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Die chromosomale Architektur und ihr Einfluss auf die Genexpression in nativen und gentechnisch veränderten Bakterien
No full textResearch in recent years has yielded new insights into the influence of chromosomal architecture at different levels on bacterial gene regulation and expression. On the topological level, chromosome compaction can bring distant genes or regions in spatially proximity, suggesting a regulatory concept of co-expression of distant genes. DNA supercoiling, which is highly dynamic and one of the major factors in nucleoid formation, can have a significant influence on gene expression by modulatory effects on transcription. Furthermore, the replication-induced copy number effect increases the expression of genes by transiently increasing the number of gene copies during replication. However, how this impacts the organism on a systemic level (global gene expression) has not been shown yet. In this work, the influence of the replication-induced copy number effect on gene expression in Escherichia coli has been investigated. It was previously shown, that genes closer to the replication origin (oriC) are higher expressed during the exponential phase compared to the stationary phase. This effect decreases with increasing distance to the oriC. In the course of this work, it was demonstrated that this expression pattern is due to the copy number effect instead of the strategic positioning of genes regulated by global transcription factors. Furthermore, the regulatory impact of the replication-induced copy number effect was determined for individual genes. It could be shown that around 40% of the genes are predominantly copy number regulated, suggesting an important role of the copy number effect for gene regulation and expression in E. coli. In addition, the influence of the copy number effect on the chromosome organization was investigated. The conservation of the position of genes relative to oriC indicates a strong influence of the copy number effect on bacterial chromosome evolution, especially in fast-growing bacteria. Moreover, a genome editing tool based on CRISPR/Cas9 (CRISPR SWAPnDROP) was established for the chromosomal modifications required for these investigations. Beyond its initial purpose, this tool was designed to facilitate large chromosomal rearrangements and the transfer of chromosomal regions between bacterial species. As a proof of principle, a 151kb chromosomal region was transferred from one E. coli strain to another as well as to the biotechnology relevant Vibrio natriegens. In addition, the RP4 conjugation system of E. coli was transferred to both V. natriegens and the plant pathogen Dickeya dadantii and its functionality was demonstrated in these organisms. Furthermore, the transfer of the E. coli lac operon to V. natriegens and the transfer of the GanB ORF from D. dadantii to E. coli demonstrate successful gain of function genome edits using CRISPR SWAPnDROP.Die Forschung der letzten Jahre hat neue Erkenntnisse über den Einfluss der chromosomalen Architektur auf die bakterielle Genregulation und -expression erbracht. Auf der topologischen Ebene kann die Chromosomenkompaktierung weit voneinander entfernte Gene oder Regionen in räumliche Nähe bringen, was auf ein Regulierungskonzept der Koexpression entfernter Gene hindeutet. DNA-Supercoiling, das hochdynamisch und einer der Hauptfaktoren bei der Nukleoidbildung ist, kann die Genexpression durch modulierende Effekte auf die Transkription erheblich beeinflussen. Außerdem erhöht der durch die Replikation hervorgerufene Kopienzahl-Effekt die Expression von Genen, indem er die Anzahl der Genkopien während der Replikation vorübergehend erhöht. Wie sich dies auf den Organismus auf einer systemischen Ebene (globale Genexpression) auswirkt, wurde jedoch noch nicht untersucht. In dieser Arbeit wurde der Einfluss des Kopienzahl-Effekts auf die Genexpression in Escherichia coli untersucht. Frühere Publikationen haben gezeigt, dass Gene, die näher am