1,713 research outputs found

    Chromatin Structure in the Cellular Slime Mold Dictyostelium discoideum

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    The structure of Dictyostelium discoideum chromatin has been studied by the following techniques: electron microscopy, staphylococcal nuclease digestion, acrylamide gel electrophoresis, sucrose gradient centrifugation, and melting. The basic unit of chromatin is the nucleosome, which is a particle 98.6 angstrom in diameter. Approximately 50% of the chromatin is protected from nuclease digestion, but this decreases when protease activity is not inhibited. The nucleosome contains 187 base pairs of DNA, including a 137-base-pair core and a 50-base-pair linker. The monomer nucleosome has an s20,w value of 11.5 S on isokinetic sucrose gradients. When the chromatin is melted, four transitions are observed, at 54.5 degrees, 66.7 degrees, 74.9 degrees, and 79.7 degrees. The structure of Dictyostelium chromatin is very similar to that seen in higher eukaryotes

    The role of ADP-ribosylation in regulating DNA interstrand crosslink repair

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    ADP-ribosylation by ADP-ribosyltransferases (ARTs) has a well-established role in DNA strand break repair by promoting enrichment of repair factors at damage sites through ADP-ribose interaction domains. Here we exploit the simple eukaryote Dictyostelium to uncover a role for ADP-ribosylation in regulating DNA interstrand crosslink repair and redundancy of this pathway with non-homologous end-joining (NHEJ). In silico searches identify a protein that contains a permutated macrodomain (Aprataxin/APLF-and-PNKP-Like protein; APL). Structural analysis reveals permutated macrodomains retain features associated with ADP-ribose interactions and APL is capable of binding poly-ADP-ribose through its macrodomain. APL is enriched in chromatin in response to cisplatin, an agent that induces DNA interstrand crosslinks (ICLs). This is dependent on the macrodomain of APL, and the ART Adprt2, indicating a role for ADP-ribosylation in the cellular response to cisplatin. Although adprt2− cells are sensitive to cisplatin, ADP-ribosylation is evident in these cells due to redundant signalling by the DSB-responsive ART Adprt1a, promoting NHEJ-mediated repair. These data implicate ADP-ribosylation in DNA ICL repair and identify NHEJ can function to resolve this form of DNA damage in the absence of Adprt2

    The Actinome of Dictyostelium discoideum in Comparison to Actins and Actin-Related Proteins from Other Organisms

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    Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes

    The transcription factor Spores Absent A is a PKA dependent inducer of Dictyostelium sporulation

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    Abstract Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation

    Sun-1, a regulator of the nuclear shape in Dictyostelium discoideum

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    In worms, flies and mammals, the nucleus is attached to the cytoskeleton by binding of the SUN domain proteins to the KASH domain proteins in the outer nuclear membrane that connects the nucleus to either Factin or microtubules. The association of the SUN domain proteins with both the nuclear lamina and the KASH domain proteins is attributed as a molecular bridging complex required for intracellular positioning and migration of the nucleus. In this study, we investigated the role of Sun-1 and interaptin, a SUN and a KASH domain protein in Dictyostelium discoideum in nuclear positioning. In marked contrast to the model proposed for higher eukaryotes, Sun-1 and interaptin localized to the nuclear envelope in a competitive fashion, which may be due to a competitive binding to a yet unknown partner. Distinct from the higher eukaryotes, which engage the nuclear lamina for INM retention of the SUN domain proteins, an alternative mechanism may be considered for D. discoideum that lacks lamins. We provided evidence that Sun-1 can be immobilized in the INM by binding to chromatin probably via its N-terminus. The association of Sun-1 with chromatin may not only contribute to the formation of a bridging complex, but also control the juxtaposition of the nucleus and centrosome, as the truncation of the Sun-1 N-terminus disconnected the nucleus and the centrosome. Consequently, the disconnection may lead to chromosome instability as indicated by: (1) Nuclear envelope deformations (2) Enlargement of the nuclear and cell size (3) Tendencies for aneuploidy and (4) Amplification of the centrosome number. These data suggest that Sun-1 may regulate the nuclear shape, chromosome stability and the connection of the nuclei to the centrosomes

    The Carboxy-Terminal Domain of Dictyostelium C-Module-Binding Factor Is an Independent Gene Regulatory Entity

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    The C-module-binding factor (CbfA) is a multidomain protein that belongs to the family of jumonji-type (JmjC) transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF) motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD). An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo

    The actinome of Dictyostelium amoebae

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    The highly conserved protein actin is the building block in the cytoskeleton of eukaryotic cells and provides a structural framework known as the microfilament system.The molecular principle of actin-based amoeboid movement was so successful in evolution that it was kept nearly identically from lower (e.g. amoebae) to higher (e.g. neutrophils) eukaryotes.To understand this type of cellular movement one has first to identify and to characterize the proteins which play a major role during the dynamic rearrangement of actin. The collection of actin isoforms, of actin-variants and actin related proteins (Arps) in a given cell is known as the 'actinome' whose number of proteins can be quite different from one organism to the next. Therefore, the present work describes studies on the actinome of thesocial amoeba Dictyostelium discoideum, compares the findings with actinomes from other organisms, and discusses similarities and alterations that might have happened during evolution. D. discoideum is among the oldest organisms which exhibit actin-based amoeboid movement, the genome is completely sequenced and the system can be easily studied by molecular and biochemical approaches. The study was started using bioinformatics and the computational methods provided a global view on the D. discoideum actinome. It turned out that the D. discoideum genome conprises a total of 33 actin and 8 Arp genes, seven actin genes are putative pseudogenes. Interestingly, there are 17 distinguishable actin genes which code for identical proteins. Phylogenetic analyses helped to understand the putative duplication events during evolution. Modelling of the three-dimensional structures showed that the typical actin-fold, the ATP-binding pocket, and other functional domains are highly conserved. Homologues of the members of the D. discoideum actinome across various model organisms clearly demonstrated which amino acids in conserved domains are of special importance. All Arp subfamilies that are found in mammals are also present in D. discoideum. Two of the actin related proteins, Arp5 and Arp6, were selected for molecular and cellular studies. Using fluorescently labeled fusion proteins first data indicated that both Arps are present also in the nucleus, suggesting an involvement in chromatin reorganization.Das hochkonservierte Protein Aktin ist die grundlegende Einheit des Zytoskeletts in eukaryontischen Zellen und formt das als Mikrofilamentsystem bezeichnete intrazelluläre Gerüst. Das molekulare Prinzip der amoeboiden Fortbewegung basiert auf Aktin und war so erfolgreich in Lauf der Evolution, dass es nahezu unverändert geblieben ist von niederen (z.B. Amoeben) zu höheren (z.B. Neutrophilen) Eukaryonten. Um diesen Typ der zellulären Forbewegung verstehen zu können, muss man zuerst die Proteine identifizieren und charakterisieren, die eine Hauptrolle bei den dynamischen Umbauvorgängen im Aktin Zytoskelett spielen. Alle Aktine, Aktin-Isoformen und Aktin-verwandten Proteine ('actin related proteins, 'Arps') werden als 'Aktinom' zusammengefasst, dessen Proteinspektrum sich in den verschiedenen Organismen stark unterscheiden kann. Die vorliegende Arbeit sollte deshalb das Aktinom der sozialen Amoebe Dictyostelium discoideum charakterisieren, die Ergebnisse mit den Aktinomen anderer Organismen vergleichen, und Ähnlichkeiten bzw. Unterschiede, die während der Evolution aufgetreten sind, diskutieren. D. discoideum gehört zu den ältesten Organismen, deren amoeboide Fortbewegung auf Aktin beruht, das Genom ist vollständig sequenziert, und das Modell-System kann für molekulare und biochemische Versuchsansätze verwendet werden. Bioinformatik und Rechner-basierte Methoden führten zu einem weitgehenden Einblick in das D. discoideum Aktinom. Es zeigte sich, dass das D. discoideum Genom 33 Aktin- und 8 Arp-Gene enthält, 7 Aktin-Gene wurden als vermutliche Pseudogene eingestuft. Interessanterweise wurden 17 unterscheidbare Aktin-Gene gefunden, die für identische Proteine kodierten. Phylogenetische Analysen haben geholfen, die vermutlichen Genduplikationen im Lauf der Evolution zu verstehen. Die Modellierung der dreidimensionalen Strukturen gab wichtige Einblicke, inwieweit die typische Aktinstruktur, die ATP-Bindungstasche, und andere funktionelle Domänen erhalten geblieben waren. Homologe der Proteine aus dem D. discoideum Aktinom in anderen Modellorganismen demonstrierten ganz klar, welche Aminosäuren in konservierten Domänen von besonderer Bedeutung sind. Darüber hinaus konnte festgestellt werden, dass alle Arp Unterfamilien in D. discoideum auch in Säugern vorhanden sind. Zwei der Aktin-verwandten Proteine, nämlich Arp5 und Arp6, wurden für weitergehende molekulare und zelluläre Untersuchungen ausgewählt. Mit Hilfe von GFP-markierten Fusionsproteinen konnte gezeigt werden, dass beide Arps auch im Nukleus vorhanden und dort wohl an der Umorganisation des Chromatins beteiligt sind
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