Dissertations / Theses on the topic 'Saccharomyces cerevisiae genome codes'
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Rowley, Neil K. "Studies on the Saccharomyces cerevisiae genome." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361615.
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Greig, Duncan. "Sex, species and Saccharomyces cerevisiae." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301401.
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Novarina, D. "MECHANISMS PRESERVING GENOME INTEGRITY IN SACCHAROMYCES CEREVISIAE." Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/215589.
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The integrity of the genome is continuously jeopardized by endogenous reactive byproducts of cellular metabolism and genotoxic insults by environmental agents, as well as by the DNA transactions (replication, transcription and recombination) required for cell survival and proliferation. Failure of the mechanisms deputed to the maintenance of genome integrity leads to genome instability, which is a hallmark of cancer and a driving force of tumorigenesis. To fully understand the mechanisms leading to genome instability and the cellular pathways counteracting them, three basic tasks must be achieved: i) identify all the genes implicated in the control of genome integrity; ii) unravel their biological role; iii) define the mechanistic molecular details of the processes in which they are implicated. This thesis describes work performed in the budding yeast Saccharomyces cerevisiae to explore the genome stability landscape at all these three levels. This model system is extremely useful for two main reasons: a) its high genetic tractability allows the application of genome-wide genetic screenings; b) the large conservation of the genome integrity pathways allows to extend the findings obtained in yeast to other eukaryotic organisms.
We performed a genome-wide screen, based on the overexpression of the DDC2 DNA damage checkpoint gene in the yeast deletion collection, to identify genome stability genes on the basis of spontaneous accumulation of endogenous DNA damage in the corresponding mutant strains. Our screen identified several genes implicated in the control of genome integrity, highlighting, in particular, a key role for pathways protecting against oxidative stress. We present here the preliminary characterization of a new genome integrity gene, VID22.
We also investigated the mechanisms counteracting a newly discovered source of genome instability, namely ribonucleotides (rNTPs) incorporated in genomic DNA during replication. We uncovered a role for RNase H enzymes, template switch pathways and Pol ζ translesion polymerase in protecting from misincorporated rNTPs. Given that mutations in any of the three human RNase H2 subunits were proven to cause Aicardi-Goutiéres Syndrome, these results might contribute to shed light on the complex and largely unknown pathogenetic mechanism of this rare genetic disease.
Finally, we studied the molecular details underlying the role of Rad9 mediator protein in DNA damage checkpoint activation, exploring the dynamics of Rad9 dimerization, chromatin binding, CDK-dependent phosphorylation and checkpoint activation in G1 and M phases of the cell cycle; in particular, we characterized an M-phase specific pathway for checkpoint activation which is relies on Rad9-Dpb11 interaction.
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SHANMUGAN, MUTHU KUMAR. "EXPLORING GENOME INTEGRITY PATHWAYS IN SACCHAROMYCES CEREVISIAE." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229912.
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Genomic DNA is under constant attack from both endogenous and exogenous DNA damaging agents like reactive oxygen species which include O2, H2O2, OH, reactive carbonyl species, alkylating agents such as estrogen and cholesterol metabolites, radiations (like UV, x-rays and gamma rays) and mutagenic chemicals. Moreover, threats to DNA integrity can also come from DNA metabolism such as replication, transcription and recombination. In order to survive and faithfully transmit the genetic material to the progeny, cells must detect the damage and activate repair mechanisms and, if the damage cannot be repaired, trigger the apoptotic program. All these processes, which are collectively known as DNA damage response (DDR), are coordinated by surveillance mechanisms often called DNA damage checkpoint, which temporarily halt or slow down cell cycle progression to provide enough time for DNA repair. The failure of the DNA damage response and other mechanisms deputed to the maintenance of genome integrity leads to a condition called “Genome Instability”, consisting in the accumulation of damage, genomic aberrations, such as mutations, gross chromosomal rearrangements and chromosome loss. Genome instability is a hallmark of cancer and a driving force in tumorigenesis.
We exploit budding yeast Saccharomyces cerevisiae as a model system for studies on genome maintenance pathways which are highly conserved throughout evolution from yeast to human. Despite recent advances in the field, genome integrity pathways are not yet fully understood and not all the genes involved have been identified. We developed a screening strategy, based on the overexpression of DDC2, a critical DNA damage checkpoint gene in the contest of a yeast deletion collection, in order to identify genes controlling genome integrity on the basis of spontaneous accumulation of endogenous DNA damage. We identified several genes and pathways associated with genome integrity maintenance, among which are many genes induced in peroxisome biogenesis and mitochondria structure and function, as well as several uncharacterized ORFs.
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Bleackley, Mark Robert. "Transition metal tolerance and the Saccharomyces cerevisiae genome." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/30821.
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Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also leads to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. The yeast Saccharomyces cerevisiae has been used as model organism for the investigation of these systems and a majority of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. Traditionally, genomic studies in metal homeostasis focus on the response to one, or in some cases two, metals. Here, I have used high density yeast arrays of a S. cerevisiae deletion collection to study the genes required for tolerance to six transition metals in parallel and I have used this data to examine the role of genes not only in the homeostasis of individual metals but to also gain insight into cellular transition metal homeostasis as a whole. Genes and pathways with novel function in the homeostasis of a particular metal have been identified along with the systems that function with broad spectrum metal specificity. Data generated in this screen has also be combined with previously published data sets that examine different aspects of yeast biology in an attempt to delve deeper in to the cellular machinery that allows yeast, and potentially the cells of other organisms, to maintain the balance between metal ions as essential nutrients as opposed to toxic moieties. Metallochaperones represent a relatively recent emerging class of proteins that play a central role in maintaining this balance. As part of the analysis of the high density array screens, putative chaperones have been identified. Additionally a yeast 2 hybrid screen using a cytoplasmic domain of the S. cerevisiae high affinity iron transporter Ftr1p has been performed to with the goal of discovering candidate iron chaperones. As a whole, the research discussed in this thesis has shed light on a number of new features of the homeostatic mechanisms that function in S. cerevisiae and will provide the basis for further investigation into the interactions between cells and metal ions eventually leading to implications in human health and disease.
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Minchell, Nicola E. "DNA topological stress during DNA replication in Saccharomyces cerevisiae." Thesis, University of Sussex, 2019. http://sro.sussex.ac.uk/id/eprint/81222/.
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DNA topological stress impedes normal DNA replication. If topological stress is allowed to build up in front of the replication fork, the fork rotates to overcome the stress, leading to formation of DNA pre-catenanes. The formation of DNA pre-catenanes is therefore a marker of DNA topological stress. In this study, I have examined how transcription linked DNA topological stress impacts on fork rotation and on endogenous DNA damage. Transcription, similar to replication, affects the topology of the DNA; and collision between the two machineries is likely to lead to high levels of DNA topological stress. I found that the frequency of fork rotation during DNA replication, increases with the number of genes present on a plasmid. Interestingly, I also found that this increase in pre-catenation is dependent on the cohesin complex. Cohesin and transcription are known to be linked, as transcription leads to the translocation of cohesin along budding yeast DNA away from its loading sites. Cohesin plays a major role in establishing chromosomal structure, influencing gene expression and genetic inheritance. In this work, I have analysed the relationship between cohesin and the generation of topological stress and found that topological stress associated with cohesin can lead to DNA replication stress and DNA damage.
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Cook, Kristen. "Regulation of Genome-Wide Transcriptional Stress Responses in Saccharomyces cerevisiae." Thesis, Harvard University, 2011. http://dissertations.umi.com/gsas.harvard:10032.
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In response to osmotic shock in Saccharomyces cerevisiae the MAP kinase Hog1 coordinates a large-scale transcriptional stress response, rapidly producing hundreds of copies of specified transcripts. Many of the most highly induced genes are bound and regulated by a transcription factor, Sko1, but lack the canonical binding site for this factor. We use ChIP-seq to demonstrate a stress-specific binding mode of Sko1. In stress, Sko1 binds to promoters in close proximity to Hog1, and another Hog1-regulated transcription factor, Hot1. This mode of Sko1 binding requires the physical presence of Hog1, but not Hog1 phosphorylation of Sko1. We identify candidate Sko1 and Hot1 binding motifs that predict co-localization of Sko1, Hot1, and Hog1 at promoters. We then demonstrate a role for Sko1 and Hot1 in directing Hog1-associated RNA Pol II to target genes, where Hog1 is present with the elongating polymerase. We suggest a possible model for Hog1 reprogramming of transcription in the early stages of the osmotic stress response. We then determine the extent and structure of the Hog1 controlled transcriptional program in a related stress, damage to the cell wall. We find that Sko1 and Hot1 have different apparent thresholds for activation by Hog1. In addition, in cell wall damage, Hog1 regulates an additional transcription factor, Rlm1, that is not involved in other Hog1 regulated stress responses. This factor is activated by the coincidence of a signal from Hog1 with that of another MAP kinase, Slt2.
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Coissac, Éric. "Analyse structurale et fonctionnelle du genome de la levure saccharomyces cerevisiae." Paris 6, 1996. http://www.theses.fr/1996PA066520.