Replikationsursprung (oriC) liegen, in der exponentiellen Phase stärker exprimiert werden als in der stationären Phase. Dieser Effekt nimmt mit zunehmender Entfernung zum Replikationsursprung ab. Im Zuge dieser Arbeit konnte nachgewiesen werden, dass dieses Expressionsmuster auf den Kopienzahl-Effekt zurückzuführen ist und nicht auf die strategische Positionierung von Genen, die durch globale Transkriptionsfaktoren reguliert werden. Darüber hinaus wurde der regulatorische Einfluss des Kopienzahl-Effekts für einzelne Gene bestimmt. Es konnte gezeigt werden, dass etwa 40% der Gene überwiegend über die Kopienzahl reguliert sind, was auf eine wichtige Rolle des Kopienzahl-Effekts für die Genregulation und -expression in E. coli hinweist. Darüber hinaus wurde der Einfluss des Kopienzahl-Effekts auf die Chromosomenorganisation untersucht. Die Konservierung der Position von Genen relativ zum Replikationsursprung deutet auf einen starken Einfluss des Kopienzahl-Effekts auf die bakterielle Chromosomenevolution hin, insbesondere bei schnell wachsenden Bakterien. Darüber hinaus wurde ein auf CRISPR/Cas9 basierendes Genome Editing Tool (CRISPR SWAPnDROP) etabliert, das für die erforderlichen chromosomalen Veränderungen dieser Untersuchungen benötigt wurde. Dieses Tool wurde außerdem entwickelt, um große chromosomale Veränderungen und den Transfer von chromosomalen Regionen zwischen Bakterienarten zu ermöglichen. Als "proof of principle" wurde eine 151kb große chromosomale Region sowohl zwischen zwei E. coli-Stämmen als auch von E. coli auf das biotechnologisch relevante Bakterium Vibrio natriegens übertragen. Zusätzlich wurde das RP4-Konjugationssystem von E. coli sowohl auf V. natriegens als auch auf das Pflanzenpathogen Dickeya dadantii übertragen und seine Funktionalität in diesen Organismen nachgewiesen. Abschließend konnten mit der Übertragung des lac-Operons von E. coli auf V. natriegens und der Übertragung des GanB ORFs von D. dadantii auf E. coli erfolgreiche "gain of function" Genomeditierungen gezeigt werden
Ein experimenteller Rahmen zur Untersuchung des Einflusses der Promotorarchitektur und des genomischen Kontextes auf die Genexpression
No full textTranscription is a fundamental process of gene expression. Information stored in DNA is transcribed into different types of mobile RNA, which play a role in various essential processes of the cell, e.g. translation. However, cells do not need all the information stored in their DNA at the same time. Therefore, the process of transcription gets regulated by a plethora of mechanisms. One frequently discussed but poorly understood mechanism of transcription regulation is DNA supercoiling [Travers and Muskhelishvili, 2005]. Whereby, the process of transcription itself affects the DNA-topology up- and downstream of the transcription machinery as described in the twin supercoiling domain model [Liu and Wang, 1987]. This phenomenon is called Transcription Coupled DNA Supercoiling (TCDS). It has also been shown that genes react individually to changes in DNA supercoiling and that there is a selection pressure on adapting to the DNA supercoiling levels emitted by neighbouring gene expression [Sobetzko, 2016]. The system in which promoters react to changes in DNA supercoiling is as diverse as there are promoters; notably, some promoters seem not to respond to DNA supercoiling at all. Thus, this raises the question as to which elements within different promoter types cause them to respond to TCDS so differently. In this thesis, I built a pipeline to investigate the effects of TCDS and DNA supercoiling on promoters. Firstly, I created a plasmid toolbox, which allows modular assembly of transcription units. The central feature of this toolbox is the flexibility to test different arrangements of multiple transcription units. I achieved this by adapting the well established Modular Cloning (MoClo) standard [Weber et al., 2011] and build my toolbox around it. I thus created a system that works on both its own and is compatible with the existing standard MoClo protocol.
