TY - JOUR A1 - Schindler, Julia A1 - Richter, Tobias A1 - Mar, Raymond T1 - Does generation benefit learning for narrative and expository texts? A direct replication attempt JF - Applied Cognitive Psychology N2 - Generated information is better recognized and recalled than information that is read. This so‐called generation effect has been replicated several times for different types of material, including texts. Perhaps the most influential demonstration was by McDaniel et al. (1986, Journal of Memory and Language, 25, 645–656; henceforth MEDC). This group tested whether the generation effect occurs only if the generation task stimulates cognitive processes not already stimulated by the text. Numerous studies, however, report difficulties replicating this text by generation‐task interaction, which suggests that the effect might only be found under conditions closer to the original method of MEDC. To test this assumption, we will closely replicate MEDC's Experiment 2 in German and English‐speaking samples. Replicating the effect would suggest that it can be reproduced, at least under limited conditions, which will provide the necessary foundation for future investigations into the boundary conditions of this effect, with an eye towards its utility in applied contexts. KW - expository texts KW - generation effect KW - learning KW - narrative texts KW - replication Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224496 VL - 35 IS - 2 SP - 559 EP - 564 ER - TY - THES A1 - Adenugba, Akinbami Raphael T1 - Functional analysis of the gene organization of the pneumoviral attachment protein G T1 - Funktionelle Analyse der Genorganisation des pneumoviralen Attachment-Protein G N2 - The putative attachment protein G of pneumonia virus of mice (PVM), a member of the Pneumoviruses, is an important virulence factor with so far ambiguous function in a virus-cell as well as in virus-host context. The sequence of the corresponding G gene is characterized by significant heterogeneity between and even within strains, affecting the gene and possibly the protein structure. This accounts in particular for the PVM strain J3666 for which two differing G gene organizations have been described: a polymorphism in nucleotide 65 of the G gene results in the presence of an upstream open reading frame (uORF) that precedes the main ORF in frame (GJ366665A) or extension of the major G ORF for 18 codons (GJ366665U). Therefore, this study was designed to analyse the impact of the sequence variations in the respective G genes of PVM strains J3666 and the reference strain 15 on protein expression, replication and virulence. First, the controversy regarding the consensus sequence of PVM J3666 was resolved. The analysis of 45 distinct cloned fragments showed that the strain separated into two distinct virus populations defined by the sequence and structure of the G gene. This division was further supported by nucleotide polymorphisms in the neighbouring M and SH genes. Sequential passage of this mixed strain in the cell line standardly used for propagation of virus stocks resulted in selection for the GJ366665A-containing population in one of two experiments pointing towards a moderate replicative advantage. The replacement of the G gene of the recombinant PVM 15 with GJ366665A or GJ366665U, respectively, using a reverse genetic approach indicated that the presence of uORF within the GJ366665A significantly reduced the expression of the main G ORF on translational level while the potential extension of the ORF in GJ366665U increased G protein expression. In comparison, the effect of the G gene-structure on virus replication was inconsistent and dependent on cell line and type. While the presence of uORF correlated with a replication advantage in the standardly used BHK-21 cells and primary murine embryonic fibroblasts, replication in the murine macrophage cell line RAW 264.7 did not. In comparison, the GJ366665U variant was not associated with any effect on replication in cultured cells at all. Nonetheless, in-vivo analysis of the recombinant viruses associated the GJ366665U gene variant, and hence an increased G expression, with higher virulence whereas the GJ366665A gene, and therefore an impaired G expression, conferred an attenuated phenotype to the virus. To extend the study to other G gene organizations, a recombinant PVM expressing a G protein without the cytoplasmic domain and for comparison a G-deletion mutant, both known to be attenuated in vivo, were studied. Not noticed before, this structure of the G gene was associated with a 75% reduction in G protein expression and a significant attenuation of replication in macrophage-like cells. This attenuation was even more prominent for the virus lacking G. Taking into consideration the higher reduction in G protein levels compared to the GJ366665A variant indicates that a threshold amount of G is required for efficient replication in these cells. In conclusion, the results gathered indicated that the expression levels of the G protein were modulated by the sequence of the 5’ untranslated region of the gene. At the same time the G protein levels modulated the virulence of PVM. N2 - Das mutmaßliche „attachment“ Protein G des Pneumonievirus der Maus (PVM), einem Mitglied des Genus Pneumovirus, ist ein bedeutender Virulenzfaktor, mit allerdings noch nicht vollständig verstandener Funktion. Dabei zeichnet sich die Sequenz des G-Gens