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Dans le cadre d'une collaboration internationale, la sequence nucleotidique du genome de la levure saccharomyces cerevisiae a ete entierement determinee. Au sein de ce projet, nous avons sequence un fragment d'adn de 39411 paires de bases correspondant aux regions telomerique et subtelomerique gauches du chromosome vii. Cette region comporte dix-huit phases ouvertes de lectures longues de plus de cent codons et une de soixante-seize. Parmi elles, six correspondent a des genes precedemment identifies : adh4, fzf1, hxk2, rtg2, hfm1 et pde1. La deletion de cinq autres a ete realisee (ygl257c, ygl255w, ygl250w, ygl249w et ygl246c). Deux de ces deletions (ygl250w et ygl246c) provoquent un retard de croissance sur milieu complet a une temperature de 28c. La phase ouverte ygl261c est un nouveau membre de la famille des seripauperines. Les caracteristiques de cette famille ont ete intensement etudiees. Cette etude montre qu'une pression de selection doit s'exercer sur cette famille afin de maintenir un de ces membres localise sur chacun des seize chromosomes. La deuxieme partie de cette these est consacree a l'analyse exhaustive des duplications de genes chez s. Cerevisiae et dans quatres organismes eucaryotes et bacteriens. Nos resultats montrent que le niveau de duplication de l'ensemble des genomes etudies est equivalent. Une etude plus approfondie demontre l'importance des telomeres dans les mecanismes de duplication chez la levure. Ces resultats suggerent qu'ils pourraient jouer le role de guides dans les processus de recombinaison ectopique.
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Teixeira, Maria Teresa. "Organisation du noyau et analyse fonctionnelle du genome de saccharomyces cerevisiae." Paris 11, 2000. http://www.theses.fr/2000PA112033.
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La structure du noyau semble etre hautement organisee : de nombreux facteurs nucleaires sont adresses a des domaines discrets, en concordance avec leur role localise. Cette regionalisation spatiale et fonctionnelle est particulierement bien etablie dans le cas du nucleole, site d'assemblage des sous-unites ribosomiques et dans le cas du pore nucleaire, site du trafic nucleo-cytoplasmique. Ce travail de these a pour but de participer a la caracterisation moleculaire de certains constituants du noyau impliques dans cette organisation fonctionnelle en prenant comme modele experimental la levure saccharomyces cerevisi. Dans une phase initiale du travail, nous nous sommes focalises dans l'etude d'une proteine constitutive du pore nucleaire, nup145p, impliquee dans l'export des arnm du noyau vers le cytoplasme. Nous avons montre que nup145p est clive in vivo en deux domaines, n- et c- nup145p, qui agissent de facon independante au niveau du pore nucleaire. En particulier, le c- nup145p s'associe avec d'autres nucleoporines pour former un sous-complexe du pore nucleaire implique dans l'export des arnm du noyau vers le cytoplasme, dans la distribution des pores nucleaires au sein de l'enveloppe nucleaire et dans l'integrite du nucleole. La maturation post-traductionelle de nup145p est essentielle a la localisation et l'activite du n-nup145p. Cette proteolyse specifique est autocatalytique et le site catalytique reside dans le n-nup145p. Nous avons egalement participe a l'analyse fonctionnelle du genome de saccharomyces cerevisi au sein du programme eurofan. 417 souches de levure individuellement deletees pour chacun des genes de fonction inconnue (collection euroscarf) ont ete analysees de facon independante pour la distribution de trois marqueurs nucleaires. Ainsi, nous avons selectionne 14 genes candidats potentiellement impliques dans l'organisation de l'architecture nucleaire et/ou dans le transport nucleo-cytoplasmique.
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Amai, Takamitsu. "Development of genome editing technology of mitochondrial DNA in Saccharomyces cerevisiae." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263707.
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Powers, Ralph Wilson. "Genome-wide screens reveal that reduced TOR signaling extends chronological and replicative life span in S. cerevisiae /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/5044.
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Leadbitter, Matthew. "Genome-wide study to investigate the organisation of global genome nucleotide excision repair in Saccharomyces cerevisiae." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/54442/.
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Driscoll, R. "Saccharomyces cerevisiae proteins Rtt109p and Esc2p : two novel regulators of genome stability." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598656.
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I reveal that Saccharomyces cerevisiae Rtt109p promotes genome stability and resistance to DNA-damaging agents, and that it does this by functionally cooperating with the histone chaperone Asf1p to maintain normal chromatin structure. Furthermore, I show that, as for Asf1p, Rtt109p is required for histone H3 acetylation on lysine 56 (K56) in vivo. Moreover I show that Rtt109p directly catalyzes this modification in vitro in a manner that is stimulated by Asf1p. These data establish Rtt109p as a member of a new class of histone acetyltransferases and show that its actions are critical fro cell survival in the presence of DNA damage during S phase. In the second part of this thesis, I reveal that cells deleted for Saccharomyces cerevisiae ESC2 exhibit synthetic sickness when combined with deletions of many genes involved in maintaining genomic stability. Moreover, I show that esc2Δ mutant cells exhibit increased recombination frequency and increased relocalisation of recombination repair protein Rad52p. Furthermore, I show that esc2Δ cells are hypersensitive to the DNA alkylating agent methane methylsulphonate and accumulate cruciform structures during replication after treatment with this drug. I therefore propose that Esc2p functions during S-phase to counteract formation of these pathological cruciform structures, thereby maintaining genomic stability. Taken together, the discovery of two more factors involved in maintaining genome stability suggests there may be more, as yet, undiscovered factors and provides further evidence for the complex nature of the cell mechanisms employed to promote genome stability.
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Huang, Meng-Er. "Contribution a l'etude du genome de la levure saccharomyces cerevisiae : chromosome 10." Paris 7, 1993. http://www.theses.fr/1993PA077268.
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Ce travail a ete effectue dans le cadre d'un programme de la communaute europeenne. Il comprend trois parties: 1) construction d'un contig du chromosome x de s. Cerevisiae, 2) sequencage du fragment de 41 kb, 3) analyse de la sequence de ce fragment. Un contig de 250 clones recombinants contenant l'adn du chromosome x a ete assemble en progressant a l'aide de ribosondes t3 et t7. Sa validite a ete confirmee par diverses approches. Une carte de restriction ecori de l'ensemble du contig a ete etablie. Un fragment de 41 kb a ete sequence en utilisant une methode aleatoire de fractionnement a l'aide d'un sequenceur abi 373a. 16 phases de lecture ouverte (plo) nouvelles ont ete identifiees. 13 plo ne montrent pas ou peu similitude avec d'autres proteines et 3 plo indiquent des similitudes significatives. Parmi ces dernieres plo, l'une presente une forte similitude avec les proteines des helicases, une seconde represente vraisemblablement la premiere proteine du canal cl# identifiee chez la levure. Par ailleurs, 2 nouveaux loci d'arnt ont ete identifies
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PORCU, GIAMPIERO. "Genome wide analysis of effects of protein farnesylation inhibition on saccharomyces cerevisiae." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2008. http://hdl.handle.net/2108/559.
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Farmaci anticancro basati sull’inibizione della farnesil-transferasi (FTIs) sono in grado di inibire la prenilazione di Ras e la crescita tumorale in un gran numero di tumori, come evidenziato durante gli studi preclinici. Tuttavia, se testati in studi clinici, gli FTIs hanno dimostrato di avere effetti anti-tumorali solo se usati in combinazione con altri farmaci. Nonostante una decade di studi, il meccanismo d’azione tramite cui gli FTIs agiscono sulle cellule tumorali rimane sconosciuto, tanto più che è evidente che tali farmaci agiscono su molteplici vie e varie proteine prenilate oltre a Ras.
Nel presente studio è stata determinata la risposta primaria in lievito all’ FTase Inhibitor I (FTI-I) a livello genomico, usando tale composto a concentrazioni con cui la sopravvivenza cellulare e la farnesilazione di Ras sono scarsamente modificate. Il profilo di espressione genomico di cellule wild-type evidenzia un effetto dell’ FTI-I sulla trascrizione di geni coinvolti nella regolazione della polimerizzazione e depolimerizzazione dei microtubuli, nella segregazione cromosomica, e nella resistenza multipla ai farmaci (multi drug resistance, MDR). Il profilo chimico risultante dal trattamento con FTI-I dei ceppi di lievito della banca di delezione EUROSCARF ha confermato tali risultati. In aggiunta essa implica le chinasi attivate da p-21 (PAKs) nella resistenza all’ FTI-I. Complessivamente, il presente studio sottolinea l’importanza di esaminare la risposta MDR nell’uso clinico degli FTIs e indica le chinasi PAK come target secondari da usare nella terapia degli FTIs in combinazione con altri farmaci.Anticancer agents based on inhibition of farnesyl-transferase (FTIs) inhibit Ras prenylation and tumour growth in a wide range of malignancy in preclinical studies. However, when tested in clinical trials, FTIs showed their beneficial effects only when used in combinatorial therapies. Despite a decade of studies, how FTIs promote morphological reversion remains unclear as they clearly impact multiple pathways and/or other prenylated proteins than Ras. Here, we combined genomic approaches to assess the primary yeast response to FTase inhibitor I (FTI-I) by using this compound at doses at which yeast viability and Ras farnesylation is poorly affected. Genomic expression profiling of wild-type cells show an FTI-I action in changing transcription of genes involved in microtubule polymerization/depolymerization and chromosome stability and in multi drug resistance (MDR). Chemical profiling of the EUROSCARF yeast deletion collection confirmed these data. In addition, it implicates the p-21 activated kinases (PAKs) in FTI-I resistance. Overall this study highlight the importance of carefully address the multi drug resistance response when using FTIs in clinical therapy and indicates PAKs as second target to use in combinatorial therapies.
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Lantermann, Alexandra. "Comparison of Genome-Wide Nucleosome Positioning Mechanisms in Schizosaccharomyces pombe and Saccharomyces cerevisiae." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-118784.
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Pegram, Kirsty Elizabeth. "The role of FOB 1 sumoylation in maintaining genome stability in saccharomyces cerevisiae." Thesis, Imperial College London, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.528293.
