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Charakterisierung von lipid droplet-Regulatoren der Fruchtfliege <i>Drosophila melanogaster</i> / Characterization of lipid droplet regulators of the fruit fly <i>Drosophila melanogaster</i>Thiel, Katharina 31 May 2012 (has links)
No description available.
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Analysis of phage-type RNA polymerase driven transcription in Physcomitrella patens and ArabidopsisRichter, Uwe 22 January 2014 (has links)
In der vorliegenden Arbeit wurde der spezifische Einfluss verschiedener kernkodierter phagentypischer RNA Polymerasen auf die organelläre Genexpression in Physcomitrella und die mitochondrial Genexpression in Arabidopsis untersucht. Während das Fehlen von AtRpoTm in Arabidopsis und PpRpoTmp1 in P. patens lethal ist, wurden Insertionsmutanten für PpRpoTmp2 und AtRpoTmp einer detailierten Untersuchung unterzogen. Sowohl PprpoTmp2, als auch AtrpoTmp Pflanzen zeigten Abweichungen im Phänotyp charakteristisch für mitochondriale Dysfunktion. Identifizierte organelläre Promotoren in P. patens wurden zum Nachweis der Transkriptionsaktivität von PpRpoTmp1 und PpRpoTmp2 in vitro herangezogen. Beide Proteine besitzen die inhärente Fähigkeit zur Promotorerkennung ohne zusätzliche Kofaktoren. Die hier vorgestellten Studien unterstreichen die essentielle Bedeutung von AtRpoTm und PpRpoTmp1 für die Transkription mitochondrialer bzw organellärer Gene in Arabidopsis und P. patens. Im Gegensatz dazu können die Funktionen von AtRpoTmp und PpRpoTmp2 partiell durch andere organelläre RNAPs ersetzt werden. Phänotypische Abweichungen belegen jedoch, das AtRpoTmp und PpRpoTmp2 für die normale Entwicklung von Arabidosis bzw. P. patens essentiell sind. Veränderte Transkriptmengen in AtrpoTmp Pflanzen korrelierten mit genspezifischen Änderungen in der mitochondrialen Transkription. AtRpoTmp muss daher als essentiel für die normale Expression eines spezifischen Sets mitochondrialer Gene angesehen werden. Jedoch konnten für diese mitochondrialer Gene keine AtRpoTmp spezifischen Promotormotive mit reduzierter Aktivität identifiziert werden. Initiationsraten an allen Promotoren stromaufwärts von mitochondrialen Genen mit geringeren Transkriptmengen sind jedoch reduziert. Es erscheint daher wahrscheinlich, daß für einen Teil der mitochondrialen Gene genspezifische Elemente existieren, welche die Transkription durch AtRpoTmp dirigieren. / This study aimed to elucidate how the different transcriptional activities are facilitated in mitochondria of Arabidopsis thaliana and in both organelles of Physcomitrella patens. Insertional mutants for PprpoTmp2 and AtrpoTmp were analysed in detail. As for Arabidopsis RpoTm, knock-out of Physcomitrella RpoTmp1 was found to be lethal. Null mutant plants PprpoTmp2 and AtrpoTmp show surprisingly similar but clearly convergent phenotypical aberrations reminiscent of phenotypes reported for other mitochondrial mutants. Evidence is provided that PpRpoTmp1 and PpRpoTmp2 are functional RNA polymerases, which both posses the inherent ability to recognize organellar promoters in a minimal in vitro transcription system without the aid of additional cofactors. The data suggest that coding for two RpoT proteins one representing an enzyme with a high portion of non-specific transcriptional activity, as seen for AtRpoTmp and PpRpoTmp1 and one that can act as a single-polypeptide enzyme and recognize numerous mitochondrial promoters in vitro as AtRpoTm and PpRpoTmp2 echo convergent inventions but reflect complementing roles of these RNA polymerases in plant mitochondrial transcription. Phenotypical aberrations of rpoTmp2 plants suggest RpoTmp2 is important for normal growth and development. Altered transcript levels in AtrpoTmp were found to result from gene-specific transcriptional changes, establishing that AtRpoTmp functions in distinct transcriptional processes within mitochondria. Decreased transcription of a specific set of mitochondrial genes in AtrpoTmp was not associated with changes in the utilisation of specific promoters. Therefore AtRpoTmp function is not promoter-specific but gene-specific. This indicates that additional gene-specific elements direct the transcription of a subset of mitochondrial genes by RpoTmp.
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A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein TagsMüller, Andreas, Neukam, Martin, Ivanova, Anna, Sönmez, Anke, Münster, Carla, Kretschmar, Susanne, Kalaidzidis, Yannis, Kurth, Thomas, Verbavatz, Jean-Marc, Solimena, Michele 04 April 2017 (has links) (PDF)
Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.