durch Nukleotid-Polymorphismen und damit verbundenen Variationen in der Genorganisation und möglicherweise der Proteinstruktur sowohl zwischen als auch innerhalb von PVM-Stämmen aus. Insbesondere für den PVM-Stamm J3666 wurden zwei verschiedene Organisationen des G-Gens beschrieben: ein Polymorphismus des Nukleotids 65 des G-Genes erzeugt einen neuen „upstream Open reading frame“ (uORF), der dem eigentlichen G-ORF vorausgeht (GJ366665A), oder führt zu einer Verlängerung des eigentlichen G-ORF von G um 18 Kodons (GJ366665U). Ziel dieser Studie war es deshalb, die Auswirkung dieser Sequenzvariabilitäten der für PVM J3666 beschriebenen G-Gene im Vergleich zu dem des Referenzstamms PVM 15 bezüglich Proteinexpression, der Virusreplikation und der Virulenz zu untersuchen. Als erstes wurden die beschriebenen Sequenzunterschiede bezüglich des PVM-Stamms J3666 untersucht. Die Analyse von 45 verschiedenen klonierten Fragmenten von PVM J3666 zeigte, dass es sich bei diesem Stamm eigentlich um zwei separate Viruspopulationen handelt, die sich durch die Sequenz und Struktur des G-Genes definieren lassen. Diese Unterscheidung wird durch weitere Nukleotid-Polymorphismen in den benachbarten Genen, M und SH, gestärkt. Sequenzielle Passagierung dieses gemischten Stammes in der standardmäßig zur Virusanzucht verwendeten BHK-21-Zelllinie resultierte in einem von zwei Experimenten in der Selektion der GJ366665A-Population, das ein Hinweis auf einen moderaten Replikationsvorteil darstellt. Der Austausch des G-Gens des Referenzstamms PVM 15 durch GJ366665A oder GJ366665U mithilfe der Reversen Genetik, zeigte, dass der uORF innerhalb von GJ366665A zu einer deutlich reduzierten Expression des eigentlichen G-ORF führt. Andererseits führte die potenzielle Verlängerung des ORF in GJ366665U zu einer im gleichen Maße erhöhten Expression des G-Proteins. Dagegen war der Einfluss der G-Genorganisation auf die Virusvermehrung in Zellkultur in Abhängigkeit von Zelllinie und Zelltyp inkonsistent. Während ein uORF mit einem Replikationsvorteil in BHK-21-Zellen und primären murinen embryonen Fibroblasten korrelierte, war dies in der murinen Makrophagen-Zelllinie RAW 264.7 nicht zu beobachten. Im Vergleich dazu konnte die GJ366665U-Variante nicht mit einem Einfluss auf die Virusvermehrung in Verbindung gebracht werden. Nichtsdestotrotz, konnte die GJ366665U-Variante, und damit eine erhöhte Expression von G, mit einer gesteigerten Virulenz assoziiert werden, während die GJ366665A-Variante, d. h. eine verringerte G-Expression zur Attenuierung des Virus führte. Die Untersuchungen wurden auf weitere G-Genstrukturen, d.h. ein rekombinantes PVM, rPVM-Gt, das ein N-terminal verkürztes G-Protein exprimiert, ausgeweitet. Zum Vergleich wurde eine Deletionsmutante des kompletten G-Gens, rPVM-ΔG, mit einbezogen. Von beiden Viren war bereits bekannt, dass sie in vivo attenuiert sind. Die Organisation des Gt-Gens war mit einer um 75 % verringerten Expression des entsprechenden Proteins assoziiert, was zuvor nicht beobachtet worden war. Zugleich zeigte rPVM-Gt eine deutliche Attenuierung der Replikation in RAW 264.7-Zellen und primären Mausmakrophagen, die von der G-Deletionsmutante noch übertroffen wurde. Die im Vergleich zu der GJ366665A-Variante deutlich höhere Reduktion der G-Expression dieser beiden G-Mutanten in Betracht ziehend, scheint dies darauf hinzuweisen, dass eine bestimmte Mindestexpression von G für eine effiziente Virusvermehrung in diesen Zellen benötigt wird. Zusammenfassend deuten die erhaltenen Ergebnisse darauf hin, dass die Expression des G-Proteins durch die jeweiligen 5’ nicht-translatierte Region des Gens moduliert wird, was einen neuen Mechanismus für Negativstrang-RNA-Viren darstellt. Zugleich moduliert die Expressionsrate von G die Virulenz von PVM. KW - G glycoprotein KW - protein regulation and expression KW - Pneumoviruses KW - regulation KW - expression KW - replication KW - virulence KW - 5`-UTR KW - PVM KW - RSV Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128146 ER - TY - JOUR A1 - Leal, Andrea Zurita A1 - Schwebs, Marie A1 - Briggs, Emma A1 - Weisert, Nadine A1 - Reis, Helena A1 - Lemgruber, Leondro A1 - Luko, Katarina A1 - Wilkes, Jonathan A1 - Butter, Falk A1 - McCulloch, Richard A1 - Janzen, Christian J. T1 - Genome maintenance functions of a putative Trypanosoma brucei translesion DNA polymerase include telomere association and a role in antigenic variation JF - Nucleic Acids Research N2 - Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to off-spring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation. KW - cross-link repair KW - cell cycle KW - gene expression KW - low fidelity KW - replication KW - bypass KW - theta KW - reveals KW - binding Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230579 VL - 48 IS - 17 ER - TY - JOUR A1 - Eidel, Matthias A1 - Kübler, Andrea T1 - Wheelchair Control in a Virtual Environment by Healthy Participants Using a P300-BCI Based on Tactile Stimulation: Training Effects and Usability JF - Frontiers in Human Neuroscience N2 - Tactile stimulation is less frequently used than visual for brain-computer interface (BCI) control, partly because of limitations in speed and accuracy. Non-visual BCI paradigms, however, may be required for patients who struggle with vision dependent BCIs because of a loss of gaze control. With the present study, we attempted to replicate earlier results by Herweg et al. (2016), with several