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The Saccharomyces cerevisiae HtrA orthologue, Ynm3, is a chaperone-protease that aids survival under heat stress / Das Saccharomyces cerevisiae HtrA Ortholog, Ynm3, ist eine Chaperon-Protease, die für das Überleben unter Hitzestress verantwortlich istPadmanabhan, Nirmala 03 November 2008 (has links)
No description available.
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Metabolic and developmental functions of the transcription factor Gcn4p of Saccharomyces cerevisiae / Die metabolischen und Entwicklungsfunktionen des Transkriptionsfaktors Gcn4p von Saccharomyces cerevisiaeHerzog, Britta 27 October 2010 (has links)
No description available.
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Regulation of Flo11p-dependent adhesion in <i>Saccharomyces cerevisiae</i> / Regulation der Flo11p-abhängigen Adhäsion in <i>Saccharomyces cerevisiae</i>Fischer, Claudia 02 November 2005 (has links)
FLO11 is coding for a cell surface adhesin in the baker s yeast Saccharomyces cerevisiae. Its expression is regulated by different environmental circumstances like glucose, nitrogen or amino acid limitation. Flo11p is strictly required to allow cells to react on these nutrient signals by a dimorphic switch from single growing yeast cells to multicellular complexes with adhesive phenotype. This work demonstrates that under repressed conditions the unusually large FLO11 promoter of about 3 kb contains only one MNase-sensitive site located 1.2 kb upstream of the open reading frame. This site correlates with the binding region for the repressor protein Sfl1p. Investigations with genes for components involved in chromatin establishment, maintenance or remodeling identified the histone variant H2A.Z/Htz1p as yet unknown factor that is required to keep FLO11 in a silent state. The chromatin remodeler Rsc1p and the histone acetyl transferase Gcn5p are antagonists to H2A.Z/Htz1p and are required to overcome this silent state under glucose depletion, and therefore, to switch to the adhesive growth mode or pseudohyphal development. Addition of the histidine analogue 3-aminotriazol results in amino acid starvation and restores Flo11p-dependent adhesion in rsc1 mutant cells. These cells express only low FLO11 mRNA levels suggesting that there m! ight be additional mechanisms which result in sufficient amounts of adhesin molecules. These mechanisms might be regulated on a post-transcriptional level. A possible post-transcriptional level of controlling FLO11 expression was addressed by analysing two isogenic ribosomal proteins, namely Rps26Ap and Rps26Bp. Both proteins are compounds of the small subunit of the ribosome and are involved in regulating FLO11 expression. Only Rps26Ap is an essential factor for efficient FLO11 mRNA translation. Investigations concerning the regulation of the two isogenes demonstrate a reciprocal effect on the translational level. Rps26Ap stimulates the translation of RPS26B mRNA into the protein, whereas formation of Rps26Ap is inhibited by Rps26Bp.
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Genetically Tailored Yeast Strains for Cell-based Biosensors in White BiotechnologyGroß, Annett 23 January 2012 (has links)
This work was performed in the framework of two application-oriented research projects that focus on the generation and evaluation of fluorescent Saccharomyces (S.) cerevisiae-based sensor and reporter cells for white biotechnology as well as the extension of the conventional single-cell/single-construct principle of ordinary yeast biosensor approaches. Numerous products are currently generated by biotechnological processes which require continuous and precise process control and monitoring. These demands are only partially met by physical or physiochemical sensors since they measure parameters off-line or use surrogate parameters that consequently provide only indirect information about the actual process performance. Biosensors, in particular whole cell-based biosensors, have the unique potential to near-line and long-term monitor parameters such as nutrient availability during fermentation processes. Moreover, they allow for the assessment of an analyte’s biological relevance.