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Garduño, Bertha Veronića. "Cbf1 regulates chromatin remodelling of the Saccharomyces cerevisiae genome at multiple binding sites." Thesis, University of Oxford, 1999. http://ora.ox.ac.uk/objects/uuid:be76ba21-1336-4ac8-9da3-918fd58d5908.
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The centromere binding factor 1, Cbf1, of Saccharomyces cerevisiae is a bHLH/ZIP protein which has been described as a determinant of specific chromatin structures and as a tethering factor for activators of transcription at the promoters of genes of the Methionine Biosynthesis Pathway. Deletion mutants show various phenotypes, among them methionine auxotrophy, an increased rate of chromosome loss, modifications in the growth rate and modification of the chromatin structure at MET genes. Meiosis competence also becomes greatly reduced in cbf1 cells. The sequence motif (RTCACRTG) to which Cbf1p binds is found at multiple loci through the yeast genome. This thesis shows that the chromatin structure is reorganised at multiple Cbf1p binding sites in vivo, when yeast cells are starved to enter meiosis. Extensive remodelling occurs at the MET16, MET17(25), DRS2 and GDH3 loci and at the YAL060W open reading frame, as detected by in vivo digestion of chromatin with micrococcal nuclease and indirect end-labelling. The same kind of analysis showed that the remodelling of chromatin at Cbf1p binding sites is not specific for meiosis, it occurs also in similarly starved haploid cells. The lack of methionine is a key trigger of these changes. This reorganisation of chromatin is dependent on Cbf1p, since starved cbf1 cells do not display any modification in nuclease accessibility patterns at or around Cbf1p binding sites. Mutational analysis revealed that a negative charge at a putative phosphorylation site (serine residue 226) and the DNA-bindmg activity of Cbf1p are both required for the chromatin reorganisation to occur in response to starvation. CBF1 mutants which do not reorganise chromatin were also shown to be unable to enter meiosis, suggesting that the remodelling of chromatin at multiple Cbf1p binding sites may be required to enter pre-meiotic DNA replication, since such cells arrest before the initiation of this process. In summary, the results presented in this thesis are compatible with a model in which Cbf1p plays an active role as part of a mechanism sensing the nutrient availability and regulates the reorganisation of chromatin, at multiple loci through the yeast genome, in response to starvation conditions.
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Jennings, Ezra (Ezra Gray) 1971. "Genome-wide expression and location profiling in Saccharomyces cerevisiae : experimental and graphical analysis." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/29919.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.Includes bibliographical references.
Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast. Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes. The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for co-ordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus. Understanding how DNA-binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations where these proteins function in vivo. We developed a microarray method that reveals the genome wide location of DNA-bound proteins and used this method to monitor binding of gene specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Ga14 as cells respond to changes in carbon source, and by Thi2 in the absence or presence of thiarnin. The results identify pathways that are coordinately regulated by these regulators and reveal novel functions for these regulators. Understanding a transcriptional network such as these will be useful in constructing a cellular regulatory network map. The use of microarray technology has created new challenges in data analysis for biologists. Visual displays can greatly facilitate the analysis and communication of large quantities of data. We have created a Graphical Display Suite (GDS) that consists of a collection of tools to assist in the visualization of data from genome-wide experiments in S. cerevisiae. The ODS is web-accessible, easy to use, and additional components can easily be incorporated into its interface. This suite of tools has proven to be useful in revealing important biological insights.
by Ezra Jennings.
Ph.D.
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Hui, Sheng. "The analysis of metabolism in saccharomyces cerevisiae with genome-scale gene expression data." HKBU Institutional Repository, 2005. http://repository.hkbu.edu.hk/etd_ra/640.
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Ojo, Tolulope A. "Characterization of new mutants in the 16S ribosomal subunit region of the mitochondrial genome of Saccharomyces cerevisiae /." Connect to online version, 2006. http://ada.mtholyoke.edu/setr/websrc/pdfs/www/2006/148.pdf.
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Lévesque, Nancy. "Deciphering the function of chromatin modifiers in genome regulation and maintenance in Saccharomyces cerevisiae." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43352.
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Chromatin is a dynamic structure that facilitates DNA compaction inside the cell nucleus and contributes nuclear process regulation such as transcription, DNA repair and DNA replication. Chromatin structure can be modified by several mechanisms, including the incorporation of histone variants, histone sliding or removal by ATP-dependent remodelling enzymes, addition of post-translational modifications, and addition of methylation marks on DNA. This dissertation aims to comprehend better the role of chromatin modifiers like histone acetyltransferases (HATs) in cellular processes such as genome regulation and maintenance in yeast Saccharomyces cerevisiae.
HATs play crucial roles in cells, as they are involved in transcription regulation and DNA damage response. This dissertation investigates the precise nature of the relationship between the histone acetyltransferase complexes NuA4 and picNuA4. I discovered that the smaller NuA4 counterpart picNuA4 is partially able to replace the function of the larger NuA4 when the latter is defective. Additionally, dissection of the NuA4 complex scaffolding subunit Eaf1 revealed that its C-terminus is largely required for NuA4-dependent function.
While NuA4 shares subunits with SWR1-C, an ATP-dependent chromatin-remodelling complex that replaces histone H2A for the histone variant H2A.Z, the role of shared modules in chromatin remodelling complexes remains unclear. Here I explored the role of shared modules through investigation of Swc4 and Yaf9, two members of the NuA4 and SWR1-C. Biochemical, genetic, and gene expression assays demonstrated that both Yaf9 and Swc4 similarly contributed to the shared module functions and generally behaved more like SWR1-C, but also had distinct roles. For instance, large-scale genetic interaction profiles exposed noticeable differences between Yaf9 and Swc4, where Yaf9 behaved more similarly to SWR1-C NuA4, Swc4 to NuA4, in some cases.
Upon DNA damage, several chromatin modifications occur, such as acetylation of histone H4 tails by NuA4 or methylation of H3K79 by Dot1. Herein I propose a model explaining the role of H3K79 trimethylation in the DNA damage response in the context where cells are deficient for components of the DNA damage response. I suggest that, directly or indirectly, H3K79 trimethylation inhibits the translesion synthesis pathway, an alternative repair pathway.
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Harris, M. R. "G1/S transcriptional regulation in Saccharomyces cerevisiae integrates cell cycle progression and genome stability." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1419003/.
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Saccharomyces cerevisiae provides an ideal model to study the regulation of cell cycle commitment due to the high conservation of signalling pathways and regulatory modules through to higher eukaryotes. My work investigates the interplay of cell cycle progression and arrest via the action of transcription factor regulation. Cell cycle commitment is controlled by the cyclin-dependent activation of transcription factor complexes, MBF and SBF. Here I describe the dynamics of SBF and MBF using new polyclonal anti-sera against the three key components Mbp1, Swi4 and Swi6, and their interaction with the inhibitor of SBF, Whi5, and the MBF co-repressor Nrm1. I identify epigenetic modifications that occur on histone proteins at promoters of SBF and MBF genes during the cell cycle. The histone deacetylase Rpd3 has also been investigated as to the role it plays in regulating G1/S transcription. Finally, I have identified a new class of G1/S genes, named switch genes, which are regulated independently by G1/S transcription factors during different phases of the cell cycle. Switch genes are regulated by SBF during G1 and MBF upon entry into S phase, and are enriched for dosage sensitive and replication induced G1/S genes. Switching from SBF-to-MBF allows genes to be activated in response to replication stress, via inactivation of Nrm1. In addition, through switching a potential defect in one of the transcriptional factor complexes will not result in overexpression of these genes. Detailed analysis of the prototypical switch gene TOS4 shows that it is regulated by SBF and MBF, accumulates in response to hydroxyurea, and delays cell cycle progression when over-expressed. The role Tos4 plays in the cell cycle and in response to checkpoint activation remains unclear, however, data suggests a role in modulating HDAC activity. The roles other switch genes play in response to checkpoint activation are yet to be investigated.
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MAFTAHI, MOHAMED. "Contribution au sequencage et a l'analyse fonctionnelle du genome de la levure saccharomyces cerevisiae." Paris 7, 1997. http://www.theses.fr/1997PA077134.
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La levure saccharomyces cerevisiae represente le premier organisme eucaryote a voir son genome entierement sequence grace a une collaboration scientifique a l'echelle internationale. Au sein du projet biotech, nous avons determine la sequence d'un fragment de 40 kb du chromosome xiv de la levure. L'analyse de ce fragment situe a proximite du telomere gauche a revele la presence de 24 orfs parmi lesquelles 8 correspondent a des genes connus, 8 autres orfs presentent soit des homologies significatives dans les banques de donnees, soit des motifs connus. Enfin les 8 orfs restantes sont considerees comme des genes orphelins. A la suite de notre participation au sequencage systematique, nous avons developpe une nouvelle methode de disruption appelee sep (sticky end pcr). Cette nouvelle strategie met en jeu un remplacement genique grace a l'utilisation de grandes regions d'homologies avec la sequence cible et peut etre utilisee de facon systematique pour la disruption de genes. Le principe de cette methode repose sur une fusion des regions promotrices et terminatrices separees par un site de restriction rare. Plusieurs alternatives de cette methode peuvent etre appliquees, afin de l'utiliser dans d'autres strategies comme le pop-in/pop-out ou le gap repair. Par ailleurs, la methode sep a ete utilisee pour la disruption de genes chez d'autres organismes comme la levure yarrowia lipolytica. L'un des resultats les plus interessants du projet genome de la levure est la decouverte d'un nombre important de genes inconnus appeles aussi genes orphelins. Au sein du programme eurofan, nous avons entrepris l'analyse fonctionnelle de 8 nouveaux genes (no320, no325, no333, no339, no348, no364, no384 et no388). Nous avons realise la disruption de ces genes dans deux contextes genetiques differents et en utilisant differentes methodes de disruption. On a ainsi demontre que les genes no348, no364 et no388 etaient essentiels a la vie vegetative de la levure. Par la suite, on a entrepris une analyse phenotypique des 5 autres genes non essentiels dans des conditions physiologiques standards. Ces genes ne sont impliques ni dans la conjugaison ni dans la sporulation. Par ailleurs, aucun phenotype decelable n'a ete detecte dans les conditions utilisees. Enfin, on a realise une etude transcriptionnelle dans des conditions nologiques qui a mis en evidence la presence de transcrits pour tous ces genes a l'exception de no320. Il n'a pas ete observe de correlation directe entre le cai et le taux de transcription. Par ailleurs, on a enregistre dans certains cas une variation du taux d'expression au cours de la croissance.