minor adjustments and a focus on training effects and usability. We invited 16 healthy participants and trained them with a 4-class tactile P300-based BCI in five sessions. Their main task was to navigate a virtual wheelchair through a 3D apartment using the BCI. We found significant training effects on information transfer rate (ITR), which increased from a mean of 3.10–9.50 bits/min. Further, both online and offline accuracies significantly increased with training from 65% to 86% and 70% to 95%, respectively. We found only a descriptive increase of P300 amplitudes at Fz and Cz with training. Furthermore, we report subjective data from questionnaires, which indicated a relatively high workload and moderate to high satisfaction. Although our participants have not achieved the same high performance as in the Herweg et al. (2016) study, we provide evidence for training effects on performance with a tactile BCI and confirm the feasibility of the paradigm. KW - brain-computer interface (BCI) KW - event-related-potential (ERP) KW - P300 KW - tactile KW - wheelchair control KW - tactually evoked potentials KW - replication Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-207775 SN - 1662-5161 VL - 14 ER - TY - JOUR A1 - Gressmann, Marcel A1 - Janczyk, Markus T1 - The (Un)Clear Effects of Invalid Retro-Cues JF - Frontiers in Psychology N2 - Studies with the retro-cue paradigm have shown that validly cueing objects in visual working memory long after encoding can still benefit performance on subsequent change detection tasks. With regard to the effects of invalid cues, the literature is less clear. Some studies reported costs, others did not. We here revisit two recent studies that made interesting suggestions concerning invalid retro-cues: One study suggested that costs only occur for larger set sizes, and another study suggested that inclusion of invalid retro-cues diminishes the retro-cue benefit. New data from one experiment and a reanalysis of published data are provided to address these conclusions. The new data clearly show costs (and benefits) that were independent of set size, and the reanalysis suggests no influence of the inclusion of invalid retro-cues on the retro-cue benefit. Thus, previous interpretations may be taken with some caution at present. KW - visual working memory KW - retro-cue KW - attention KW - replication Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165296 VL - 7 IS - 244 ER - TY - JOUR A1 - Wanzek, Katharina A1 - Schwindt, Eike A1 - Capra, John A. A1 - Paeschke, Katrin T1 - Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability JF - Nucleic Acids Research N2 - The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity. KW - replication KW - regulation KW - genome integrity KW - Saccharomyces cerevisiae Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170577 VL - 45 IS - 13 ER - TY - JOUR A1 - Phillips, Jane A. A1 - Chan, Angela A1 - Paeschke, Katrin A1 - Zakian, Virginia A. T1 - The Pif1 helicase, a negative regulator of telomerase, acts preferentially at long telomeres JF - PLoS Genetics N2 - Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres. KW - Saccharomyces cerevisiae telomeres KW - DNA helicase KW - Pol II KW - in vitro KW - genome instability KW - yeast telomerase KW - G-quadruplex motifs KW - elongation KW - length KW - replication Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148722 VL - 11 IS - 4 ER - TY - JOUR A1 - Jun, Kyong-Hwa A1 - Gholami, Spedideh A1 - Song, Tae-Jin A1 - Au, Joyce A1 - Haddad, Dana A1 - Carson, Joshua A1 - Chen, Chun-Hao A1 - Mojica, Kelly A1 - Zanzonico, Pat A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Szalay, Aladar A1 - Fong, Yuman T1 - A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter JF - Journal of Experimental & Clinical Cancer Research N2 - Background: Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with Tc-99m pertechnetate scintigraphy and I-124 positron emission tomography (PET). Methods: GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. Tc-99m pertechnetate scintigraphy and I-124 microPET imaging were performed. Results: GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK-45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by Tc-99m pertechnetate scintigraphy and I-124 microPET imaging 2 days after treatment. Conclusions: GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings. KW - oncolytic viral therapy KW - GLV-1 h153 KW - gastric cancer KW - human sodium iodide symporter (hNIS) KW - radioiodine therapy KW - gene therapy KW - expression KW - replication KW - stomach KW - tumors KW - surgery Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117716 SN - 1756-9966 VL - 33 IS - 2 ER - TY - JOUR A1 - Carsillo, Thomas A1 - Huey, Devra A1 - Levinsky, Amy A1 - Obojes, Karola A1 - Schneider-Schaulies, Jürgen A1 - Niewiesk, Stefan T1 - Cotton Rat (Sigmodon hispidus) Signaling Lymphocyte Activation Molecule (CD150) Is an Entry Receptor for Measles Virus JF - PLOS ONE N2 - Cotton rats (Sigmodon hispidus) replicate measles virus (MV) after intranasal infection in the respiratory tract and lymphoid tissue. We have cloned the cotton rat signaling lymphocytic activation molecule (CD150, SLAM) in order to investigate its role as a potential receptor for MV. Cotton rat CD150 displays 58% and 78% amino acid homology with human and mouse CD150, respectively. By staining with a newly generated cotton rat CD150 specific monoclonal