TY - JOUR A1 - Stoll, Sascha A1 - Feldhaar, Heike A1 - Fraunholz, Martin J. A1 - Gross, Roy T1 - Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus N2 - Background: The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C.floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals. Results: We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of Blochmannia. In adult animals the number of bacteriocytes generally decreased. Conclusions: During the life cycle of the animals the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of Blochmannia for its host in particular during metamorphosis. KW - Camponotus floridanus KW - carpenter ant Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67950 ER - TY - THES A1 - Kapustjansky, Alexander T1 - In vivo imaging and optogenetic approach to study the formation of olfactory memory and locomotor behaviour in Drosophila melanogaster T1 - In vivo Imaging und der optogenetische Ansatz zu Untersuchung der Gedächtnissbildung und lokomotorischem Verhalten bei Drosophila melanogaster N2 - Understanding of complex interactions and events in a nervous system, leading from the molecular level up to certain behavioural patterns calls for interdisciplinary interactions of various research areas. The goal of the presented work is to achieve such an interdisciplinary approach to study and manipulate animal behaviour and its underlying mechanisms. Optical in vivo imaging is a new constantly evolving method, allowing one to study not only the local but also wide reaching activity in the nervous system. Due to ease of its genetic accessibility Drosophila melanogaster represents an extraordinary experimental organism to utilize not only imaging but also various optogenetic techniques to study the neuronal underpinnings of behaviour. In this study four genetically encoded sensors were used to investigate the temporal dynamics of cAMP concentration changes in the horizontal lobes of the mushroom body, a brain area important for learning and memory, in response to various physiological and pharmacological stimuli. Several transgenic lines with various genomic insertion sites for the sensor constructs Epac1, Epac2, Epac2K390E and HCN2 were screened for the best signal quality, one line was selected for further experiments. The in vivo functionality of the sensor was assessed via pharmacological application of 8-bromo-cAMP as well as Forskolin, a substance stimulating cAMP producing adenylyl cyclases. This was followed by recording of the cAMP dynamics in response to the application of dopamine and octopamine, as well as to the presentation of electric shock, odorants or a simulated olfactory signal, induced by acetylcholine application to the observed brain area. In addition the interaction between the shock and the simulated olfactory signal by simultaneous presentation of both stimuli was studied. Preliminary results are supporting a coincidence detection mechanism at the level of the adenylyl cyclase as postulated by the present model for classical olfactory conditioning. In a second series of experiments an effort was made to selecticvely activate a subset of neurons via the optogenetic tool Channelrhodopsin (ChR2). This was achieved by recording the behaviour of the fly in a walking ball paradigm. A new method was developed to analyse the walking behaviour of the animal whose brain was made optically accessible via a dissection technique, as used for imaging, thus allowing one to target selected brain areas. Using the Gal4-UAS system the protocerebral bridge, a substructure of the central complex, was highlighted by expressing the ChR2 tagged by fluorescent protein EYFP. First behavioural recordings of such specially prepared animals were made. Lastly a new experimental paradigm for single animal conditioning was developed (Shock Box). Its design is based on the established Heat Box paradigm, however in addition to spatial and operant conditioning available in the Heat Box, the design of the new paradigm allows one to set up experiments to study classical and semioperant olfactory conditioning, as well as semioperant