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Gillet-Markowska, Alexandre. "Etude quantitative des variations structurelles des chromosomes chez Saccharomyces cerevisiae." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066233/document.
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L’accumulation de remaniements de la structure des chromosomes aussi appelés variations structurelles (SV) est un important contributeur à la transformation des cellules malignes et à la constitution d’une hétérogénéité intratumorale. Nous avons développé un outil bio-informatique qui permet désormais d’obtenir une image fine de ces SV qui se produisent dans le génome humain. Nous avons ainsi pu démontrer l’existence de SV présentes à de faibles fréquences dans différentes populations cellulaires supposées clonales montrant que les taux de formation des SV pourraient être grandement sous-estimés. Parallèlement, nous avons montré que le niveau d’instabilité des individus dépend de facteurs génétiques de prédisposition. Pour les identifier, nous avons développé des systèmes génétiques de mesure des taux de SV chez la levure qui vont nous permettre d'identifier les gènes contrôlant l'instabilité chromosomique par analyse de liaison à grande échelle. Ces régulateurs représenteront de nouveaux gènes candidats impliqués dans le développement du cancer chez l’homme, car les déterminants génétiques impliqués dans le métabolisme de l'ADN sont très conservés entre la levure et les mammifèresThe accumulation of chromosomal rearrangements also called Structural Variations (SV) is a major contributor to the transformation of tumoral cells and to the constitution of intratumoral heterogeneity. We have developed a bio-informatic tool that can now provide a sharp image of SV that occur in the human genome. We have demonstrated the existence of SV present in low proportions in different supposedly clonal cell populations showing that the rates of SV formation could be greatly underestimated. In parallel, we have shown that the level of instability of the genome depends on predisposition factors. To identify those, we have developed genetic assays to measure the rate of SV in yeast that will allow us to identify new genes controlling the stability of the genome using large scale linkage analysis. These regulators represent new gene-candidates involved in the development of cancer in human as the determinants involved in DNA metabolism are very conserved between yeast and mammals
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Tsaponina, Olga. "Regulation of ribonucleotide reductase and the role of dNTP pools in genomic stability in yeast Saccharomyces cerevisiae." Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-43978.
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Every living organism is programmed to reproduce and to pass genetic information to descendants. The information has to be carefully copied and accurately transferred to the next generation. Therefore organisms have developed the network of conserved mechanisms to survey the protection and precise transfer of the genetic information. Such mechanisms are called checkpoints and they monitor the correct execution of different cell programs. The DNA damage and the replication blocks are surveyed by the conserved Mec1-Rad53 (human ATM/ATR and Chk2, respectively) protein kinase cascade. Mec1 and Rad53 are essential for survival and when activated orchestrate the multiple cellular responses, including the activation of the ribonucleotide reductase (RNR), to the genotoxic stress. RNR is an enzyme producing all four dNTPs - the building blocks of the DNA - and is instrumental for the maintenance both proper concentration and balance of each of dNTPs. The appropriate concentration of the dNTPs should be strictly regulated since inadequate dNTP production can impede many cellular processes and lead to higher mutation rates and genome instability. Hence RNR activity is regulated at many levels, including allosteric and transcriptional regulation and the inhibition at protein level. In our research, we addressed the question of the transcriptional regulation of RNR and the consequences of dNTP malproduction in the terms of the genomic stability. In yeast S. cerevisiae, four genes encode RNR: 2 genes encode a large subunit (RNR1 and RNR3) and 2 genes encode a small subunit (RNR2 and RNR4). All 4 genes are DNA-damage inducible: transcription of RNR2, RNR3 and RNR4 is regulated via Mec1-Rad53-Dun1 pathway by targeting the transcriptional repressor Crt1 (Rfx1) for degradation; on the contrary, RNR1 gene promoter does not contain Crt1-binding sites and is not regulated through the Mec1-Rad53-Dun1 pathway. Instead, we show that intrastrand cross (X)-link recognition protein (Ixr1) is required for the proper transcription of the RNR1 gene and maintenance of the dNTP pools both during unperturbed cell cycle and after the DNA damage. Thus, we identify the novel regulator of the RNR1 transcription. Next, we show that the depletion of dNTP pools negatively affects genome stability in the hypomorphic mec1 mutants: the hyper-recombination phenotype in those mutants correlates with low dNTP levels. By introducing even lower dNTP levels the hyper-recombination increased even further and conversely all the hyper-recombination phenotypes were suppressed by artificial elevation of dNTP levels. In conclusion, we present Ixr1 as a novel regulator of the RNR activity and provide the evidence of role of dNTP concentration in the genome stability.
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Martos, Alexandre. "Relocalisation expérimentale de gènes mitochondriaux au noyau : un éclairage nouveau sur l'évolution du génome mitochondrial." Thesis, Bordeaux 2, 2012. http://www.theses.fr/2012BOR22003/document.
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Malgré la relocalisation au noyau d'une majorité des gènes du procaryote ancestral à l'origine des mitochondries, une poignée de gènes réside encore dans l'organite après près de deux milliards d'années d'évolution. Les raisons du maintien d'un génome mitochondrial sont mal comprises. Je me suis intéressé à cette problématique via des expériences de relocalisation artificielle de gènes mitochondriaux chez la levure Saccharomyces cerevisiae. Nous avons réussi, pour la première, à exprimer de manière fonctionnelle depuis le noyau le gène ATP9 qui encode une petite protéine hydrophobe essentielle au canal à protons de l'ATP synthase. Majoritairement mitochondrial chez les eucaryotes, comme S.cerevisiae, ce gène est retrouvé dans le génome nucléaire de la majorité des métazoaires, des algues vertes chlorophycées et des champignons filamenteux ascomycètes tel que Podospora anserina. Nos résultats montrent que l'hydrophobicité de la sous-unité Atp9p doit être diminuée pour qu'elle puisse être importée dans la mitochondrie depuis le cytosol. Nous avons également identifié un certain nombre d'autres adaptations pour optimiser l'expression du gène ATP9 relocalisé. Il apparaît donc que si le transfert du gène ATP9 est en principe possible chez la levure, il s'agit d'un processus très complexe. Une telle évolution n'a donc que peu de chances de se produire et d'être maintenue par la sélection naturelle, à moins que le transfert du gène ATP9 au noyau ne confère quelque avantage à l'organisme. Nous avons confirmé cette hypothèse par une étude menée chez P.anserina où nous avons montré que la relocalisation au noyau du gène ATP9, qui s'est produite naturellement au cours de l'évolution, a permis la mise en place de régulations spécifiques permettant d'ajuster les besoins en ATP synthase au cours du cycle de vie de ce champignon. Les résultats de cette étude nous amènent à introduire une nouvelle hypothèse selon laquelle les variations de contenu en gènes des génomes mitochondriaux ne sont pas influencées uniquement par des contraintes au niveau de la structure de leur produits, mais aussi par le mode de vie de l'organismeDespite the nuclear relocation of most genes of the ancestral procaryotic genome which gave birth to mitochondria, a small set of genes still remains into the organite after 2 billions years of evolution. The reasons for this maintenance of mitochondrial genome are currently not clear. I studied these questions by experimenting artificial relocations of mitochondrial genes in the yeast Saccharomyces cerevisiae. We managed, for the first, to functionally express the ATP9 gene from the nucleus, which encodes a small hydrophobic essential subunit of the proton chanel of the ATP synthase. Mostly mitochondrial within eukaryotes like S.cerevisiae, this gene can be found in the nuclear genome in most metazoans, chlorophyceans green algae and ascomycota filamentous fungi like Podospora anserina. Our results show that the hydrophobicity of the Atp9p subunit has to be decreased to be imported into the mitochondria from the cytosol. We also identified some adaptations optimizing the expression of the relocated ATP9 gene. It seems that if the ATP9 gene relocation is possible within the yeast, yet it is a complex and difficult process. Such an evolution has only few chances to occur and to be maintained by natural selection, unless it could confer some advantage to the organism. We have confirmed this hypothesis in a study made on P.anserina, in which we showed that the natural ATP9 relocation to the nucleus that appeared during its evolution allowed the setting up of specific regulations modulating the ATP synthase needs during the life-cycle of this fungus. The results presented here lead us to introduce a new hypothesis postulating that the variations of the set of genes contained in the mitochondrial genome are influenced not only by the constraints generated by their products structure, but also by the lifestyle of the organism
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Tran, Grant. "Modeling drug efficacy in the tumour microenvironment with Saccharomyces cerevisiae genome-wide screens in hypoxic conditions." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/60210.