Prototype yeast sensor and reporter strains derived from common laboratory strains were transformed with multicopy expression plasmids that mediate constitutive or inducible expression of a fluorescence reporter gene. Performance of these cells was examined by various qualitative and quantitative detection methods – representative of putative transducer technologies. Analyses were performed on the population level by microplate reader-based fluorometry and Western blot as well as on the single-cell level by fluorescence microscopy and flow cytometry. ‘Signature’ promoters that are activated or repressed during particular nutrient-limited growth conditions were selected in order to generate yeast nutrient sensor strains for monitoring the biological availability of nitrogen, phosphorus or sulphur. For each category, at least one promoter mediating at least threefold changed green fluorescence levels between sensor cells in non-limited and nutrient-limited conditions was identified. Sensor strains were evaluated in detail regarding sensitivity, analyte selectivity and the ability to restore basic fluorescence after shift from nutrient-limited to non-limited conditions (regeneration). The applicability for bioprocess monitoring purposes was tested by growth of yeast nutrient sensor cells in microalgae media and supernatants. Despite successful proof of principle, numerous challenges still need to be solved to realise prospective implementation in this field of white biotechnology.
The major drawback of plasmid-borne detection constructs is a high fluorescence variance between individual cells. By generation of a nitrogen sensor strain with a genome-integrated detection construct, uniform expression on the single-cell level and simultaneous maintenance of basic properties (ability of fluorescence induction/regeneration and lack of cross-reactivity) was achieved. However, due to the singular detection construct per cell, significantly weaker overall fluorescence was observed. The traditional single-cell/single-construct approach was expanded upon in two ways. Firstly, a practical dual-colour sensor strain was created by simultaneous, constitutive expression of a red fluorescence reporter gene in green fluorescent nitrogen sensor cells.
Secondly, an innovative cellular communication and signal amplification system inspired by the natural S. cerevisiae pheromone system and mating response was established successfully. It features the yeast pheromone alpha-factor as a trigger and alpha-factor-responsive reporter cells which express a fluorescence reporter gene from the pheromone-inducible FIG1 promoter as an output signal. The system was functional both with synthetic and cell-secreted alpha-factor, provided that recombinant cells were deleted for the alpha-factor protease Bar1p. Integration of amplifier cells which secrete alpha-factor in response to stimulation with the pheromone itself could increase the system\'s sensitivity further. Signal amplification was demonstrated for phosphorus sensor cells as a proof of concept. Therefore, the alpha-factor-based cellular communication and signal amplification system might be useful in applications that suffer from poor signal yield. Due to its modular design, the system could be applied in basically any cell-based biosensor or sensor-actor system.
Immobilisation of the generated sensor and reporter cells in transparent natural polymers can be beneficial considering biosensor fabrication. Functionality of sensor and reporter cells in calcium-alginate beads or nano-printed arrays was successfully demonstrated. For the latter setup, fluorescence scanning and software-assisted fluorescence quantification was applied as a new detection method. In an experiment using an agarose-based two-compartment setup proposed by Jahn, 2011, properties of the alpha-factor-based cellular communication and signal amplification system after immobilisation were tested. These studies provide an initial experimental basis for an appropriate geometry of miniaturised immobilisation matrices with fluorescent yeast sensor and reporter cells in prospective biosensor designs.
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Yeast mitochondrial copper metabolism: topology and role of Cox11pKhalimonchuk, Oleh 15 February 2006 (has links)
Cytochrome c oxidase (COX) is one of two known Cu-containing enzymes in mitochondria. Delivery and insertion of copper into COX are very complex processes that require multiple steps and involve a large number of assisting factors. One of the involved components is Cox11p, a copper binding protein in the inner mitochondrial membrane that is conserved from prokaryotes to eukaryotes. Cox11p is essential for respiratory growth and implicated in the assembly of the CuB site located in subunit Cox1p of COX. In the thesis the topology of Cox11p was determined and evidence for its association with the mitochondrial translation machinery is provided. The interaction of Cox11p with mitoribosomes is mediated by its single evolutionary conserved transmembrane segment and appears to be indirect and mediated by another conserved membrane protein(s). A model is proposed in which the CuB site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the mitochondrial intermembrane space. In addition the genetic and biochemical characterization of S. pombe Cox11p homologue was performed. Two versions of cox11+ gene are detected in a haploid S. pombe genome. Cells lacking either of the cox11+ copies remain respiratory competent, whereas deletion of both S. pombe cox11+ alleles appears to result in either spore lethality or in severe decrease of spores viability. Thus, both versions of SpCox11p are functional and important. In S. pombe Cox11p exists as a tandem with the mitoribosomal protein Rsm22p. This precursor protein is cleaved during mitochondrial import into two mature protein species corresponding to Rsm22p- and Cox11p-like moieties.