place learning and operant no idleness experiments. First experiments involving place learning were successfully performed in the new apparatus. N2 - Das Verständniss für die komplexen Interaktionen und Zusammenhänge, die von der molekularen Ebene bis zum Auftreten von bestimmten Verhaltensmustern führen, erfordert die interdisziplinäre Zusammenarbeit unterschiedlicher Forschungsrichtungen. Das Ziel der vorgelegten Arbeit war es einen solchen interdisziplinären Ansatz für die Erforschung und die Manipulation von Verhalten und ihm zu Grunde liegenden Mechanismen zu verwirklichen. Optisches in vivo Imaging ist eine neue, sich ständig weiterentwickelnde Methode, welche es ermöglicht, nicht nur lokale sondern auch weitläufige Aktivitäten innerhalb des Nervensystem zu untersuchen. Drosophila melanogaster stellt aufgrund der leichten genetischen Zugänglichkeit einen herausragenden experimentellen Organismus dar, bei welchem neben optischem Imaging eine ganze Reihe optogenetischer Methoden angewandt werden kann, um die neuronalen Grundlagen des Verhaltens zu erforschen. Im Rahmen dieser Arbeit wurde mit Hilfe von vier genetisch kodierten Sensoren in vivo die Dynamik der cAMP Konzentration in den horizontalen Loben des Pilzkörpers, bei Applikation unterschiedlicher physiologischer und pharmazeutischer Stimuli untersucht. Dabei wurden mehrere transgene Fliegenlinien mit Sensorkonstrukten Epac1, Epac2, Epac2K390E und HCN2 an unterschiedlichen genomischen Insertionsorten, hinsichtlich ihrer Signalqualität untersucht, eine der Linien wurde für weitere Experimente ausgewählt. Zunächst wurde an dieser die in vivo Tauglichkeit des Sensors gezeigt, indem die Konzentration von cAMP durch pharmakologische Applikationen von 8-Bromo-cAMP und Forskolin, einer Substanz welche die Aktivität von cAMP produzierenden Adenylatcyclasen stimuliert, appliziert wurden. Anschließend wurde eine Untersuchung der cAMP Dynamik als Antwort auf einen elektrischen Schock, unterschiedliche Düfte, sowie einen durch Applikation von Acetylcholin simulierten Duftstimulus durchgeführt. Vorläufige Ergebnisse bestärken das aktuelle Modell der klassischen olfaktorischen Konditionierung durch die Koinzidenzdetektion auf der Ebene der Adenylatcyclase. In einem weiteren Experiment wurde der Versuch einer optogenetischen neuronalen Aktivierung unternommen, dabei wurde basierend auf einem Laufball Paradigma eine Methode entwickelt, das Laufverhalten der Fliegen zu analysieren während ihr Gehirn durch eine Imaging-Präparation freigelegt wurde, um gezielt bestimmte durch fluoreszierende Proteine markierte Gehirnbereiche anzuregen. Erste Aufzeichnungen des Laufverhaltens bei Aktivierung der protocerebrallen Brücke, einer Substruktur des Zentralkomplexes, wurden durchgeführt. Schließlich wurde eine neue Apparatur (Shock Box) für die Konditionierung von Einzeltieren entwickelt und gebaut, das Design beruht auf dem der sogenannten Heat Box, ermöglicht jedoch klassische und semioperante olfaktorische Konditionierung zusätzlich zu der in der Heat Box möglichen räumlichen und operanten Konditionierung. Die ersten Versuche für räumliches Lernen wurden in der Apparatur durchgeführt. KW - Taufliege KW - Pilzkörper KW - Cyclo-AMP KW - Gedächtnis KW - In vivo KW - Imaging KW - Drosophila KW - Memory KW - In vivo KW - Imaging KW - Drosophila KW - Memory Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69535 ER - TY - JOUR A1 - Friedrich, Torben A1 - Rahmann, Sven A1 - Weigel, Wilfried A1 - Rabsch, Wolfgang A1 - Fruth, Angelika A1 - Ron, Eliora A1 - Gunzer, Florian A1 - Dandekar, Thomas A1 - Hacker, Joerg A1 - Mueller, Tobias A1 - Dobrindt, Ulrich T1 - High-throughput microarray technology in diagnostics of enterobacteria based on genome-wide probe selection and regression analysis N2 - The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Results: We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Conclusions: Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics. KW - Mikroarray KW - Enterobacteriaceae Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67936 ER - TY - JOUR A1 - Laisney, Juliette