TY - THES A1 - Bortfeld, Silvia T1 - Analysis of Medicago truncatula transcription factors involved in the arbuscular mycorrhizal symbiosis T1 - Analyse von Medicago truncatula Transkriptionsfaktoren, die während der arbuskulären Mykorrhiz-Symbiose eine Rolle spielen N2 - For the first time the transcriptional reprogramming of distinct root cortex cells during the arbuscular mycorrhizal (AM) symbiosis was investigated by combining Laser Capture Mirodissection and Affymetrix GeneChip® Medicago genome array hybridization. The establishment of cryosections facilitated the isolation of high quality RNA in sufficient amounts from three different cortical cell types. The transcript profiles of arbuscule-containing cells (arb cells), non-arbuscule-containing cells (nac cells) of Rhizophagus irregularis inoculated Medicago truncatula roots and cortex cells of non-inoculated roots (cor) were successfully explored. The data gave new insights in the symbiosis-related cellular reorganization processes and indicated that already nac cells seem to be prepared for the upcoming fungal colonization. The mycorrhizal- and phosphate-dependent transcription of a GRAS TF family member (MtGras8) was detected in arb cells and mycorrhizal roots. MtGRAS shares a high sequence similarity to a GRAS TF suggested to be involved in the fungal colonization processes (MtRAM1). The function of MtGras8 was unraveled upon RNA interference- (RNAi-) mediated gene silencing. An AM symbiosis-dependent expression of a RNAi construct (MtPt4pro::gras8-RNAi) revealed a successful gene silencing of MtGras8 leading to a reduced arbuscule abundance and a higher proportion of deformed arbuscules in root with reduced transcript levels. Accordingly, MtGras8 might control the arbuscule development and life-time. The targeting of MtGras8 by the phosphate-dependent regulated miRNA5204* was discovered previously (Devers et al., 2011). Since miRNA5204* is known to be affected by phosphate, the posttranscriptional regulation might represent a link between phosphate signaling and arbuscule development. In this work, the posttranscriptional regulation was confirmed by mis-expression of miRNA5204* in M. truncatula roots. The miRNA-mediated gene silencing affects the MtGras8 transcript abundance only in the first two weeks of the AM symbiosis and the mis-expression lines seem to mimic the phenotype of MtGras8-RNAi lines. Additionally, MtGRAS8 seems to form heterodimers with NSP2 and RAM1, which are known to be key regulators of the fungal colonization process (Hirsch et al., 2009; Gobbato et al., 2012). These data indicate that MtGras8 and miRNA5204* are linked to the sym pathway and regulate the arbuscule development in phosphate-dependent manner. N2 - Die Leguminose Medicago truncatula (gehört zur Gattung des Schneckenklees) ist in der Lage sowohl eine Symbiose mit stickstofffixierenden Bakterien (Rhizobien), als auch mit Mykorrhiza-Pilzen einzugehen. Der Mykorrhiza-Pilz Rhizophagus irregularis dringt in die Wurzelrindenzellen ein und bildet Strukturen aus, die als Arbuskeln bezeichnet werden. Diese ermöglichen den Transfer von Nährstoffen, wie Phosphat in die Wurzelzellen. Die Pflanze liefert hingegen bis zu 20 % ihrer Photosyntheseprodukte an den Pilz. Da die Lebenszeit der Arbuskeln nur wenige Tage beträgt, können Wurzelrindenzellen mehrere Arbuskeln nacheinander beherbergen. Somit können neben arbuskelhaltigen, auch nicht-arbuskelhaltige Zellen in kolonisierten Wurzeln auftreten. Die nicht-arbuskelhaltigen Zellen beeinträchtigen die Sensitivität von Genregulationsanalysen, wenn die Genregulation während der Mykorrhiza-Symbiose anhand von ganzen kolonisierten Wurzeln untersucht wird. In dieser Arbeit konnte eine Zelltyp-spezifische Analyse der Genregulation von arbuskelhaltigen und nicht-arbuskelhaltigen Zellen durchgeführt, und eine Erhöhung der Sensitivität erreicht werden. Mittels Laser Capture Microdissection wurden Zellen aus Gefrierschnitten von Wurzeln isoliert. Aus den so gewonnen Zellen konnte RNA von ausreichender Qualität und Quantität extrahiert werden, um das Transkriptom der beiden Zelltypen mittels Mikroarrayhybridisierung zu untersuchen. Transkriptionsfaktoren (TFs) spielen wahrscheinlich eine Schlüsselrolle in der Umprogrammierung von Wurzelzellen während der Mykorrhiza-Symbiose. Daher wurde die Genregulation von TF-Genen in den zwei Zelltypen gezielt untersucht. Anhand von quantitativer RT-PCR und Promoter-Reporter-Fusionen wurde die differentielle Expression von mehreren TF-Transkripten in den verschiedenen Zelltypen bestätigt. Die Charakterisierung eines potentiellen GRAS TF (MtGRAS8) konnte eine stark Symbiose- und Phosphat-abhängige Induktion von Transkripten bestätigt werden. Mutanten mit verringerter MtGras8 Transkriptmenge wiesen eine verringerte Arbuskelzahl und deformierte Arbuskeln auf. MtGras8 scheint daher an der Arbuskelentwicklung beteiligt zu sein. Vorherige Experimente zeigten, dass MtGras8 Transkripte, von der Phosphat-regulierten MikroRNA5204* geschnitten werden (Devers et al., 2011). Dies konnte durch Überexpression der MikroRNA5204* in vivo bestätigt werden. Weiterhin konnten Protein-Protein-Interaktionen von MtGras8 mit bekannten GRAS TFs (NSP1, NSP2, RAM1) nachgewiesen und daraus eine Verbindung zu bekannten Symbiose-induzierten Signalkaskaden geschlossen werden. In dieser Arbeit wurde erstmals die Umprogrammierung von nicht-arbuskelhaltigen Zellen untersucht und neue Regulationselemente für die Kontrolle der Arbuskelentwicklung, wie MtGRAS8 und MikroRNA5204*, charakterisiert. KW - arbuskuläre Mykorrhiza-Symbiose KW - Transkriptionsfaktoren KW - Zelltyp-spezifisch KW - Transkriptomanalyse KW - arbuscular mycorrhizal symbiosis KW - transcription factors KW - cell type-specific KW - transcriptome analysis Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-70664 ER - TY - GEN A1 - Bentele, Kajetan A1 - Saffert, Paul A1 - Rauscher, Robert A1 - Ignatova, Zoya A1 - Bluethgen, Nils T1 - Efficient translation initiation dictates codon usage at gene start T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 912 KW - codon usage KW - mRNA structure KW - translation Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441337 SN - 1866-8372 IS - 912 ER - TY - GEN A1 - Baumann, Tobias A1 - Arndt, Katja Maren A1 - Müller, Kristian M. T1 - Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. Results: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. Conclusions: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 983 KW - cohesive ends KW - DNA cleavage KW - genetic vectors KW - modified primers KW - molecular methods KW - polymerase chain reaction KW - recombinant Escherichia coli KW - restriction enzymes Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431085 SN - 1866-8372 IS - 983 ER - TY - GEN A1 - Barbosa Pfannes, Eva Katharina A1 - Anielski, Alexander A1 - Gerhardt, Matthias A1 - Beta, Carsten T1 - Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells N2 - Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 239 KW - cyclic-gmp KW - dictyostelium-discoideum KW - ena/vasp proteins KW - osmotic-stress KW - chemotaxis KW - phosphorylation KW - amp KW - cytoskeleton KW - oscillations KW - chemoattractant Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-94984 SP - 1456 EP - 1463 ER -