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Androgenic properties of the dietary supplement 5α‑hydroxy‑laxogeninBeer, Carolin, Keiler, Annekathrin M. 22 February 2024 (has links)
Dietary supplements sold for anabolic benefits or performance enhancement often contain substances, which are nonapproved and might lack quality controls. With regard to athletes, the inclusion of substances or methods in the prohibited list of the World Anti-Doping Agency is based on medical or scientific evidence. 5α-hydroxy-laxogenin is a synthetic spirostane-type steroid, which is contained in dietary supplements and advertised as anabolic agent. To date, evidence is missing on anabolic or androgenic activity of 5α-hydroxy-laxogenin. We investigated its androgenic potential in two in vitro bioassays. While no activity was observed in the yeast androgen screen, 5α-hydroxy-laxogenin was able to trans-activate the androgen receptor in human prostate cells in a dose-dependent manner. Interestingly, a biphasic response was observed with antagonistic properties at lower concentrations and agonistic effects at higher concentrations tested. The demonstrated androgenic properties of the higher concentrations demonstrate that further investigations should focus on the safety as well as on potential anabolic effects of 5α-hydroxy-laxogenin. This is of interest with regard to abuse for doping purposes.
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Mathematical modelling of DNA replicationBrümmer, Anneke 30 September 2010 (has links)
Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren. / Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.
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A proteome-wide screen utilizing second generation sequencing for the identification of lysine and arginine methyltransferase protein interactionsWeimann, Mareike 13 September 2012 (has links)
Proteinmethylierung spielt eine immer größere Rolle in der Regulierung zellulärer Prozesse. Die Entwicklung effizienter proteomweiter Methoden zur Detektion von Methylierung auf Proteinen ist limitiert und technisch schwierig. In dieser Arbeit haben wir einen neuen Hefe-Zwei-Hybrid-Ansatz (Y2H) entwickelt, der Proteine, die miteinander wechselwirken, mit Hilfe von Sequenzierungen der zweiten Generation identifiziert (Y2H-Seq). Der neue Y2H-Seq-Ansatz wurde systematisch mit dem Y2H-Seq-Ansatz verglichen. Dafür wurde ein Bait-Set von 8 Protein-Arginin-Methyltransferasen, 17 Protein-Lysin-Methyltransferasen und 10 Demethylasen gegen 14,268 Prey-Proteine getestet. Der Y2H-Seq-Ansatz ist weniger arbeitsintensiv, hat eine höhere Sensitivität als der Standard Y2H-Matrix-Ansatz und ist deshalb besonders geeignet, um schwache Interaktionen zwischen Substraten und Protein-Methyltransferasen zu detektieren. Insgesamt wurden 523 Wechselwirkungen zwischen 22 Bait-Proteinen und 324 Prey-Pr oteinen etabliert, darunter 11 bekannte Methyltransferasen-Substrate. Netzwerkanalysen zeigen, dass Methyltransferasen bevorzugt mit Transkriptionsregulatoren, DNA- und RNA-Bindeproteinen wechselwirken. Diese Daten repräsentieren das erste proteomweite Wechselwirkungsnetzwerk über Protein-Methyltransferasen und dienen als Ressource für neue potentielle Methylierungssubstrate. In einem in vitro Methylierungsassay wurden exemplarisch mit Hilfe massenspektrometrischer Analysen die methylierten Aminosäurereste einiger Kandidatenproteine bestimmt. Von neun getesteten Proteinen waren sieben methyliert, zu denen gehören SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP und WDR42A. Wahrscheinlich sind viele Methylierungssubstrate im Netzwerk vorhanden. Das vorgestellte Protein-Protein-Wechselwirkungsnetzwerk zeigt, dass Proteinmethylierung sehr unterschiedliche zelluläre Prozesse beeinflusst und ermöglicht die Aufstellung neuer Hypothesen über die Regulierung Molekularer Mechanismen durch Methylierung. / Protein methylation on arginine and lysine residues is a largely unexplored posttranslational modification which regulates diverse cellular processes. The development of efficient proteome-wide approaches for detecting protein methylation is limited and technically challenging. We developed a novel workload reduced yeast-two hybrid (Y2H) approach to detect protein-protein interactions utilizing second generation sequencing. The novel Y2H-seq approach was systematically evaluated against our state of the art Y2H-matrix screening approach and used to screen 8 protein arginine methyltransferases, 17 protein lysine methyltransferases and 10 demethylases against a set of 14,268 proteins. Comparison of the two approaches revealed a higher sensitivity of the new Y2H-seq approach. The increased sampling rate of the Y2H-seq approach is advantageous when assaying transient interactions between substrates and methyltransferases. Overall 523 interactions between 22 bait proteins and 324 prey proteins were identified including 11 proteins known to be methylated. Network analysis revealed enrichment of transcription regulator activity, DNA- and RNA-binding function of proteins interacting with protein methyltransferases. The dataset represents the first proteome-wide interaction network of enzymes involved in methylation and provides a comprehensively annotated resource of potential new methylation substrates. An in vitro methylation assay coupled to mass spectrometry revealed amino acid methylation of candidate proteins. Seven of nine proteins tested were methylated including SPIN2B, DNAJA3, QKI, SAMD3, OFCC1, SYNCRIP and WDR42A indicating that the interaction network is likely to contain many putative methyltransferase substrate pairs. The presented protein-protein interaction network demonstrates that protein methylation is involved in diverse cellular processes and can inform hypothesis driven investigation into molecular mechanisms regulated through methylation.