A. G. C. A1 - Braasch, Ingo A1 - Walter, Ronald B. A1 - Meierjohann, Svenja A1 - Schartl, Manfred T1 - Lineage-specific co-evolution of the Egf receptor/ligand signaling system N2 - Background: The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system. Results: We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution. Conclusions: Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr ligands. This fish-specific occurrence of Egf receptor specialization offers unique opportunities to study the functions of different Egf receptor-ligand combinations and their biological outputs in vertebrates. Furthermore, our results strongly support the use of homologous ligands in future studies, as sufficient cross-specificity is very unlikely for this ligand/receptor system. KW - Epidermaler Wachstumsfaktor-Rezeptor KW - epidermal growth factor receptor KW - Egfr KW - teleost fishes Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67922 ER - TY - JOUR A1 - Brocher, Jan A1 - Vogel, Benjamin A1 - Hock, Robert T1 - HMGA1 down-regulation is crucial for chromatin composition and a gene expression profile permitting myogenic differentiation N2 - Background: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable overexpression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours. KW - HMG-Proteine KW - High mobility group Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67914 ER - TY - JOUR A1 - Teutschbein, Janka A1 - Haydn, Johannes M. A1 - Samans, Birgit A1 - Krause, Michael A1 - Eilers, Martin A1 - Schartl, Manfred A1 - Meierjohann, Svenja T1 - Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins N2 - Background: Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation. Methods: Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated. Results: Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration. Conclusion: Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes. KW - Melanoma Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67900 ER - TY - JOUR A1 - Vainshtein, Yevhen A1 - Sanchez, Mayka A1 - Brazma, Alvis A1 - Hentze, Matthias W. A1 - Dandekar, Thomas A1 - Muckenthaler, Martina U. T1 - The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays N2 - Background: Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results: The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions: ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see “Additional Files” section) and at: http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/ KW - Microarray KW - ICEP KW - IronChip Evaluation Package Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67869 ER - TY - JOUR A1 - Schmid, Benjamin A1 - Schindelin, Johannes A1 - Cardona, Albert A1 - Longair, Martin A1 - Heisenberg, Martin T1 - A high-level 3D visualization API for Java and ImageJ N2 - Background: Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set. Results: Here we present a platform-independent framework based on Java and Java 3D for accelerated rendering of biological images. Our framework is seamlessly integrated into ImageJ, a free image processing package with a vast collection of community-developed biological image analysis tools. Our framework enriches the ImageJ software libraries with methods that greatly reduce the complexity of developing image analysis tools in an interactive 3D visualization environment. In particular, we provide high-level access to volume rendering, volume editing, surface extraction, and image annotation. The ability to rely on a library that removes the low-level details enables concentrating software development efforts on the algorithm implementation parts. Conclusions: Our framework enables biomedical image software development to be built with 3D visualization capabilities with very little effort. We offer the source code and convenient binary packages along with extensive documentation at http://3dviewer.neurofly.de. KW - Visualisierung KW - Java 3D KW - ImageJ KW - framework Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67851 ER - TY - THES A1 - Halder, Partho T1 - Identification and characterization of synaptic proteins