antibody expression of CD150 was confirmed in cotton rat lymphoid cells and in tissues with a pattern of expression similar to mouse and humans. Previously, binding of MV hemagglutinin has been shown to be dependent on amino acids 60, 61 and 63 in the V region of CD150. The human molecule contains isoleucine, histidine and valine at these positions and binds to MV-H whereas the mouse molecule contains valine, arginine and leucine and does not function as a receptor for MV. In the cotton rat molecule, amino acids 61 and 63 are identical with the mouse molecule and amino acid 60 with the human molecule. After transfection with cotton rat CD150 HEK 293 T cells became susceptible to infection with single cycle VSV pseudotype virus expressing wild type MV glycoproteins and with a MV wildtype virus. After infection, cells expressing cotton rat CD150 replicated virus to lower levels than cells expressing the human molecule and formed smaller plaques. These data might explain why the cotton rat is a semipermissive model for measles virus infection. KW - immune suppression KW - SLAM CDW150 KW - cellular receptor KW - wild-type KW - glycoproteins KW - replication KW - morbilliviruses KW - CD46 KW - strains KW - spread Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115178 SN - 1932-6203 VL - 9 IS - 10 ER - TY - THES A1 - Bernardi, Tina Sibylla T1 - Die Rolle des Zytoskeletts für die Replikation des Masernvirus - insbesondere seiner Komponenten Aktin und Tubulin T1 - The role of the cytosceleton in the replication of the measles virus - in particular its components actin and tubulin N2 - Für die Replikation des Masernvirus wird den Komponenten des Zytoskeletts eine wichtige Rolle zugeschrieben. Die vorliegende Arbeit zeigt, dass Aktin in polymerisierter Form vorliegen muss, um das Budding zu ermöglichen. Die Beeinflussung der ersten Schritte des Replikationszyklus konnte für Aktin, vor allem aber für Tubulin nachgewiesen werden, so dass ein Transport des viralen Genoms zum Ort seiner Replikation entlang der Mikrotubuli möglich wäre. N2 - The components of the cytosceleton play an important role in the replication process of the measles virus. This paper shows that actin has to be in polymerized form to enable budding. Its influence on the first steps of the replication cycle is shown for actin and in particular tubulin, enabling a transport of the viral genome along the microtubule to the location of its replication. KW - Masern KW - Replikation KW - Zytoskelett KW - Aktin KW - Tubulin KW - measles KW - replication KW - cytosceleton KW - actin KW - tubulin Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-17345 ER - TY - THES A1 - Auth, Tanja T1 - Funktionelle Analyse der Interaktion und Lokalisation von Replikationsfaktoren und replikationsrelevanten Proteinen in Mauszellen T1 - Functional analysis of the interaction and localization of replicationfactors and replicationrelevant proteins in murine cells N2 - Zeil dieser Arbeit war die Identifikation von Proteinen, die mit Bestandteilen des für die DNA-Replikation essentiellen präreplikativen Komplexes in der Maus wechselwirken. Hierbei konnten Interkationen des Heterochromatin Proteins 1a mit den Replikationsfaktoren ORC1, ORC2 und CDC6 sowohl in Two Hybrid-Studien als auch in Immunpräzipitationen gezeigt werden. Darüberhinaus konnten signifikante Kolokalisationen dieser Proteine mit HP1a an heterochromatischen Regionen in murinen NIH3T3-Zellen nachgewiesen werden. Ebenfalls konnte hier erstmals eine Lokalisation von HP1a am Centrosom demonstriert werden. Ein siRNA-vermittelter Knock Down von HP1a zeigte jedoch keinen direkten Einfluß auf die Replikation. Es konnte hingegen gezeigt werden, daß ein Knock Down von HP1a in signifikatnen Defekten in der Cytokinese und einer deutlich verlangsamten Zellproliferation resultiert. So konnten häufig multinukleäre Zellen und eine Arretierung in der G1-Phase beobachtet werden. Weiterhin wurde der Einfluß der Phosphorylierung von HP1a durch die Casein Kinase II mithilfe von Phosphorylierungsmutanten untersucht. Im Gegensatz zu Drosophila-Zellen zeigten sich in murinen Zellen jedoch keine Auswirkungen dieser Mutationen auf die Lokalisation von HP1a an Heterochromatin. Wieterhin konnten Interaktionen des Replikationsinhibitors Geminin mit den Replikationsproteinen ORC1, ORC2 und CDC7 sowohl im Two Hybrid-System als auch in Immunpräzipitationen gezeigt werden, die unterschiedliche Zellzyklusabhängigkeiten aufwiesen. In murinen NIH3T3-Zellen zeigte eine Knock Down von Geminin jedoch im Gegensatz zu anderen Zellinien keinen Einfluß auf die Replikation. In weiteren Teilen dieser Arbeit konnten Interaktionen des Retinoblastoma Proteins mit ORC2 und MCM7 sowohl in Two Hybrid- als auch in Immunpräzipitations-Experimenten gezeigt werden. Darüberhinaus wies Pescadillo Interaktionen mit den Replikationsproteinen ORC2, ORC6, MCM2, MCM3, MCM6 und CDC45 im Two Hybrid-System und Interaktionen mit MCM2 und MCM3 in Biolumineszenz-Resonanzenergietransfer-Experimenten auf. Eine Kolokalisation von Pescadillo und ORC6 in den Nukleoli läßt auf eine Funktion beider Proteinen bei der Ribosomen Biogenese schließen. Es konnten ebenfalls Interaktionen der Untereinheit E1 des humanen Papillomavirus Subtyp 11 mit den Replikationsfaktoren ORC2,3,4,5,6, MCM2, MCM3, MCM6, CDC6, CDC7, CDT1, HP1a, Rb und Pescadillo im Two Hybrid-System beobachtet werden. N2 - The identification of proteins, which