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Hypoxia, the state of reduced oxygen, is a microenvironment found in many solid tumours and is correlated with an increased risk in patient mortality. This is due to an increase in resistance to radiotherapy and chemotherapy as well as a decrease in drug efficacy. The mechanisms and cellular factors (gene products) associated with this reduced chemotherapeutic efficacy in hypoxia remains unclear. This research looks to identify cellular processes and pathways that cancerous cells are able to exploit in order to survive and thrive in this microenvironment. The eukaryotic model baker’s yeast Saccharomyces cerevisiae combined with a genome-wide approach was used to screen the yeast knockout collection for specific genotypes that are sensitive to the hypoxic environment alone, and in combination with commonly used chemotherapeutics. Pathways and processes identified in these screens include transcriptional regulation, cytoskeleton maintenance, ribosomal biogenesis, macromolecular complex assembly and the heat shock response. The combination of heat and hypoxia was found to result in a synergistic effect that drastically affected cell fitness. DNA-damaging chemotherapeutics screened in hypoxic conditions showed reduced efficacy. Genotypes most sensitive to drugs in the hypoxic environment fall into Gene Ontology (GO) terms categorized in the response to the specific mechanism of the drug. This includes DNA repair processes such as homologous repair, post-replicative repair and mismatch repair. The mechanistic specificity uncovered in these screens suggests that the hypoxic environment exacerbates drug-specific stresses, and the identified genotypes highlight gene products and pathways critical for these responses. Cell survival and success in this microenvironment therefore requires adaptations to these exacerbated stresses, a phenomenon successfully accomplished by resistant tumour cells. This research contributes to our understanding of cellular biology under this cancer microenvironment, and provides data to highlight the challenges in using chemotherapeutics to treat tumours.Pharmaceutical Sciences, Faculty of
Graduate
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Schlecht, HeÌleÌ€ne. "Investigating the correlation between genome location, ectopic recombination and chromosome organisation during meiosis in Saccharomyces cerevisiae." Thesis, University of Sheffield, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398663.
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Harbison, Christopher T. 1973. "Genome-wide analysis of transcriptional expression programs, regulatory networks and Cis-regulatory sequences in Saccharomyces cerevisiae." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/28933.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.
Historically, knowledge of gene-specific transcription has been accumulated by the study of the individual genetic and physical interactions between transcriptional regulators and the genes they regulate, often requiring considerable time and effort. Microarray technology now enables investigation of gene expression at the level of the entire genome, allowing researchers access to rich datasets and promising new levels of depth in the understanding of transcriptional regulation. Our lab has made use of these technologies both to measure the levels of all mRNA transcripts within a population of cells, as well as to locate the regions within the genome that are bound by transcriptional regulators. Such studies not only allow for the functional annotation of both genes and regulators, but can also provide clues about the identity of the regulatory regions within DNA, the structure of global regulatory networks and the regulation of DNA-binding proteins. These and other insights are presented here based on our genome-wide studies of transcriptional regulation in the yeast Saccharomyces cerevisiae.
by Christopher T. Harbison.
Ph.D.
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Jones, Hope. "Genetic Characterization and Analysis of Cis and Trans-elements That Facilitate Genome Stability in Saccharomyces cerevisiae." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/193584.
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Chromosomal fragile sites are specific loci associated with a high frequency of breakage and recombination. A cell's ability to repair and/or replicate through a lesion is prerequisite to the maintenance of genomic stability. An improved understanding of fragile site biology and its contribution to replication defects and genomic instability is critical for prevention, intervention, and diagnosis of genetic diseases such as cancer. This work seeks to identify and characterize both trans and cis fragile sites associated elements involved in instability onset and progression. An array of Saccharomyces cerevisiae isogenic DNA repair deficient mutants were utilized to identify genes contributing to the stability or instability of a natural fragile site ~ 403 kb from the left telomere on chromosome VII. Findings suggest that the RAD52 epistasis group, the MRX complex, non-homologous end-joining (NHEJ) pathways, MUS81 and SGS1 helicases, translesion polymerases, and a majority of the post replication repair (PRR) proteins are all required for faithful replication of the 403 fragile site and likely other fragile sites as well. In contrast I found that MMS2, previously thought to be specific to the PRR pathway, is required to prevent the fusion of repetitive elements within the 403 site. mgs1 (homolog of the human Werner helicase interacting protein, WHIP) and pol3-13 (a subunit of the DNA polymerase delta) mutants also exhibited reduced instability in checkpoint deficient cells. These findings suggest previously uncharacterized function of Mgs1, Pol3 and Mms2 in regulation of genome regions at risk of replication damage. We further find the presence of inverted repeats (IR) are sufficient to induce instability. Two IR's proximal to the 403 site consistently fuse to generate acentric and dicentric chromosomes involving the 403 fragile site and a newly identified site on chromosome VII as well. The frequency of fusion events is aggravated by chromatin traffic stressors such as tRNA transcription induced fork stalling and replisome termination regions.
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Biteau, Nicolas. "Faisabilité du séquençage systématique d'un chromosome : stratégies et exploration du génome de Saccharomyces cerevisiae." Bordeaux 2, 1993. http://www.theses.fr/1993BOR28241.
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Coi, Anna Lisa. "A genome based approach to characterize genes involved in yeast adaptation to Sherry-like wines’ biological ageing." Thesis, Montpellier, SupAgro, 2014. http://www.theses.fr/2014NSAM0005/document.
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La fermentation œnologique et le vieillissement oxydatif des vins sous voile représentent des modes de vie très contrastés qui sont effectués par deux lignées différentes de souches de levures de l'espèce Saccharomyces cerevisiae. Dans cette thèse, nous avons comparé le génome de souches de levures de voile à celui de levures de vin afin de détecter leurs spécificités. Nous tout d'abord sélectionné 16 souches (8 levures de vin et 8 levures de voile) isolées en France, Hongrie, Italie et Espagne, pour séquencer leur génome sur une plateforme Illumina (HiSeq2000). Nous avons également développé un ensemble de souches de vin et de voile haploïdes pour l'évaluation moléculaire de différentes cibles. Nous avons également mis au point un milieu synthétique mimant le vin à cette fin. A partir de la comparaison des séquences du génome nous avons établi une phylogénie qui montre que les levures de voile représentent un groupe spécifique de levure, différentes des levures de vin, puis à partir de différentes méthodes (analyse en composantes principale, diversité nucléotidique et D de Tajima) nous avons identifié des régions divergentes. Ces régions variantes comprennent des gènes remplissant plusieurs fonctions clé associées à la croissance en voile. En particulier, des variations alléliques ont été rencontrées chez les levures de voile pour plusieurs gènes impliqués dans la régulation de l'expression de FLO11 tels que les voies MAP kinase, ou des voies Ras/cAMP/PKA, ainsi que pour plusieurs gènes impliqués dans l'homéostasie des cations divalents de métaux de transition tels que le zinc, le cuivre ou le fer. La comparaison des transcriptomes d'une levure de voile et d'une levure de vin sur notre milieu synthétique a révélé des différences d'expression pour les floculines (FLO1, 5, 8, 11) ainsi que pour le transport des hexoses, mais a également suggéré que la levure de voile P3-D5 était en situation de carence en zinc et en inositol par rapport à la levure de vin, tandis que la levure de vin K1 exprimait certains gènes suggérant des défauts mitochondriaux. L'impact de la variation allélique de plusieurs gènes a été évalué dans le phénotype de voile: le transporteur de zinc à haute affinité Zrt1 ainsi que la pyruvate décarboxylase majeure Pdc1Wine fermentation and flor ageing are performed by two groups of the yeast Saccharomyces cerevisiae, with very different lifestyles. In this thesis we have studied the genome of flor yeast in comparison to wine yeast in order to unravel their specificities. We have first selected 16 strains (8 wine and 8 flor) from France, Hungary, Italy and Spain in order to sequence their genome sequence on an Illumina HiSeq2000 platform. Three flor strains and two wine strains were haploidized in order to obtain a set of haploid flor strains for the molecular evaluation of different targets. We developed as well a synthetic media mimicking wine for that purpose. From the genome sequence we have drawn a phylogeny that showed that flor yeasts represent a specific lineage of yeast, different from the wine strains lineage, and identified divergent regions. These regions contain genes involved in key functions and several associated with velum growth. Remarkably, many genes involved in FLO11 regulation such as MAP kinase, or Ras/PKA pathways were mutated among flor strains and many variations were encountered in genes involved in metal homeostasis such as zinc and divalent metal transporters. A transcriptome analysis comparing one flor and one wine yeast on our wine synthetic media revealed expression differences associated to floculins and hexose transport, but also suggested that flor yeast P3-D5 face a zinc and inositol deficiency, whereas wine yeast K1 presented mitochondrial defects. The impact of allelic variation of several genes coding for the high affinity zinc transporter (ZRT1), and the major pyruvate decarboxylase (PDC1) has been evaluated in order to assess their role in the flor phenotype
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Mischo, Hannah. "Disengaging Polymerases : Transcriptional termination by RNA polymerase II in Saccharomyces cerevisiae and the maintenance of genome integrity." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.514968.
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Colby, E. R. "Creating site-specific abasic sites in the genome of Saccharomyces cerevisiae to analyse replication-associated lesion bypass." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1419675/.