of Drosophila melanogaster using monoclonal antibodies of the Wuerzburg Hybridoma Library T1 - Identifikation und Charakterisierung von synaptischen Proteinen von Drosophila melanogaster mit Hilfe von monoklonalen Antikörpern der Würzburger Hybridoma-Bibliothek N2 - For a large fraction of the proteins expressed in the human brain only the primary structure is known from the genome project. Proteins conserved in evolution can be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and characterized with the aim to identify the target antigen. The mAb ab52 was found to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15 (epidermal growth factor receptor pathway substrate clone 15) to be a strong candidate. Another mAb from the library, aa2, was already found to recognize EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and 2D electrophoretic separations revealed similar patterns, hence indicating that both antigens could represent the same protein. Finally absence of the wild-type signal in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52 antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for applications like immunoprecipitation (IP). It has already been submitted to the Developmental Studies Hybridoma Bank (DSHB) to be easily available for the entire research community. The mAb na21 was also found to be an IgM. It recognizes a membrane associated antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to biochemically purify the endogenously expressed protein from the tissue, gave promising results but could not be completed due to lack of time. Thus biochemical purification of the protein seems possible in order to facilitate its identification by mass spectrometry. Several other mAbs were studied for their staining pattern on cryosections and whole mounts of Drosophila brains. However, many of these mAbs stained very few structures in the brain, which indicated that only a very limited amount of protein would be available as starting material. Because these antibodies did not produce signals on Western blots, which made it impossible to enrich the antigens by electrophoretic methods, we did not attempt their purification. However, the specific localization of these proteins makes them highly interesting and calls for their further characterization, as they may play a highly specialized role in the development and/or function of the neural circuits they are present in. The purification and identification of such low expression proteins would need novel methods of enrichment of the stained structures. N2 - Für einen Großteil der Proteine, die im menschlichen Gehirn exprimiert werden, ist lediglich die Primärstruktur aus dem Genomprojekt bekannt. Proteine, die in der Evolution konserviert wurden, können in genetischen Modellsystemen wie Drosophila untersucht werden. In dieser Doktorarbeit werden monoklonale Antikörper (mAk) aus der Würzburger Hybridoma Bibliothek produziert und charakterisiert, mit dem Ziel, die erkannten Proteine zu identifizieren. Der mAk ab52 wurde als IgM typisiert, das auf Western Blots ein zytosolisches Protein von Mr ~110 kDa erkennt. Das Antigen wurde durch zwei-dimensionale Gelelektrophorese (2DE) als einzelner Fleck aufgelöst. Massenspektrometrische Analyse dieses Flecks identifizierte dass EPS-15 (epidermal growth factor receptor pathway substrate clone 15) als viel versprechenden Kandidaten. Da für einen anderen mAk aus der Bibliothek, aa2, bereits bekannt war, dass er EPS-15 erkennt, wurden die Western-Blot-Signale der beiden Antikörper nach 1D und 2D Trennungen von Kopfhomogenat verglichen. Die Ähnlichkeit der beiden Muster deuteten darauf hin, dass beide Antigene dasselbe Protein erkennen. Das Fehlen des Wildtyp-Signals in homozygoten Eps15 Mutanten in einem Western Blot mit mAk ab52 bestätigten schließlich, dass EPS-15 das Antigen zu mAk ab52 darstellt. Demnach erkennen beide mAk, aa2 und ab52, das Drosophila Homolog zu EPS-15. Da mAk aa2 ein IgG ist, dürfte er für Anwendungen wie Immunpräzipitation (IP) besser geeignet sein. Er wurde daher bereits bei der Developmental Studies Hybridoma Bank (DSHB) eingereicht, um ihn der ganzen Forschergemeinde leicht zugänglich zu machen. Der mAk na21 wurde ebenfalls als IgM typisiert. Er erkennt ein Membran assoziiertes Antigen von Mr ~10 kDa auf Western Blots. Aufgrund der Membranassoziierung des Proteins war es nicht möglich, es in 2DE aufzulösen und da es sich um ein IgM handelt, war eine Anreicherung des Antigens mittels IP nicht erfolgreich. Vorversuche zur biochemischen Reinigung des endogenen Proteins aus Gewebe waren Erfolg versprechend, konnten aber aus Zeitmangel nicht abgeschlossen werden. Daher erscheint eine biochemische Reinigung des Proteins für eine Identifikation durch Massenspektrometrie möglich. Eine Reihe weiterer mAk wurden hinsichtlich ihrer Färbemuster auf Gefrierschnitten und in Ganzpräparaten von Drosophila Gehirnen untersucht. Allerdings färbten viele dieser mAk sehr wenige Strukturen im Gehirn, so dass nur eine sehr begrenzte Menge an Protein als Startmaterial verfügbar wäre. Da diese Antikörper keine Signale auf Western Blots produzierten und daher eine Anreicherung des Antigens durch elektrophoretische Methoden ausschlossen, wurde keine Reinigung versucht. Andererseits macht die spezifische Lokalisation dieser Proteine sie hoch interessant für eine weitere Charakterisierung, da sie eine besonders spezialisierte Rolle in der Entwicklung oder für die Funktion von neuralen Schaltkreisen, in denen sie vorkommen, spielen könnten. Die Reinigung und Identifikation solcher Proteine mit niedrigem Expressionsniveau würde neue Methoden der Anreicherung der gefärbten Strukturen erfordern. KW - Taufliege KW - Synapse KW - Proteine KW - Monoklonaler Antikörper KW - synaptische Proteine KW - monoklonale Antikörper KW - Drosophila melanogaster KW - synaptic proteins KW - monoclonal antibodies Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67325 N1 - korrigierte Ausgabe der Arbeit aus dem Jahr 2022 unter: https://doi.org/10.25972/OPUS-27020 ER - TY - THES A1 - Duraphe, Prashant T1 - Identification and characterization of AUM, a novel human tyrosine phosphatase T1 - Identifizierung und Charakterisierung von AUM, einer neuen humanen Tyrosin-Phosphatase N2 - Protein Phosphatasen werden aufgrund der Aminosäuresequenzen ihrer aktiven Zentren in drei große Familien unterteilt. In einer neu entdeckten Familie von Phosphatasen ist das aktive Zentrum durch die Sequenz DXDX(T/V) charakterisiert. Diese Aspartat-abhängigen Phosphatasen gehören zu der Superfamilie der Hydrolasen vom Haloazid Dehalogenase(HAD)-Typ, einer evolutionär konservierten und ubiquitär verbreiteten Enzymfamilie. Bislang konnten 58 menschliche HAD Enzyme durch Datenbankanalysen identifiziert werden. Ihre Funktionen sind jedoch nach wie vor nur rudimentär verstanden. Im Rahmen dieser Arbeit wurde zunächst das Komplement aller menschlichen HAD Phosphatasen durch Datenbank-Recherchen erfasst. Zusammen mit phylogenetischen Analysen gelang es, eine zum damaligen Zeitpunkt unbekannte, putative Phosphatase zu identifizieren, die eine vergleichsweise hohe Sequenz-Homologie zu der Zytoskelettregulierenden HAD Phosphatase Chronophin aufweist. Dieses neuartige Enzym wurde kloniert und mit biochemischen und zellbiologischen Methoden charakterisiert. Auf der Basis dieser Befunde bezeichnen wir dieses neuartige Protein als AUM (actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase).Mittels Northern blot, real-time PCR und Western blot Analysen konnte gezeigt werden, dass AUM in allen untersuchten menschlichen und murinen Geweben exprimiert wird. Die höchste Expression konnte in Hodengewebe nachgewiesen werden. Durch immunohistochemische Untersuchungen konnte gezeigt werden, dass AUM spezifisch in reifenden Keimzellen mit einem Expressionsmaximum zum Zeitpunkt der Spermiogenese exprimiert wird. Um die Substratpräferenz von AUM zu charakterisieren, wurde zunächst ein peptidbasierter in vitro Phosphatase-Substrat-Screen durchgeführt. Hierbei wurden 720 aus menschlichen Phosphoproteinen abgeleitete Phosphopeptide untersucht. Interessanterweise dephosphorylierte AUM ausschließlich Phosphotyrosin (pTyr)-enthaltende Peptide. Nur 17 