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Analyse von Komponenten der organellären Transkriptionsmaschinerien aus Arabidopsis thaliana und Nicotiana tabacumBohne, Alexandra-Viola 21 August 2009 (has links)
Die Gesamtheit mitochondrialer Gene sowie ein Teil der plastidären Gene photosynthetischer Eukaryoten wird durch kernkodierte Phagentyp-RNA-Polymerasen transkribiert. In der vorliegenden Arbeit wurden unter Verwendung eines homologen in vitro-Transkriptionssystems, die spezifischen Funktionen der Phagentyp-RNA-Polymerasen RpoTm, RpoTp und RpoTmp aus Arabidopsis untersucht. Während RpoTmp keine Präferenz für die angebotenen Promotoren zeigte, transkribierten RpoTm und RpoTp eine überlappende Gruppe mitochondrialer und plastidärer Promotoren vielfältiger Architektur. RpoTm und RpoTp präsentierten eine Kofaktor-unabhängige Fähigkeit zur Promotorerkennung bei Angebot superhelikaler DNA-Matrizen. Eine selektive Promotornutzung sowie die Unfähigkeit zur spezifischen Transkription linearer Promotormatrizen in vitro implizieren die Assoziation zusätzlicher, in die Promotorerkennung und/oder DNA-Aufschmelzung involvierter Kofaktoren in vivo. Die in vitro-Erkennung mitochondrialer Promotoren durch eine plastidäre Phagentyp-RNA-Polymerase (und umgekehrt) sowie weitere Ähnlichkeiten der Transkriptionsapparate der Mitochondrien und Plastiden, wie die strukturelle Organisation ihrer Promotoren und die phylogenetische Herkunft ihrer kernkodierten Transkriptasen inspirierte in planta Studien zur spezifischen Transkription eines mitochondrialen Promotors in den Plastiden. Hierzu wurde die Expression des nptII-Reportergens unter Kontrolle des mitochondrialen PatpA-Promotors aus Oenothera in transplastomischen Tabakpflanzen analysiert. Die durchgeführten Studien belegen eine korrekte Transkription des mitochondrialen PatpA-Promotors durch eine plastidäre Phagentyp-RNA-Polymerase in in vitro-Transkriptionsassays sowie in transplastomischen Tabakpflanzen. Diese Resultate enthüllen weitere unerwartete Ähnlichkeiten der organellären Genexpression, die aufschlussreiche evolutionäre Einblicke erlauben und verbesserte Anwendungen zur Manipulation plastidärer Genome ermöglichen könnten. / All mitochondrial and a subset of plastidial genes of photosynthetically active eukaryotes are transcribed by nuclear-encoded, phage-type RNA polymerases. In this study, a homologous in vitro transcription system was used to define the specific functions of Arabidopsis phage-type RNA polymerases RpoTm, RpoTp and RpoTmp in organellar transcription. RpoTmp displayed no significant promoter specificity, whereas RpoTm and RpoTp were able to accurately initiate transcription from overlapping subsets of mitochondrial and plastidial promoters of diverse architecture. RpoTm and RpoTp thereby demonstrated an intrinsic capability to recognize promoters on supercoiled DNA templates without the aid of protein cofactors. A selective promoter recognition by the phage-type RNAPs in vitro and the inability to recognize promoters on linear templates imply that auxiliary factors are required for efficient initiation of transcription and/or DNA melting in vivo. Crosswise recognition of organellar promoters by the phage-type RNA polymerases in vitro as well as other similarities of the mitochondrial and plastidial transcription machineries such as promoter structures and the phylogenetic origin inspired in planta studies to investigate specific transcription of a mitochondrial promoter in plastids. Therefore, the expression of an nptII reporter gene under control of the mitochondrial PatpA promoter from Oenothera was analyzed in transplastomic tobacco plants. The data presented here demonstrate the faithful recognition of the mitochondrial PatpA promoter by a plastid RNA polymerase both in in vitro transcription assays and in transplastomic tobacco plants. These findings disclose further unexpected similarities of the organellar gene expression systems which deliver interesting evolutionary insights and might facilitate improved applications for chloroplast genome engineering.