interact with members of the for the initiation of DNA replication necessary prereplicative complex in murine cells was of greatest interest for this work. Thereby, interactions of the heterochromatin protein 1a with the replication proteins ORC1, ORC2 and CDC6 were demonstrated in the two-hybrid system as well as in immunoprecipitations. Furthermore, significant colocalisation of these proteins with HP1a could be observed at heterochromatic regions in murine NIH3T3 cells. In NIH3T3 cells HP1a is also localisized on the centrosome. Knock down of HP1a by siRNAs showed however no direct influence on DNA- replication. Though, a knock down of HP1a resulted in significant defects in cytokinesis and reduced cell proliferation. Thus, frequntly cells with several nuclei and an arrest of the cells in G1 phase could be observed. Furthermore, the influence od phosphorylation of HP1a by Casein kinase II was analysed with phosphorylationssite In contrast to Drosophila cells there were no differences of the localization of Hp1a on heterochromatin in murine NIH3T3 cells. Also interactions of the replication inhibitor Geminin with the replication factors ORC1, ORC2 und CDC7 were found in two-hybrid analyses as well as in immunoprecipitations, which showed several cell cycle dependence. However, in murine NIH3T3 cells a knock down of Geminin showed in contrast to several other cell lines no influences on DNA replication. In a furthe part of this work interactions of the Retinoblastoma protein with ORC2 and MCM7 were observed in two-hybrid experiments as well as in immunoprecipitations.Furthermore, Pescadillo showed interaction with the replication proteins ORC2, ORC6, MCM2, MCM3, MCM6 and CDC45 in the two-hybrid system as well as interactions with MCM2 and MCM3 in bioluminescence resonance energytransfer experiments. The coloalization of Pescadillo and ORC6 in nucleoli points to a function of these proteins in ribosome biogenesis. In a further part of this work there were also interactions of the protein E1 of the human Papilloma virus 11 with the replication proteins ORC2,3,4,5,6, MCM2, MCM3, MCM6, CDC6, CDC7, CDT1, HP1a, Rb and Pescadillo observed in two-hybrid screenings. KW - Maus KW - Replikation KW - Proteine KW - Replikation KW - Heterochromatin Protein 1 KW - Geminin KW - Retinoblastoma Protein KW - Pescadillo KW - replication KW - heterochromatin protein 1 KW - Geminin KW - retinobalstoma protein KW - Pescadillo Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-13082 ER - TY - THES A1 - Schulz, Carsten T1 - Analyse des Replikationsprozesses der Maus T1 - Analysis of the murine replication process N2 - Die Initiation der DNA-Replikation ist in Eukaryonten ein hochkonservierter Prozess. Zuerst bindet der „origin recognition complex“ (ORC) an Replikationsstartpunkte chromosomaler DNA und stellt das Startsignal für die Assemblierung des präreplikativen Komplexes (pre-RC) dar. Anschließend assoziieren die Initiationsfaktoren CDC6 und CDT1 mit dem ORC. Durch die Rekrutierung des MCM-Komplexes wird der pre-RC schließlich vervollständigt. Die Aktivität der CDC7/DBF4-Kinase und die Anlagerung von CDC45 lizensiert den Origin für die DNA-Replikation. Ein Ziel dieser Arbeit war, den vollständigen murinen ORC rekombinant darzustellen. Um den gesamten Komplex durch Copräzipitation zu isolieren, wurden ORC1, 3, 4, 5 und 6 als Wildtyp-Proteine und ORC2 mit einer N-terminalen Poly-His-Domäne mit Hilfe von Baculoviren koexprimiert. Nach der Aufreinigung konnten, mit Ausnahme von ORC3, alle ORC-Untereinheiten in den Elutionsfraktionen immundetektiert werden. Eine Gelfiltration der Fraktionen ließ auf die Isolierung eines 450 kD großen Komplexes schließen, der mindestens fünf der sechs ORC-Untereinheiten enthielt. Dies zeigt, dass der murine ORC als Holokomplex rekombinant isoliert werden kann. In einem weiteren Teil dieser Arbeit sollte die Rolle des MCM-Komplexes bei der Termination der DNA-Replikation am 3'-Ende muriner rDNA-Transkriptionseinheiten untersucht werden. Durch polare Replikationsgabelbarrieren im 3'-Bereich der ribosomalen Gene wird über die Kontrahelikaseaktivität von TTF-I die Bewegungsrichtung der Replikation auf die Richtung der Transkription limitiert. In dieser Arbeit sollte festgestellt werden, ob dies auch bei der murinen MCM4/6/7-Helikase der Fall ist. Um MCM4/6/7-Hexamere zu isolieren, wurden die Untereinheiten MCM4 und 7 in Wildtyp-Form und MCM6 mit einem N-terminal fusionierten HA-Tag mittels Baculoviren koexprimiert. Zur Durchführung der Kontrahelikasestudien musste die Helikaseaktivität der isolierten Komplexe ermittelt werden. Bereits mit kurzen partiell doppelsträngigen M13-Substraten (17 nt) zeigte sich eine geringere Entwindungsfähigkeit als in der Literatur beschrieben. Bei weiteren Helikasestudien wurden DNA-Substrate (30 nt) mit einem 5'-Überhang sowie SSB bzw. RPA eingesetzt. Zwar konnte so eine Steigerung der Helikaseaktivität von MCM4/6/7 verzeichnet werden, jedoch fand diese nicht in ausreichendem Maße statt. Zudem war das entwundene Oligonukleotid einem Abbau unterworfen, dessen Ursache nicht aufgeklärt werden konnte. Aufgrund der zu geringen Helikaseaktivität im Hinblick auf die TTF-I-Kontrahelikasestudien wurden diese Arbeiten eingestellt. Ein weiterer Aspekt dieser Arbeit war der Transport von MCM-Proteinen in den Zellkern. Der MCM-Komplex ist in fast allen Organismen konstitutiv im Zellkern lokalisiert. Die Überexpression einzelner