In the second part of this thesis, I established an experimental pipeline using synthetic σ70-promoters to investigate the influence of DNA supercoiling on transcription. The experimental setup allowed precise changes in parts of the promoter and at the same time created a library of these promoters. Using this pipeline to investigate the spacer region of the promoter, I was able to confirm that the spacer influences the promoter strength. Further, I showed that the promoter spacer has only a limited effect on the supercoiling sensitivity of a promoter. I also showed that a 5‘-TGTG-3‘ motif in the spacer region could lower transcription by enhancing RNA-polymerase (RNAP)-binding. Moreover, the experimental setup also showed the constraints of using the DNA-relaxing drug novobiocin on a plasmid-based system. Hence, to further investigate the effects of TCDS on neighbouring transcription, I applied an optogenetically-controllable promoter to the previously established pipeline. Finally, I began to explore the possibility of integrating my experimental promoter setup into any genomic position. As such, a CRISPR/Cas9-based homologous re-combination system was developed further to make it modular and compatible with the Modular Cloning protocol. I could show the first features of this system to work.Die Transkription ist ein grundlegender Prozess der Genexpression. Die in der DNA gespeicherten Informationen werden in mobile RNA transkribiert, die bei verschiedenen essenziellen Prozessen der Zelle, wie z.B. der Translation, eine Rolle spielen. Die Zellen benötigen jedoch nicht alle in der DNA gespeicherten Informationen gleichzeitig. Daher wird der Prozess der Transkription durch eine Vielzahl von Mechanismen reguliert. Ein häufig diskutierter, aber nicht vollständig verstandener Transkriptionsregulator ist das DNA Supercoiling [Travers and Muskhelishvili, 2005]. Wobei der Transkriptionsprozess selbst die DNA-Topologie vor und hinter der Transkriptionsmaschinerie beeinflusst, wie im twin supercoiling domain-Modell von Liu and Wang [1987] beschrieben. Dieses Phänomen wird als Transkriptions-gekoppeltes DNA-Supercoiling (TCDS) bezeichnet. Darüber hinaus reagieren die Gene individuell auf Veränderungen des DNA-Supercoilings und es besteht ein Selektionsdruck für Gene sich an die DNA-Supercoiling-Niveaus, welche durch das benachbarte Expressionsverhalten emittiert werden, anzupassen [Sobetzko, 2016]. Die Reaktionen der Promotoren auf Veränderungen im DNA-Supercoiling sind so vielfältig wie die Promotoren selbst. Insbesondere scheinen einige Promotoren nicht auf Änderungen im DNA-Supercoiling zu reagieren. Dies führt zu der Frage was die Vielfalt der Promotoren dazu bringt so unterschiedlich auf TCDS zu reagieren. Im Laufe dieser Arbeit wurde ein Versuchsprozess entwickelt, um die Auswirkungen von DNA-Supercoiling und TCDS auf Promotoren zu untersuchen. Zuerst wurde dafür eine Plasmid-Toolbox erstellt, die den modularen Aufbau von Transkriptionseinheiten ermöglicht. Das zentrale Merkmal dieser Toolbox ist die Flexibilität, verschiedene Anordnungen derselben Teile zu testen. Dies wurde erreicht, indem der gut etablierte MoClo-Standard adaptiert wurde und die Toolbox um diesen herum aufgebaut wurde. Dadurch wurde ein System geschaffen, welches eigenständig funktioniert, mit dem bestehenden MoClo-Standard kompatibel ist und ihn erweitert. Im zweiten Teil dieser Arbeit wurde ein Experimentaufbau entwickelt in welchem mit Hilfe von synthetischen σ70-Promotoren, der Einfluss von DNA-Supercoiling auf die Transkription untersucht werden kann. Der Versuchsaufbau ermöglichte präzise Veränderungen in Teilen des Promotors und schaffte gleichzeitig eine library dieser Promotoren. Mit Hilfe dieser Experimente zur Untersuchung der spacer-Region des Promotors konnte bestätigt werden, dass der spacer die Stärke des Promotors beeinflusst. Außerdem konnte gezeigt werden, dass der spacer die Supercoiling-Empfindlichkeit nur geringfügig beeinflusst. Ferner konnte gezeigt werden, dass ein 5‘-TGTG-3‘-Motiv im spacer die Transkription durch eine verstärkte RNAP-Bindung senken könnte. Der Versuchsaufbau zeigte jedoch die Einschränkungen bei der Verwendung der DNA relaxierenden Chemikalie Novobiocin mit einem plasmid basierten System. Deshalb – und um die Auswirkungen von TCDS auf die benachbarte Transkription weiter zu untersuchen, wurde ein optogenetisch kontrollierbarer Promotor in den bereits etablierten Versuchsaufbau integriert. Schließlich wurden die ersten Schritte unternommen, um einen Weg zu finden, den Promoter-Test in jede beliebige genomische Stelle integrieren zu können. Dafür wurde ein auf CRISPR/Cas9 basierendes, homologes Rekombinationssystem weiterentwickelt und modularisiert, welches DNA-Fragmente nahtlos ins Genom integrieren kann
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Impact of global gene regulators in E. coli on cellular proteome and metabolic function