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A theoretical model on the role of lateral gene transfer in the evolution of endosymbiotic genomesMunoz, Víctor Hugo Anaya 05 January 2012 (has links)
Laterale Gentransfer wurde zuerst von Schwartz und Dayhoff (1978) entdeckt, die es aber als eine Exzentrizität werteten und als solche ignorierten. Später, als mehrere DNS- und Eiweißsequenzen sequenziert und raffiniertere Phylogenien rekonstruiert wurden, hat die Rolle an Relevanz gewonnen, die der laterale (oder horizontale) Gentransfer in der evolutionären Geschichte von lebendigen Organismen gespielt hat. Außerdem existiert auch zwischen Endosymbionten und Zellkernen statt. Ich habe ein theoretisches Modell entwickelt, das den lateralen Gentransfer zwischen Endosymbionten und dem Zellkern repräsentiert. Das Modell erforscht die Bedeutung des Fehlens von Rekombination in den Organellen (Muller’s Ratchet) sowie Abweichungen von Muller’s Ratchet in Form der non-symmetrical homologous recombination in Gentransfermechanismen. Ich habe zum einen Zellkern-Inkompatibilitäten, die aus der Übertragung eines Gens resultieren, und zum anderen Zyto- und Zellkern-Inkompatibilitäten zwischen den mutierten endosymbiotischen Genomen und dem modifizierten Zellenkern untersucht. Die Ergebnisse zeigen, dass unter bestimmten Bedingungen die Existenz oder Nicht-Existenz von Rekombination die gleiche Wirkung haben können. Es zeigte sich auch, dass Rekombination, wenn sie vorkommt und wenn sie nicht symmetrisch ist, starke Auswirkungen auf die Allelenfrequenz einer Population haben kann. Es wurde auch klar, dass es eine starke Beziehung zwischen dem Zellkern und endosymbiotischen Genomen gibt, und dass das evolutionäre Schicksal des einen größtenteils von den evolutionären Kräften abhängig ist, die das andere beeinflussen. Wenn man Zellkern- und Cyto-Zellkerninkompatibilitäten in das Modell einführt, dann zeigen die Ergebnisse, dass die Inkompatibilitäten, die der laterale Gentransfer produziert hat, möglicherweise eine ähnliche Rolle im Speziationsmechanismus spielen könnten wie die Inkompatibilitäten zwischen Mitochondrien und Zellkernen in verschiedenen Nasonia-Arten. / Lateral gene transfer has played a key role in the evolution of living beings. This process was first acknowledged in 1978 by Schwartz and Dayhoff but considered a relatively infrequent eccentricity and ignored. Later on, as DNA and protein sequences accumulated and more refined phylogenies were reconstructed, the contribution of lateral (or horizontal) gene transfer to the evolutionary history of living organisms gained relevance. Besides, gene transfer is known to occur not only between independent organisms but also, and more frequently between endosymbionts including eukaryotic organelles. I developed a theoretical model to study the lateral gene transfer process between cell organelles (but extendible to other endosymbionts) and the cell nucleus. The model explores the role of the lack of recombination in the organelles (Muller''s ratchet) as well as deviations from Muller''s ratchet in the form of non-symmetrical homologous recombination in relation with the gene transfer process. Also, nuclear incompatibilities resulting from the inclusion of a transferred gene, and cyto-nuclear incompatibilities between the mutant endosymbiotic genomes and the modified nuclear genome are investigated. The results obtained show that under certain circumstances the existence recombination or its non-existence produce the same results, and that deviations from symmetry in the recombination process might have important effects on the frequency of different alleles. It is also clear that there is a strong relation between nuclear and endosymbiotic genomes, and that the evolutionary fate of one largely depends on the forces affecting the other. When nuclear and cyto-nuclear incompatibilities are introduced in the model, the results show that lateral gene transfer-induced incompatibilities could potentially play a role in the speciation process similar to the one produced by mitochondria in the Nasonia species.
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Mechanismus der Neuentstehung von Peroxisomen in Saccharomyces cerevisiae / Mechanism of the formation of peroxisomes in Saccharomyces cerevisiaeOkuda, Kenichi 05 October 2010 (has links)
No description available.
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Studies on AP-1 Sorting Function and Regulation of Membrane BindingRadhakrishnan, Karthikeyan 16 January 2007 (has links)
No description available.
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Investigation of SNARE function in the early endosomal compartment / Untersuchung der Funktion von SNARE Proteinen im frühendosomalen KompartimentBethani, Ioanna 29 April 2009 (has links)
No description available.
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A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein TagsMüller, Andreas, Neukam, Martin, Ivanova, Anna, Sönmez, Anke, Münster, Carla, Kretschmar, Susanne, Kalaidzidis, Yannis, Kurth, Thomas, Verbavatz, Jean-Marc, Solimena, Michele 04 April 2017 (has links)
Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.
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PIP₂ determines length and stability of primary cilia by balancing membrane turnoversStilling, Simon, Kalliakoudas, Theodoros, Benninghoven-Frey, Hannah, Inoue, Takanari, Falkenburger, Björn H 08 April 2024 (has links)
Primary cilia are sensory organelles on many postmitotic cells. The ciliary membrane is continuous with the plasma membrane but differs in its phospholipid composition with phosphatidylinositol 4,5-bisposphate (PIP₂) being much reduced toward the ciliary tip. In order to determine the functional significance of this difference, we used chemically induced protein dimerization to rapidly synthesize or degrade PIP₂ selectively in the ciliary membrane. We observed ciliary fission when PIP₂ was synthesized and a growing ciliary length when PIP₂ was degraded. Ciliary fission required local actin polymerisation in the cilium, the Rho kinase Rac, aurora kinase A (AurkA) and histone deacetylase 6 (HDAC6). This pathway was previously described for ciliary disassembly before cell cycle re-entry. Activating ciliary receptors in the presence of dominant negative dynamin also increased ciliary PIP₂, and the associated vesicle budding required ciliary PIP₂. Finally, ciliary shortening resulting from constitutively increased ciliary PIP₂ was mediated by the same actin – AurkA – HDAC6 pathway. Taken together, changes in ciliary PIP₂ are a unifying point for ciliary membrane stability and turnover. Different stimuli increase ciliary PIP₂ to secrete vesicles and reduce ciliary length by a common pathway. The paucity of PIP₂ in the distal cilium therefore ensures ciliary stability.
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