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Novel regulators of trafficking in the yeast Golgi-endosomal systemGravert, Maike 09 October 2006 (has links) (PDF)
Over the past few years a large amount of work has provided growing insight into the molecular mechanisms that direct post-Golgi trafficking events in the budding yeast Saccharomyces cerevisae. However, a key event in this process, the formation of secretory vesicles at the Golgi and sorting of cargo into these transport carriers, remains poorly understood. It has been demonstrated that phosphatidylinositol 4-phosphate (PI(4)P) generated by the PI(4)-kinase Pik1p plays an essential role in maintenance of Golgi secretory function and morphology. Up to now relatively few targets of Pik1/PI(4)P signaling at the Golgi have been identified and it thus remains elusive how Pik1p mediates its essential function in Golgi secretion. During my thesis work, I used synthetic genetic array analysis (SGA) of a temperature-sensitive mutant allele of PIK1 (pik1-101) in order to gain better understanding of Pik1p function at the TGN and to isolate new regulators of post-Golgi transport in yeast. I identified a total of 85 genes, whose deletion resulted in a synthetic growth defect when combined with the pik1-101 mutation. 21 isolated deletion mutants were used for further analysis, several of which were found to share common trafficking phenotypes with the pik mutant. A striking result of the screen was the finding that Pik1p interacts genetically with several components of a potential post-translational modification pathway referred to as “urmylation pathway”. In addition, a novel, previously uncharacterized subunit of the Transport protein particle (TRAPP) complex was isolated as genetic interactor of Pik1p, suggesting a function for the TRAPP complex in a Pik1p dependent trafficking pathway. Using tandem affinity purification, I could also demonstrate that TRAPP shows previously unknown interactions with other regulators of post-Golgi transport. The second part of this thesis describes the development of a new visual screening approach. Recent work indicates that secretory cargo in yeast can be transported to the cell surface via at least two different exocytic branches. Upon block of one pathway cargo can be partially redistributed into the other pathway. This partial redundancy of exocytic pathways provides one explanation why genetic screens in the past were largely unsuccessful in identifying the molecular machinery that directs vesicle budding and cargo sorting at the TGN. I collaborated in the development of a novel screening method that was devised to circumvent this problem. The method took advantage of the systematic yeast knockout array and was based on the assumption that a defect in cargo sorting and cell surface transport could be detected as intracellular accumulation of a GFP-tagged model cargo. The suitability of our approach for identifying regulators of secretory transport has been demonstrated in a small-scale pilot study that will be presented in this thesis. The screening method proofed to be applicable on a genome-wide scale and can now be used for the screening of additional markers. This novel approach provides an entry point to the comprehensive study of TGN sorting.
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Zur Funktion des MPH1-Gens von Saccharomyces cerevisiae bei der rekombinativen Umgehung von replikationsarretierenden DNA-Schäden / On the function of the MPH1 gene from Saccharomyces cerevisiae in recombinational bypass of replication arresting DNA lesionsSchürer, Anke 22 January 2004 (has links)
No description available.
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