No full textAlmost sixty yearsafter the determination of the DNA helix structure and ten years after the human genome project gene regulation remains still obscure in many aspects. Although many regulatory interactions of regulatory proteins are known as well as their metabolic impact (e.g. lac operon), holistic concepts of regulation are still mainly uncovered or attributed to single effectors. Nevertheless, certain concepts were already revealed showing for example boolean like logics in the organization of the Escherichia coli regulatory network also found in neuronal networks and computer chips supporting the view of an information processing system. According to the last example transcription is mostly regarded as the complex interaction of regulators and targets integrated in the transcriptional regulatory network (TRN). Each regulator to target connection was inferred by deletion of the regulator, complementation in trans e.g. on a plasmid and confirmed by in vitro studies in a minimal factor context. The studies were performed under certain environmental conditions and are therefore in principle only valid under these conditions. The TRN can therefore be considered as the sum of possible regulatory interactions known down to the present day. The architecture of the network is mainly considered as hierarchical. Within the hierarchy, comprehensive regulatory overlaps were found called DORs. These overlaps exhibit more or less specific functions. We investigated the regulatory overlaps of major regulators (σ-factors and NAPs) regulons creating so called couplons. Gene ontology analysis showed an enrichment of specific metabolic function compared to random subsets taken from individual regulons. Moreover, the presence of highly overlapping regulons implies the reciprocal regulation of the involved regulators. These regulators are in turn organized in a heterarchically organized network of major regulators, constructing a decision making organization coordinating downstream regulation. This is supported by noise damping feedforward connections to couplons on the next hierarchy level most permeable for consistent signal sent by the upstream heterarchical network. However the majority of genes is not yet included in the TRN but also show complex transcription patterns. We show indications, that these genes may not be integrated into the TRN in a digital way. Here digital refers to a on-off target behavior dependent on a one to one relation of regulator and target. It has been shown, that supercoilii ing of the DNA is besides the TRN a global regulator of transcription driving growth related metabolic pathways and controlling anabolism/catabolism ratios. However the analysis was based on pure microarray data lacking relevance for the proteome composition. Therefore we performed a proteome analysis based on the experimental design of previous microarray experiments and find a strong correlation of transcriptome and proteome with a temporal delay of 20 minutes. Moreover, we find the same metabolic pathways activated on the proteomics level, proving the functional relevance of supercoiling changes. Analysing different gene sets under altered supercoiling conditions we found a diametrally opposed transcription pattern of TRN regulated genes and the complementary set of isolated genes during altered supercoiling supporting previous observations of two distinct types of information (TRN and gene neighborhood) executing regulatory control. However, we find regulators reacting on supercoiling similarly to isolated genes connecting supercoiling sensitive isolated genes with TRN regulators and in fact the TRN. Moreover, major TRN regulators also constituting major structural proteins like HU, FIS or H-NS show non-random distribution of binding sites. These proteins are also involved in buffering supercoiling dynamics and therefore coordinate gene regulation and chromosome topology. Although protein binding sites may play a structural role, like matS sites for the terminus macrodomain formation, or HU for transcription foci formation, the chromosome compaction itself has major effects on regulatory control. HU mutants have shown to create the chromosomal replication origin/terminus dependent transcription differences. The genes in the origin region were more active than in the wild type and the opposite was found for the terminus. In accordance with the HU transcription pattern also the binding sites of σ-factors show clear spatial gradients. Especially the antagonists σ70 and σ38 show a diametral gradient from origin to terminus indicating a spatial coding of temporal gene expression patterns. In this study we describe a spatio-temporal transcription program based on gradients of different DNA features monitored throughout the growth cycle of Escherichia coli. We show that gene order is conserved in the γ-Proteobacteria implementing a temporal program in space starting from origin of replication in exponential phase to the terminus in stationary phase. We finally propose that supercoiling, GC-content, gene orientation and binding site distributions coordinate of the spatio-temporal gene expression program
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