exogener MCM-Proteine zeigte allerdings, dass nur MCM2 und 3 mit Hilfe ihrer ihrer NLS-Motive in den Kern transportiert werden, während dies bei MCM4 bis 7 nicht erfolgt. Two-Hybrid-Studien unserer Arbeitsgruppe ließen auf paarweise Wechselwirkungen der MCM4 bis 7-Untereinheiten mit MCM2 bzw. MCM3 schließen. Deshalb wurden EGFP-MCM-Proteine zusammen mit Wildtyp-MCM-Proteinen in Mauszellen koexprimiert. Dabei zeigte sich, dass MCM2 die Proteine MCM4, 6 und 7 in den Kern transportiert, während MCM3 nur MCM5 in den Zellkern einschleust. Weitere Interaktionen zwischen MCM6 und 4 sowie zwischen MCM6 und 7 konnten bei MCM4/6/7-Aufreinigungen beobachtet werden. Zuletzt wurde noch die Lokalisation von CDT1 in der OBR-Region des murinen rDNA-Cistrons untersucht. Bislang wurde nur in S. cerevisiae eine sequenzspezifische ORC-Bindung an ACS-Bereiche identifiziert. In unserer Arbeitsgruppe konnte im murinen rDNA-Cluster stromaufwärts des Transkriptionsstartpunktes ein Origin charakterisiert und die Bindungstelle verschiedener Initiatorproteine um die Position -2500 eingegrenzt werden. Die Assoziation von CDT1 mit derselben Region würde die Assemblierung eines pre-RC in dem untersuchten Bereich zusätzlich bestätigen. Zur Umsetzung von ChIP-Studien wurden CDT1-Antikörper hergestellt. Um die Assemblierung von CDT1 mit dem Origin in Abhängigkeit des Zellzyklus zu untersuchen, wurden FM3A-Mauszellen in früher G1-, später G1-, G1/S-, S- und in der G2/M-Phase arretiert. Die Auswertung der ChIP-Analysen, die den zu analysierenden Bereich von -2837 bis -1820 umspannten, zeigte, dass CDT1 ausschließlich während der G1-Phase mit dem Chromatin assoziiert ist. Dies ist konsistent mit der Aktivität von CDT1 während des Zellzyklus in Säugern. Der höchste Anteil an DNA-gebundenem CDT1 konnte in dem Bereich -2519 bis -2152 festgestellt werden. Eine Sequenzanalyse des OBR der murinen rDNA lieferte keine Homologie zu anderen bekannten Origins. Jedoch wurden diverse DNA-Strukturelemente, wie z.B. HSS, DUEs oder CpG-Inseln, sowie verschiedene Protein-Bindungsstellen gefunden, die potentiellen Einfluss auf die Festlegung des murinen OBR haben könnten. N2 - The eukaryotic initiation of DNA replication is a highly conserved process. In the first step the binding of the "origin recognition complex" (ORC) to replication origins of chromosomal DNA acts as a start signal for the assembly of the pre-replicative complex (pre-RC). Subsequently the initiation factors CDC6 and CDT1 associate with ORC. The following recruitment of the MCM complex finally completes the pre-RC. The activity of the CDC7/DBF4 kinase and the binding of CDC45 license the origin and allow the initiation of DNA replication. One aim of this work was to purify a recombinant murine ORC. In order to isolate the entire complex by co-precipitation, wild-type ORC1, 3, 4, 5 and 6 and ORC2 that contained a His6 epitope tag at its N-terminus were co-expressed by means of baculoviruses. After the following purification, all ORC subunits with the exception of ORC3 were detected in the isolated fractions by immunoplotting. Analysis of the fractions after gel filtration indicated the isolation of a large complex corresponding to a molecular mass of 450 kD, which contained at least five of the six ORC subunits. Therefore, we assume that the recombinant murine ORC was isolated as a holo-complex. A further part of this work deals with the role of the MCM complex in the termination of DNA replication at the 3' end of murine rRNA transcription units. Polar replication fork barriers in the 3' region of ribosomal genes limit progression of DNA replication to the same direction as rDNA transcription by the contrahelicase activity of TTF-I. In this study it should be determined whether murine MCM4/6/7 helicase activity is impaired in the same manner. In order to isolate hexameric MCM4/6/7 complexes wild-type MCM4 and MCM7 proteins and MCM6 with an N-terminal fused HA epitope tag were coexpressed by means of baculoviruses. For contrahelicase experiments, the helicase activity of the isolated complexes had to be obtained. The unwinding activity of short partial duplex M13-substrates (17 nt) was lower than described in literature. For subsequent contrahelicase studies DNA substrates (30 nt) with a 5' tail as well as SSB or RPA were applied. Though it was possible to increase the helicase activity of MCM4/6/7 by forked DNA substrates, this was not sufficient for our studies. Moreover, the unwound oligonucleotide was subjected to degradation, the cause of which could not be explained. The work on this topic was stopped since the helicaseactivity of MCM4/6/7 was insufficient regarding further TTF-I contrahelikase studies. A further aspect of this work was the transport of MCM proteins into the nucleus. The MCM complex is constitutively located in the nucleus of almost all organisms. However, the overexpression of individual ectopically expressed MCM proteins showed that only MCM2 and 3 are transported into the nucleus due to their NLS motives, while this is not possible for MCM4 to 7. Two-hybrid studies of our group suggested pairwise interactions of MCM4, 6, 5 and 7 with MCM2 and/or MCM3. Therefore, various EGFP-MCM proteins were coexpressed together with wild-type MCM proteins in mice cells. It was shown that MCM2 directs MCM4, 6 and 7 into the nucleus, whereas MCM3 transfers only MCM5 into the nucleus. This behaviour reflects possible pairwise