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Abasic sites are thought to be one of the most frequently formed lesions within cells. They are particularly dangerous during DNA replication as they can block the progression of replication forks. Such stalled forks have the potential to collapse, which can impact genome stability and therefore cell survival. To complete replication in the presence of abasic sites, cells use DNA damage tolerance pathways that can bypass abasic sites without their repair. The in vivo study of DNA damage tolerance is complicated by the temporal and spatial nature of naturally occurring damage. To understand the molecular details of these pathways, it is useful to have systems that can recapitulate events ideally at single replication fork resolution. Plasmids harbouring damage site-specifically have been introduced into cells, allowing the study of the genetic control and mutagenic bypass at these sites. However it is uncertain if events observed fully reflect those occurring in the context of chromatin. I have developed a system where abasic sites can be formed site-specifically at a known location in the Saccharomyces cerevisiae genome. By creating these lesions during the G1-phase of the cell cycle, upon release into S-phase, the response of the DNA replication fork can be analysed. Their formation has been characterised with respect to their site-specific targeting, distribution, effects on replication and activation of the DNA damage checkpoint. This system can now be used to analyse the damage bypass response by looking at the recruitment of proteins to the fork by chromatin immuno-precipitation and live-cell fluorescence microscopy. Additionally, replication forks undergoing abasic site bypass can sbe cross-linked to identify novel factors involved. These techniques will provide further insight into DNA damage tolerance events occurring at abasic sites.
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Wu, Jingyan. "A Genome-wide Analysis to Identify and Characterize Novel Genes Involved in tRNA Biology in Saccharomyces cerevisiae." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1429197786.
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Tuck, Alex Charles. "Genome-wide identification of non-canonical targets of messenger RNA synthesis and turnover factors in Saccharomyces cerevisiae." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11719.
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Pervasive transcription is widespread amongst eukaryotic genomes, and produces long noncoding RNAs (lncRNAs) in addition to classically annotated transcripts such as messenger RNAs (mRNAs). LncRNAs are heterogeneous in length and map to intergenic regions or overlap with annotated genes. Analogous to mRNAs, lncRNAs are transcribed by RNA polymerase II, regulated by common transcription factors, and possess 5’ caps and perhaps 3’ poly(A) tails. However, lncRNAs perform distinct functions, acting as scaffolds for ribonucleoprotein complexes or directing proteins to nucleic acid targets. The act of transcribing a lncRNA can also affect the local chromatin environment. Furthermore, whereas mRNAs are predominantly turned over in the cytoplasm, both nuclear and cytoplasmic pathways reportedly participate in lncRNA degradation. In this study, I address the question of when and how lncRNAs and mRNAs are distinguished in the cell. Messenger RNAs interact with a defined series of protein factors governing their production, processing and decay, and I hypothesised that lncRNAs might be similarly regulated. I therefore sought to determine which mRNA-binding proteins, if any, also bind lncRNAs. I reasoned that this would reveal the point at which lncRNAs and mRNAs diverge, and how differences in their biogenesis and turnover equip them for different roles. I selected factors from key stages of mRNA metabolism in Saccharomyces cerevisiae, and identified their transcriptome-wide targets using CRAC (crosslinking and analysis of cDNAs). CRAC can detect interactions with low abundance transcripts under physiological conditions, and reveal where within each transcript a protein is bound. Analyses of binding sites in mature mRNAs and intron-containing pre-mRNAs revealed the order in which the tested factors interact with mRNAs, and which region they bind. The poly(A)-binding protein Nab2 bound throughout mRNAs, consistent with an architectural role, whereas the cytoplasmic decay factors Xrn1 and Ski2 bound to poly(A) tails, which might act as hubs to coordinate turnover. The RNA packaging factors Tho2 and Gbp2, and nuclear surveillance factors Mtr4 and Trf4 bound abundantly to intron-containing premRNAs, indicating that they act during or shortly after transcription. The tested factors bound lncRNAs to various extents. LncRNA binding was most abundant for Mtr4 and Trf4, moderate for Tho2, Gbp2, the cap binding complex component Sto1, and the 3’ end processing factors Nab2, Hrp1 and Pab1, and lowest for Xrn1, Ski2 and the export receptor Mex67. This suggests that early events in lncRNA and mRNA biogenesis are similar, but unlike mRNAs, most lncRNAs are retained and degraded in the nucleus. Analyses of two documented classes of lncRNA, cryptic unstable transcripts (CUTs) and stable unannotated transcripts (SUTs), revealed some differences. SUTs were most similar to mRNAs, with canonical cleavage and polyadenylation signals flanking their 3’ ends, and poly(A) tails bound by the poly(A)-binding protein Pab1. CUTs lacked these characteristics, and in comparison to SUTs bound more abundantly to Mtr4 and Trf4 and less so to Ski2, Xrn1 and Mex67. Furthermore, CUTs accumulated upon Hrp1 depletion, suggesting that Hrp1 functions non-canonically to promote CUT turnover. Mtr4, Trf4 and Nab2 also bound abundantly to promoter-proximal RNA fragments generated from ~1000 protein coding genes. These fragments possessed short oligo(A) tails (hallmarks of nuclear surveillance substrates), were not bound to cytoplasmic factors, and apparently correspond to a population of ~150-200 nt promoter-proximal lncRNAs. Notably, CRAC analyses of Mtr4 and Sto1 targets in yeast subjected to a media shift revealed widespread changes in the abundance and surveillance of mRNAs, promoter-proximal transcripts and CUTs, which at many loci were arranged in a complex transcriptional architecture. Overall, the transcriptome-wide binding analyses presented here reveal that lncRNAs diverge from mRNAs prior to export, and are predominantly retained in the nucleus. Transcript fate is apparently determined during 3’ end processing, with CUTs diverging from mRNAs early in transcription via a distinct termination pathway coupled to rapid turnover, and SUTs diverging during or shortly after cleavage and polyadenylation, making them more stable and perhaps prone to escape to the cytoplasm. Promoter-proximal transcripts might arise from termination associated with an early checkpoint in Pol II transcription. The diverse behaviours of lncRNAs arise from their association with distinct subsets of RNA binding proteins, some of which perform different roles when bound to different types of transcript. In conclusion, my results provide the foundation for a mechanistic understanding of how distinct classes of non-coding Pol II transcripts are produced, and how they can perform diverse functions throughout the nucleus.
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Treu, Laura. "A genomic and transcriptomic approach to characterize oenological Saccharomyces cerevisiae strains." Doctoral thesis, Università degli studi di Padova, 2012. http://hdl.handle.net/11577/3422965.
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Genus Saccharomyces includes a large number of microorganisms that are important for industrial applications such as the production of fermented beverages, biofuel and baking. Natural selection combined with domestication applied selective pressures to the genome of this yeast producing large numbers of different strains with specialized phenotypes.
During the last decades thousand of strains have been phenotypically characterized but correlation between phenotype and genotype is not yet completely unveiled. Genome sequence analysis is a crucial step to obtain a general description of gene content and highlight differences between strains. In this study the homozygous derivatives of four ecotypical Saccharomyces cerevisiae strains isolated from Raboso and Prosecco fermented grape bunch have been successfully sequenced using next generation sequencing, and a variety of tools have been used and developed to solve the complex task of genome finishing.
A detailed overview of gene expression in different winemaking and laboratory strains has also been performed using SOLiD RNA-seq. Samples growth in synthetic wine media on controlled bioreactors have been collected during fermentation process. Our results revealed a transcriptional fingerprint characterizing oenological strains adaptation to stressful environment. A comparison between differences in promoter sequences between strains and their downstream effect on gene expression have been performed and the results show a higher influence of tandem repeat variability respect to mutations on transcription factor binding sites. Finally using statistical analysis we correlate the genetic traits of strains with their metabolic properties and we obtained a global overview of fermentation performances in the different genetic groups.Il genere Saccharomyces comprende un gran numero di microrganismi di interesse tecnologico, utilizzati ad esempio per la produzione di bevande fermentate, biocarburanti e per la panificazione. La selezione naturale unita alla domesticazione ha determinato una pressione selettiva che ha modificato il genoma di questi lieviti producendo un ampio numero di ceppi diversi con fenotipi specializzati. Negli ultimi anni centinaia di ceppi sono stati caratterizzati dal punto di vista fenotipico ma una correlazione tra il fenotipo e il genotipo non è stata ancora completamente chiarita. L’analisi del sequenziamento genomico è un passo cruciale per ottenere una descrizione globale del contenuto genico e per evidenziare le differenze tra i ceppi. In questo studio sono stati sequenziati i genomi degli omozigoti derivati da quattro ceppi ecotipici di S. cerevisiae isolati da grappoli fermentati di Prosecco e Raboso Piave utilizzando sequenziatori di Nuova Generazione. Numerosi strumenti informatici sono stati utilizzati e sviluppati per adempire al complesso compito del finishing. Inoltre una dettagliata panoramica dell’espressione genica in 5 ceppi di vinificazione e 1 di laboratorio è stata effettuata utilizzando la tecnica RNA-seq con la metodologia SOLiD. I lieviti sono stati cresciuti in mosto sintetico in bioreattori controllati e dei campioni sono stati prelevati durante il processo fermentativo. I risultati hanno rivelato un profilo trascrizionale caratteristico dell’adattamento dei ceppi enologici allo stress dell’ambiente di vinificazione. Un confronto tra le differenze nelle sequenze promotoriali tra i ceppi e il successivo effetto a catena sull’espressione genica è stato considerato e i risultati evidenziano una maggior influenza della variabilità delle tandem repeat rispetto alle mutazioni sui siti di binding dei fattori di trascrizione. Infine utilizzando dei modelli statistici siamo riusciti a correlare le caratteristiche genetiche dei ceppi con le loro proprietà metaboliche e ad avere una visione globale dell’abilità di fermentazione dei diversi ceppi.
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Peter, Jackson. "Dissection de la relation génotype-phénotype par des études d'association chez Saccharomyces cerevisiae." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAJ064/document.
