-terminal tails.

At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 4: Manuscript; Paper 5: Manuscript

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

The yeast FBP1 and PCK1 genes and the gluconeogenic enzymes that they encode, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, are subject to multiple levels of regulation by glucose. It has been reported that transcriptional repression of these genes is exceptionally sensitive to glucose, being triggered by glucose concentrations of less than 0.005% (0.25 mM). It was shown here that in addition at transcriptional repression, the FBP1 and PCK1 and mRNAs are destabilised about 2-fold upon addition of the same low levels of glucose. Low levels of the fermentable sugars fructose or sucrose also stimulated this effect but galactose did not. This destabilisation was lost in a triple hxk1, hxk2, glk1 mutant, but was not triggered by addition of 2-deoxyglucose. The data suggests that sugar phosphorylation and further metabolism of glucose is required to trigger this response. Analysis of metabolic mutants showed that mutations in the upper part of the glycolytic pathway abolish the destabilisation of the FBP1 mRNA. Differences were shown to exist between the regulatory pathways that mediate glucose-stimulated mRNA decay and transcriptional repression. Models which might account for the mechanisms by which rapid decay of the gluconeogenic mRNAs is triggered are discussed. A strategy based on gene fusions with the stable PGK1 mRNA was designed in order to map cis-acting regions which influence PCK1 mRNA stability. A fusion mRNA containing the PCK1 mRNA protein coding region was not destabilised upon addition of low levels of glucose. It was therefore suggested that glucose-stimulated mRNA decay might in some way be dependent upon translation initiation via an interaction with the 5'-leader.

Studies were made of 2 mum based chimaeric plasmid copy number in Saccharomyces cerevisiae. A plasmid (pAYE56) containing three selectable genes in yeast (yeast LEU2, bacterial CAT and HSV-1 - TK) was constructed to reflect changes in copy number. Yeast transformants could be grown under three selection regimes and plasmid copy number estimated. During selective growth for the LEU2 gene there are about 20 plasmids per cell. This increases to about 100 during selective growth for the TK gene and furthermore the copy number can be controlled by the stringency of selection. Simultaneous selection for the TK and CAT genes may lead to a further increase (160 copies). Two models are proposed to account for these increases. The amplification model proposes plasmid replication without cell growth whilst the selection model suggests that plasmid copy number varies greatly in a population of transformants and cells with a high copy number are selected for growth under the TK/CAT selection conditions. Whilst the mechanism of copy number increase is unclear, an attempt was made to relate the expression of a heterologous gene (Human alpha2-IFN) to gene dosage using the promotion and secretion signals of the alpha-factor gene. Production of intracellular alpha2-IFN was unaffected by copy number whilst secreted material showed a 100 fold increase over a ten fold increase in gene dosage. Attempts were made to isolate plasmid copy number mutants. After mutagenesis (of cells or plasmid) transformants were selected under conditions for simultaneous over-expression of the TK and CAT genes. Mutants capable of growth under these conditions were obtained. In one group the mutant phenotype was lost upon curing but did not return upon retransformation. In a second group a chromosomal mutation was isolated. Plasmid copy number estimates indicated that this was unchanged however. Alternative strategies are discussed for the isolation of mutants.