interactions within MCM subcomplexes. Further interactions of MCM6 with MCM4 and MCM7 were observed at MCM4/6/7 purifications described above. Further the cell cycle-dependent localization of CDT1 in the OBR region of the murine rRNA transcription unit was examined. So far only in S. cerevisiae a sequence-specific ORC assembly at the ACS region has been identified. In our group a murine OBR has been characterized upstream to the rDNA transcription start site und the binding site of various initiation factors has been localized around position -2500. The association of CDT1 within the same region would confirm the assembly of a pre-RC. For ChIP studies monospecific antibodies against CDT1 have been produced. In order to examine the cell cycle-dependent assembly of CDT1 within the initiation site, FM3A cells were arrested in early G1 phase, in late G1 phase, at the G1/S transition, in S phase and in the G2/M phase. The evaluation of the ChIP studies, which encompassed the range from -2837 to -1820 revealed, however, that CDT1 is associated with the chromatin exclusively during the G1-Phase. This is consistent with the activity of CDT1 during the cell cycle in mammalian cells. The major part of DNA-bound CDT1 was observed within the range -2519 to -2152. The sequence of the murine OBR exhibits no homology to other origins described in the literature. But sequence analysis revealed various DNA structure elements, e.g. HSS, DUEs or CpG islands, as well as several protein binding sites of potential importance for determination of the murine OBR. KW - Maus KW - Replikation KW - Replikationsursprung KW - Replikation KW - Initiation KW - ORC KW - MCM KW - CDT1 KW - replication KW - initiation KW - ORC KW - MCM KW - CDT1 Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-11199 ER - TY - THES A1 - Stürmer, Andrea T1 - Interaktionen und Lokalisationen der Replikationsproteine der Maus T1 - Interactions and Localizations of murine Replication Proteins N2 - Die Initiation der DNA-Replikation in Eukaryonten ist ein hochkonservierter Prozess, der in drei Stufen unterteilt werden kann. Im ersten Schritt bindet der „origin recognition complex“ (ORC) an Replikationsorigins innerhalb chromosomaler DNA, wodurch eine Assemblierung des präreplikativen Komplexes an diesen Startpunkten ausgelöst wird. An den ORC lagern sich anschließend die Proteine CDC6 und RLF-B/CDT1 an, die beide schließlich für die Rekrutierung des heterohexameren MCM-Komplexes verantwortlich sind. Durch die Aktivität der Kinase CDC7/DBF4 wird der Origin für den Start der DNA-Replikation lizenziert, sobald die finale Anlagerung des Initiationsfaktors CDC45 den präreplikativen Komplex vervollständigt hat. Ein Ziel der vorliegenden Arbeit war es, das komplexe Netzwerk von Protein-Protein-Interaktionen zwischen den verschiedenen Initiationsfaktoren durch FRET-Studien aufzuklären. Es konnten Interaktionen zwischen MCM5 und MCM3, MCM5 und MCM7, ORC5 und MCM7, sowie CDT1 und MCM6 in vivo nachgewiesen werden. Die vorliegende Arbeit hatte weiterhin die Untersuchung der intrazellulären Lokalisation der sechs murinen MCM-Proteine in Fibroblasten-Zellen der Maus zum Ziel.Lokalisationsstudien der EGFP-gekoppelten MCM-Proteine zeigten, dass die Proteine EGFP-MCM4, MCM4-EGFP, MCM4-NLS-EGFP, EGFP-MCM5, MCM5-EGFP, MCM5-NLS-EGFP, und EGFP-MCM7 u.a. am Centrosom lokalisiert sind. Durch Immunfluoreszenz-Färbung mit Antikörpern gegen eine konservierte Domäne aller sechs MCM-Untereinheiten sowie mit spezifischen MCM3- und MCM6-Antikörpern konnte eine centrosomale Lokalisation auch für die endogenen Proteine nachgewiesen werden. Zusätzlich zu den Lokalisationsanalysen konnte über Immunpräzipitationsstudien gezeigt werden, dass MCM3 und MCM6 mit dem centrosomalen Protein g-Tubulin präzipitierbar sind. Die Tatsache, dass alle Untereinheiten des MCM-Komplexes mit dem Centrosom assoziiert sind, deutet darauf hin, dass die MCM-Proteine am Centrosom als Multiproteinkomplex gebunden sind. Da MCM3 und MCM6 auch in allen Mitose-Stadien an das Centrosom gebunden sind, kann von einer funktionellen Aufgabe dieser Proteine während der Zellteilung ausgegangen werden. Im letzten Teil dieser Arbeit sollte die Funktion der MCM-Proteine am Centrosom durch „knock-down“ des Proteins MCM3 mittels RNA-Interferenz-Studien untersucht werden. Ziel war, ein induzierbares MCM3-siRNA-exprimierendes System zu etablieren. Das gezielte An- und Abschalten der MCM3siRNA-Transkription sollte durch das TetOn-System ermöglicht werden. Bei diesem System wird durch Zugabe von Doxycyclin die Transkription aktiviert, bei Abwesenheit von Doxycyclin wird sie abgeschaltet. Auf dieser Basis wurde der Einfluss von Doxycyclin auf das Wachstumsverhalten der MCM3siRNA-exprimierenden Zelllinie untersucht. Im Vergleich zu NIH/3T3-Zellen und NIH/3T3-TetOn-Zellen konnte eine deutlich reduzierte Proliferation bei Behandlung der Zellen mit Doxycyclin beobachtet werden. Diese Ergebnisse deuten auf eine durch Produktion von MCM3siRNA verursachte Störung des Zellwachstums hin. Zusätzlich beeinflusst die durch Doxycyclin induzierte Synthese von MCM3siRNA die Zellzyklusverteilung. So befinden sich nach Doxycyclinbehandlung mehr Zellen in der G2/M-Phase als in unbehandelten, asynchronen NIH/3T3-Zellen. Die MCM3-Proteinmenge wurde nach 19 Tagen Doxycyclinbehandlung fast vollständig durch die produzierte MCM3siRNA herunterreguliert. Um einen möglichen Einfluss der MCM3siRNA auf andere