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Un objectif central en biologie est de comprendre la relation entre le génotype et le phénotype. Afin de disséquer les bases génétiques de la diversité phénotypique, il est nécessaire de disposer d’une collection de données génomiques d’un grand nombre d’individus d’une même espèce. Dans ce but, mes travaux de thèse se basent sur l’étude des séquences génomiques ainsi que des données phénotypiques de 1011 isolats naturels de la levure Saccharomyces cerevisiae. Dans un premier temps, je me suis intéressé à la description de la variation génétique et phénotypique pour dresser un portrait précis de l’histoire évolutive de cette espèce. Les données de phénotypage nous ont permis de réaliser des études pangénomiques d’association génotype-phénotype avec une puissance jusque là inégalée chez Saccharomyces cerevisiae. Je me suis par la suite penché sur l’évaluation des paramètres influençant le pouvoir de détection d’une telle approche, d’en apprécier limites pour tenter de les contournerElucidating the genetic origin of phenotypic diversity among individuals within the same species is essential to understand evolution. Using whole genome sequences of 1,011 Saccharomyces cerevisiae isolates, my work sought to describe intraspecific genetic variation and investigate of its phenotypic consequences. Doing so, I obtained a precise view of the evolutionary history of S. cerevisiae. Phenotypic characterization provided the opportunity to perform genotype-phenotype genome-wide association studies with unprecedented power. I then focused on the evaluation of the parameters influencing genome-wide association studies, the appreciation of the limits of such an approach, and ways to circumvent them
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TIAN, GUO-LIANG. "Recherches sur la structure et l'organisation du genome mitochondrial de la levure saccharomyces douglasii et d'un genome mitochondrial chimerique s. Douglasii et s. Cerevisiae." Paris 6, 1993. http://www.theses.fr/1993PA066260.
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Nous avons etabli la carte physique et genetique du genome mitochondrial de la levure saccharomyces douglasii. La capacite codante de ce genome est tres semblable a celle du genome mitochondrial de s. Cerevisiae. Les sequences nucleotidiques des genes coxi, aap1, oli2 et cytb ont ete etablies. La structure des genes morceles (coxi et cytb) est differente par rapport a ceux correspondants chez s. Cerevisiae (notamment la presence des nouveaux introns). La comparaison structurale des genomes mitochondriaux de s. Douglasii et s. Cerevisiae a revele la translocation d'un segment genomique d'environ 15 kb modifiant l'ordre des genes. La comparaison d'un point de vue evolutif de ces deux genomes nous a permis de mettre en evidence des regles nouvelles qui gouvernent la fixation des mutations exoniques et introniques. L'etude d'un recombinant yl4 fait deuxieme partie de cette these. Ce recombinant possede le genome nucleaire sauvage de s. Cerevisiae et le genome mitochondrial chimerique s. Douglasii et s. Cerevisiae. L'etude du phenomene concernant la restauration de la compatibilite nucleo-mitochondriale observe chez cette souche nous a permis de mettre en evidence d'une activite maturasique trans-specifique de la proteine codee par l'intron ai5b du gene coxi de s. Cerevisiae
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Schulze, Julia Maria. "Genome-wide analysis of chromatin modification patterns and their functional associations with major cellular processes in Saccharomyces cerevisiae." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/29154.
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Chromatin is a nucleoprotein complex packaging DNA inside the cell nucleus and is of crucial relevance for genome regulation. Its structure is highly dynamic undergoing post-translational modifications, replacement by histone variants, and ATP-dependent remodelling. My dissertation aims to better understand the regulation of chromatin by studying the structure and function of a chromatin modifier, mapping chromatin modifications at a genome-wide scale, and linking modification patterns to cellular functions in the model organism Saccharomyces cerevisiae. Multiple chromatin modifying and transcription complexes contain a YEATS domain, and their misregulation has been implicated in the development of cancer. This study recognizes the evolutionary conservation of the YEATS domain from yeast to human, presents its structural composition as well as its function in depositing the histone variant H2A.Z. Besides histone variants, histone modifications determine chromatin structure and often co-occur in certain genomic regions. Histone H3 lysine 79 methylation is one such modification that adds an additional level of complexity being either mono-, di- or trimethylated (H3K79me1, me2, me3). In this work, I show that these methylation states are functionally not redundant as previously proposed, and that H3K79 di- and trimethylation are associated with different regions of the genome. In contrast to H3K79me3, H3K79me2 marks M/G1 cell cycle regulated genes and its levels change during the cell cycle. The trigger for trimethylation of H3K79, as well as for trimethylation of lysine 4 on histone H3 (H3K4me3), is monoubiquitination of histone H2B (H2BK123ub). The map of H2BK123ub that I present herein demonstrates its role as upstream regulator for H3K79me3 and H3K4me3 on a genome-wide scale. Removal of the transient H2BK123ub mark is facilitated by the deubiquitinases Ubp8 and Ubp10. I herein reveal that they mainly act at distinct genomic loci. While Ubp8 removes H2BK123ub at sites enriched for H3K4me3, Ubp10 functions at those marked by H3K79me3. Finally, my thesis describes chromatin signatures of multiple modifications, and finds the combination of H2BK123ub, H3K4me3, and H3K36me3 to be specific for highly transcribed genes, including those containing introns. In this context, I show evidence that link the histone H2B ubiquitin ligase Bre1 to mRNA splicing.
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Chu, Hui-Yi. "Genome-wide Investigation of Cellular Functions for tRNA Nucleus-Cytoplasm Trafficking in the Yeast Saccharomyces cerevisiae." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1343397048.
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Jain, Shveta. "Genome-wide analysis of kinases and phosphatases reveal an essential MAP kinase involved in pexophagy in Saccharomyces cerevisiae." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC IP addresses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1453663.
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Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed July 28, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references.
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Dauban, Lise. "Organisation du génome par le complexe cohésine chez la levure Saccharomyces cerevisiae." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30100.
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La cohésine est un complexe protéique conservé dans l'évolution composé d'un anneau capable d'embrasser l'ADN et de protéines auxiliaires régulant son association avec l'ADN. D'une part, la cohésine confère la cohésion des chromatides sœurs nécessaire à leur ségrégation, d'autre part elle établit et maintient des boucles de chromatine. Ces boucles sont requises pour la formation de domaines topologiques, l'expression génique et la stabilité du génome. Cependant les mécanismes régissant leur formation ne sont pas entièrement élucidés. Selon le modèle d'extrusion de boucles, la cohésine capturerait des boucles de petites tailles et les élargirait en extrudant l'ADN à travers son anneau. Dans ce modèle, la taille des boucles dépendrait à la fois du temps de résidence des cohésines sur l'ADN et de leur processivité. Étudier la régulation des cohésines est donc fondamental pour comprendre la biologie des chromosomes. Dans cette étude nous avons montré que les bras des chromosomes mitotiques de la levure Saccharomyces cerevisiae étaient organisés sous forme de boucles de chromatine dépendantes des cohésines. Nous avons étudié le rôle des sous-unités régulatrices des cohésines, Pds5, Wpl1 et Eco1 dans la formation de ces boucles. Nos données montrent que Pds5 inhibe leur expansion, via Wpl1 et Eco1. Comme décrit chez les mammifères, Wpl1 les abolit en dissociant les cohésines des chromosomes. En revanche, nos résultats suggèrent qu'Eco1 entraverait la translocation des cohésines sur l'ADN, nécessaire pour l'agrandissement des boucles. Nous avons ensuite analysé le rôle de ces protéines dans l'organisation de l'ADN ribosomique (ADNr), séquence enrichie en cohésines, hautement transcrite et isolée du reste du génome. Pds5 semble avoir un rôle central dans l'organisation de cette séquence, qui ne dépendrait pas de Wpl1 ou d'Eco1. Afin d'analyser de manière fine les réorganisations spatiales de l'ADNr, nous avons développé une analyse d'image dédiée permettant de sonder l'organisation de cette fibre en trois dimensions. Nous avons révélé une structure sous-jacente de l'ADNr composée d'une succession de domaines organisés spatialement par les cohésines. Cette étude ouvre des perspectives vers une meilleure compréhension de la régulation des cohésines dans l'organisation du génomeCohesin is an evolutionary-conserved complex composed of a ring capable of DNA entrapment and of auxiliary proteins regulating its association with DNA. On the one hand, cohesin confers sister chromatid cohesion required for their proper segregation and on the other hand it establishes and maintains chromatin looping. Chromatin loops are crucial for assembly of topological domains, gene expression and genome stability. However, mechanisms driving their establishment remain to be elucidated. According to loop extrusion model, cohesin would capture small loops and enlarge them by extruding DNA throughout its ring. This model predicts that loop size would depend on both cohesin residence time on DNA and on its processivity. Deciphering cohesin regulation is thus fundamental to understand chromosome biology. In this study, we showed that mitotic chromosome arms of yeast Saccharomyces cerevisiae are organised in cohesin-dependent chromatin loops. We studied the role of cohesin regulatory subunits Pds5, Wpl1 and Eco1 on loop establishment. Our data show that Pds5 inhibits loop expansion via Wpl1 and Eco1. As previously described in mammals, Wpl1 counteracts loop expansion by dissociating cohesin from DNA. Our results suggest that Eco1 would inhibit cohesin translocation on DNA, required for loop expansion. We then studied how these proteins contribute to the organisation of the ribosomal DNA array (rDNA), a cohesin-rich, highly transcribed sequence segregated away from the rest of the genome. Our data point toward a central role for Pds5 in organising this genomic region, independently of Wpl1 and Eco1. To study in detail rDNA spatial organisation, we developed a dedicated image analysis to assess its organisation in three dimensions. We have unveiled an underlying organisation for rDNA, made by a succession of small domains spatially organised by cohesin. This study opens large perspectives towards a better understanding of cohesin regulation in genome organisation
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Lee, Ming-Ta. "Analysis of genome stability in mutants defective for the SUMO isopeptidase Smt4/Ulp2 /." Diss., UC access only, 2009. http://proquest.umi.com/pqdweb?index=6&did=1907279801&SrchMode=2&sid=2&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1270053784&clientId=48051.
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Thesis (Ph. D.)--University of California, Riverside, 2009.Includes abstract. Includes bibliographical references (leaves 213-243). Issued in print and online. Available via ProQuest Digital Dissertations.
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Humphryes, Neil. "Global genome nucleotide excision repair factors and the ubiquitin-proteasome system regulate the DNA damage response in Saccharomyces cerevisiae." Thesis, Cardiff University, 2010. http://orca.cf.ac.uk/55468/.
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Delahodde, Agnès. "Activites arn-maturasique et adn-endonucleasique de deux proteines introniques homologues du genome mitochondrial de la levure saccharomyces cerevisiae." Paris 6, 1988. http://www.theses.fr/1988PA066185.
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Dans les mitochondries de levure saccharomyces cerevisiae, l'intron bi4 du gene codant pour le cytochrome b et l'intron ai4 du gene codant pour la sousunite i de la cytochrome oxydase possedent une phase de lecture ouverte qui peut-etre traduite en deux proteines homologues. Adaptation des codons uga, ava et cun de ces phases de lecture au code genetique universel de maniere a etudier leur fonction apres traduction dans le cytoplasme de levure ou dans escherichia coli. Chez la levure, les proteines traduites dans le cytoplasme penetrent dans la mitochondrie en associant le cote n terminal des presequences d'adressage nitochondriale. La proteine p27bi4 est necessaire pour l'excision des introns bi4 et ai4 alors que la proteine p28ai4 est toxique pour e. Coli. , peut couper specifiquement l'adn double brin a la sequence correspondant a la fusion de deux exons
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Louvet, Olivier. "Analyse fonctionnelle de l'ORF YBR264c identifié lors du séquençage systématique du génome de la levure Saccharomyces Cerevisiae." Bordeaux 2, 1998. http://www.theses.fr/1998BOR28557.
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D'Angiolo, Melania Jennifer. "Étude des mécanismes moléculaires de l'évolution du génome chez la levure bourgeonnante Saccharomyces cerevisiae." Electronic Thesis or Diss., Université Côte d'Azur, 2021. http://theses.univ-cotedazur.fr/2021COAZ6001.
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L'évolution du génome consiste en la modification progressive de ce dernier au fil du temps et résulte de la variation des gènes dans leur ensemble, des mutations, des recombinaisons et échanges génétiques entre populations. L'essor des technologies de séquençage de nouvelle génération ainsi que la réduction de leur coût ont permis d’augmenter le nombre de génomes disponibles, permettant d’élucider les mécanismes moléculaires impliqués dans leur évolution. Dans ce travail, j'ai utilisé la levure bourgeonnante Saccharomyces cerevisiae pour étudier deux aspects fondamentaux de l'évolution du génome: l'origine des introgressions inter-espèces et la diversité des télomères.Une introgression est une insertion de matériel génétique provenant d’une population dans une autre. Ce phénomène naît d'événements d'hybridation suivis de rétrocroisements répétés avec l'une des populations parentales. Dans la première partie de ma thèse, j'ai étudié une lignée de S. cerevisiae isolée à partir des eaux usées de la production d'huile d'olive (Alpechin), qui comprend d'abondantes introgressions de l'espèce sœur S. paradoxus, ainsi qu’une souche hybride S. cerevisiae/S. paradoxus caractérisée par des nombreuses régions de perte d'hétérozygotie (LOH). J'ai établi une carte génétique détaillée des LOHs dans la souche hybride et comparé leur position aux introgressions dans les souches d'Alpechin pour déterminer leurs relations. J'ai constaté que LOHs et introgressions se chevauchaient et provenaient de l’ascendance de S. paradoxus, indiquant que les introgressions dans la lignée d’Alpechin découlent directement des LOHs. J'ai proposé un modèle pour expliquer l'origine des introgressions chez la levure selon lequel les LOHs permettent aux hybrides inter-espèces de surmonter leur stérilité et j’ai validé la fiabilité de ce postulat à l'aide d’approches expérimentales et informatiques.Dans la deuxième partie de ma thèse, j'ai caractérisé la diversité des télomères chez S. cerevisiae et l’effet d’un stress télomérique sur le fitness cellulaire. Premièrement, j'ai estimé la longueur des télomères dans plus de 900 souches isolées à travers le monde et constaté une vaste hétérogénéité, bien que les souches issues d’habitats naturels présentent des télomères plus courts que celles issues de l’agroalimentaire. J'ai ensuite réalisé une étude d'association pangénomique qui a permis d’identifier des variants génétiques susceptibles de moduler la longueur des télomères. De plus, j'ai identifié des mutations délétères dans des gènes connus pour influencer la longueur des télomères. J'ai aussi utilisé un ensemble de données phénotypiques pour déterminer si certains facteurs non génétiques sont associés à la variation de longueur des télomères, et j’ai ainsi pu constater une connexion entre le métabolisme mitochondrial et les télomères dans les souches naturelles.Deuxièmement, j'ai étudié l’effet d’un stress télomérique chronique chez des levures modifiées avec des répétitions télomériques humaines. J'ai fait évoluer ces levures «humanisées» sur plusieurs générations par le biais de lignées cellulaires non-sélectives et j’ai observé un ralentissement de la vitesse de division et de la longévité, parallèlement à une augmentation du taux de mutations. Enfin, j'ai procédé à une expérience d’évolution adaptative pour permettre l’émergence de mutations bénéfiques qui contrecarrent le déclin de fitness des levures «humanisées». Après évolution, la plupart des lignées ont retrouvé leurs caractéristiques originelles grâce à l'apparition de mutations spécifiques en lien avec la réponse aux dommages de l'ADN.Dans l'ensemble, mes travaux ont permis d’établir une nouvelle hypothèse expliquant l’origine des introgressions chez les espèces reproductivement isolées et de même ont permis de caractériser la diversité et l’instabilité des télomères à une échelle sans précédent, contribuant à l’élucidation des mécanismes moléculaires impliqués dans l’évolution des génomesGenomes are progressively modified during their evolution leading to gene content variation, recombination, mutation and genetic exchange among species/subpopulations. The advent of next-generation sequencing technologies and their cost reduction increased the number of genomes available for evolutionary studies, opening the way to understand the molecular mechanisms involved in genome evolution. In this work, I used the budding yeast Saccharomyces cerevisiae as model organism to investigate two important aspects of genome evolution: the origin of interspecies introgressions and telomere evolution.An introgression is the flow of genetic material between populations and it results from ancient hybridization events followed by repeated backcrossings with one of the parental populations. In the first part of my PhD, I studied a lineage of S. cerevisiae strains isolated from the wastewater of olive oil production (Alpechin), carrying abundant introgressions from the sister species S. paradoxus, and a natural S. cerevisiae/S. paradoxus hybrid, with 50% genome contribution from each parent, carrying abundant regions of loss-of-heterozygosity (LOH). I derived an accurate genetic map of LOHs in the hybrid and compared their position to the introgressions in the Alpechin strains, to infer their evolutionary relations. I found that LOH and introgressions overlapped and shared the same S. paradoxus ancestry, indicating that LOHs are the direct origins of introgressions in the Alpechin lineage. I proposed a model for the origin of yeast introgressions in which LOH regions allow interspecies hybrids to overcome sterility, which constitutes the main barrier to introgressions' onset in reproductively isolated species, such as yeasts, and validated the reliability of my model using experimental and computational techniques.In the second part of my PhD, I studied the extent of telomere diversity in S. cerevisiae and the outcome of chronic telomeric stress on cellular fitness. In a first project, I estimated telomere length in over 900 strains isolated around the world and observed remarkable variation. Strains isolated in wild habitats had shorter telomeres than domesticated ones. I performed a genome-wide association study that revealed novel genetic variants possibly regulating telomere length. I also pinpointed private loss-of-function mutations in known telomere length maintenance genes that could explain the very long/short telomeres of certain lineages. Moreover, I used multiple phenotypic datasets available for this collection to look for non-genetic factors associated to telomere length variation, and discovered an association between mitochondrial metabolism and telomeres in wild strains.In a second project, I performed experimental evolution of engineered yeasts synthetizing human telomeric DNA repeats at their chromosome-ends. I evolved telomere-humanized strains through mutation accumulation lines (MALs) to minimize selection, and I characterized the detrimental effects caused by telomeres' reshaping. During MALs, humanized yeasts gradually slowed their growth, shortened chronological lifespan and had higher mutation rate and genome instability. Next, I submitted MALs to adaptive evolution by multiple serial transfers (STs) of large population sizes, to map mutations that counteract their fitness decline. After multiple STs, most humanized lines recovered fitness thanks to the independent occurrence of mutations in the DNA-damage response pathway. Overall, my work contributed to elucidate the molecular mechanisms driving genome evolution, by providing a plausible model for introgression evolution in reproductively isolated species and by giving an unprecedented overview of the impact of the variation of telomere DNA length and sequence on global organismal fitness
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Mendez, Jamie Elizabeth. "Investigation of Hsf1 Interacting Partners via a Genome-wide Yeast Two-hybrid Screen." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4543.
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Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.