MCM-Proteine zu untersuchen, wurde der Protein-Level von MCM6 analysiert. Dabei wurde eine vermehrte MCM6-Expression nachgewiesen. Diese Beobachtung deutet darauf hin, dass durch Bildung von MCM3siRNA der Expressions-Level von MCM6 beeinflusst wird. Auffällig häufig lagen in MCM3-„knock-down“-Zellen mehrere Zellkerne vor. Neben Zellen mit zwei Zellkernen finden sich auch Zellen mit einer ungeraden Anzahl an Zellkernen. Demnach durchlaufen die Zellkerne in einer Zelle unterschiedliche Zellzyklusstadien. Die Phänotypen, die nach Transkription der MCM3siRNA beobachtet wurden, sind komplex und zeigen Defekte in zahlreichen Mitose-Stadien. Das Auftreten multinukleärer Zellen ist auf eine fehlende Cytokinese zurückzuführen. Die Mikrotubuli waren in den MCM3-„knock-down“-Zellen nur unzureichend organisiert, wobei sie kaum mit der Zellperipherie verankert waren. Diese Resultate weisen darauf hin, dass die MCM-Proteine neben ihrer essentiellen Rolle in der Ausbildung des präreplikativen Komplexes eine zusätzliche Funktion in der Mitose ausüben. N2 - The initiation of DNA replication is a highly conserved process which is subdivided into three steps. The first step is the binding of the “origin recognition complex” (ORC) to the replication origins in chromosomal DNA which triggers the assembly of the prereplicative complex at the origins. Subsequently the proteins CDC6 and the RLF-B/CDT1 bind to ORC which are both responsible for the recruitment of the heterohexameric MCM-complex to the prereplicative complex (preRC). The kinase CDC7/DBF4 licenses the origin after completion of the preRC by binding of the CDC45 protein. One task of this work was to dissolve the complex network of protein-protein interactions between the different initiator proteins using the method of FRET (Fluorescence Resonance Energy Transfer). Interactions were found between MCM5 and MCM3, MCM5 and MCM7, ORC5 and MCM7 as well as between CDT1 and MCM6. A further task of this work was to study the intracellular localization of the six murine MCM proteins in murine fibroblasts.In a MCM2-EGFP expressing cell population multinucleated cells occurred frequently. This indicates an incorrect cell division caused by overexpression of MCM2-EGFP and to a possible role of MCM2 in mitosis. Inspecting the intracellular localization of EGFP-fused MCM-proteins was shown, that EGFP-MCM4, MCM4-EGFP, MCM4-NLS-EGFP, EGFP-MCM5, MCM5-EGFP, MCM5-NLS-EGFP, and EGFP-MCM7 were localized in the centrosomes. In contrast, the proteins MCM2-EGFP, EGFP-MCM2, MCM3-EGFP, EGFP-MCM3, MCM6-EGFP, MCM6-NLS-EGFP, MCM7-EGFP and MCM7-NLS-EGFP are not associated with centrosomes. The localization of endogeneous MCM proteins was analyzed by immunostaining using antibodies against a conserved region among all MCM proteins and also with specific antibodies against MCM3 and MCM6. Centrosomal localization was observed for the MCM proteins and in particular for MCM3 and MCM6. In addition to localization studies, immunoprecipitations showed that MCM3 and MCM6 precipitate with the centrosomal protein g-tubulin. The fact that all MCM proteins assemble at the centrosome, indicates that the MCM proteins act as a multiprotein complex at the centrosome. Since MCM3 and MCM6 are bound to centrosomes in all stages of mitosis these proteins may play a role in the progression of cell division. In the last part of this work, the function of MCM3 at the centrosome was analyzed by protein knock-down using the method of RNA interference. To approach this, an inducible MCM3siRNA-expressing system was established. Switching the transcription of MCM3siRNA on and off was made feasible using the TetOn-System. In this system the transcription is activated by addition of doxycycline, without doxycycline the transcription is switched-off. The influence of doxycycline on cell growth of the MCM3siRNA-expressing cell line was analyzed. By comparison to NIH/3T3 cells and NIH/3T3-TetOn cells a significantly reduced proliferation rate was observed after treatment with doxycycline. These results suggest a disturbance of cell growth by MCM3siRNA production. After treatment with doxycycline more cells are accumulated in G2/M phase compared to untreated cells. The expression of MCM3 was almost completely knocked-down by treatment of cells with doxycycline for 19 days. To analyze the influence of MCM3siRNA on other MCM proteins, the protein level of MCM6 was examined. An increased MCM6 expression was observed. This implies that MCM3siRNA influences the expression level of MCM6. More nuclei were frequently observed in MCM3 knocked-down cells. Cells with two nuclei as well as cells with an impaired number of nuclei were observed, indicating that probably the nuclei pass thruogh different cell cycle stages. Phenotypes observed after expression of MCM3siRNA showed defects in multiple stages of mitosis. The presence of multinucleated cells is apparently due to a blocked cytokinesis. Microtubuli were insufficiently organized and were deficiently bound to the cellular periphery. These results indicate an essential role of the MCM proteins in mitosis besides its described role in the establishment of the prereplicative complex. KW - Replikation KW - Maus KW - Proteine KW - Interaktion KW - Replikation KW - Lokalisation KW - Interaktion KW - Proteine KW - replication KW - localization KW - interaction KW - proteins Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-9563 ER -