pTyr-Peptide (~2% aller untersuchten Peptide) fungierten als AUM-Substrate. Diese Daten legen eine hohe Substratspezifität von AUM nahe. Zu den putativen AUM Substraten gehören Proteine, die in die Dynamik der Zytoskelett-Reorganisation sowie in Tyrosin Kinasevermittelte Signalwege eingebunden sind. In Übereinstimmung mit den Ergebnissen dieses Phosphopeptid-Screens konnte mittels Phosphatase overlay assays sowie in Zellextrakten aus Pervanadat-behandelten HeLa Zellen demonstriert werden, dass AUM eine begrenzte Anzahl Tyrosin-phosphorylierter Proteinen dephosphorylieren kann.In zellulären Untersuchungen wurde die mögliche Rolle von AUM im Rahmen der durch den epidermalen Wachstumsfaktor (EGF) ausgelösten Tyrosin-Phosphorylierung in einer Spermatogonien Zelllinie (GC-1 spg-Zellen) analysiert. So konnte nachgewiesen werden, dass die Überexpression von AUM zu einer moderaten Abnahme Tyrosin phosphorylierter Proteine nach EGF-Stimulation führte. Im Gegensatz dazu löste jedoch die durch RNAInterferenz vermittelte Depletion von endogenem AUM einen robusten Anstieg Tyrosinphosphorylierter Proteine aus, zu denen auch der EGF-Rezeptor selbst zählt. Zusätzlich zu dem EGF-Rezeptor wurde die Src-Kinase im Zuge des Phosphopeptid- Screens als mögliches AUM Substrat identifiziert. Daher wurden in vitro Kinase/Phosphatase-Assays mit gereinigtem Src und AUM durchgeführt. Mit diesem Ansatz konnte erstmals gezeigt werden, dass AUM in der Lage ist, die Src-Kinase zu aktivieren, während Src AUM phosphoryliert und die AUM Phosphatase-Aktivität blockiert. Diese Ergebnisse deuten auf eine gekoppelte, wechselseitige Regulation von AUM und Src hin. Obwohl die Details dieser Regulation derzeit noch unklar sind, zeigen unsere initialen Ergebnisse, dass AUM die Src-Aktivität unabhängig von seiner Phosphatase Aktivität steigert, während Src die AUM Phosphatase-Aktivität Kinase-abhängig vermindert. Auf zellulärer Ebene sind AUM-depletierte Zellen durch Veränderungen der Aktin- Zytoskelett-Dynamik und der Zelladhäsion charakterisiert. So weisen AUM-defiziente Zellen stabilisierte Aktin Streßfasern und vergrößerte fokale Adhäsionen auf. Weiterhin sind AUMdepletierte Zellen durch ein beschleunigtes spreading auf Fibronektin gekennzeichnet. Wir haben mit AUM ein bisher nicht beschriebenes Mitglied der Familie Aspartat-abhängiger Phosphatasen entdeckt. In dieser Arbeit ist es gelungen, AUM phylogenetisch, biochemisch und zellbiologisch zu charakterisieren. Unsere Ergebnisse legen nahe, dass AUM einen wichtigen, neuartigen Regulator der Src-vermittelten Zytoskelett-Dynamik im Rahmen der Zelladhäsion und Migration darstellt. N2 - Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. KW - Tyrosin KW - Phosphatase KW - Signal transduction KW - Cell adhesion KW - Actin cytoskeleton KW - Src KW - Spermatogenesis Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-44256 ER - TY - THES A1 - Mishra, Dushyant T1 - The content of olfactory memory in larval Drosophila T1 - Olfaktorisches Gedächtnis in der Drosophila Larve N2 - An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade. N2 - Das Überleben von Tieren ist in hohem Maße abhängig von ihrer Fähigkeit zu riechen und olfaktorische Gedächtnisse zu bilden. Meine Arbeit besteht aus zwei Abschnitten, in denen ich solche Prozesse anhand von Drosophila Larven untersuche. Im ersten Abschnitt beschreibe ich verschiedene Aspekte der Geruchsprozessierung, der zweite Abschnitt betrifft Gedächtnis- und Lernprozesse. Kapitel I.1 handelt davon, wie Geruchsintensitäten in die olfaktorische Wahrnehmung von Drosophila-Larven integriert sein könnten. Zuerst beschreibe ich die Lernbarkeit verschiedener Duftstoffe abhängig von ihren Intensitäten. Anhand dieser Dosis-Wirkungs-Kurven wähle ich dann eine niedrige, eine mittlere, und eine hohe Duft-Intensität. Ich trainiere Larven mit der mittleren Duft-Intensität und teste sie entweder mit dieser mittleren Intensität, oder mit der höheren, oder mit der niedrigen Duft-Intensität. Ich beobachte, dass der Gedächtnisabruf mit der trainierten Intensität für alle verwendeten Duftstoffe am besten ist. Außerdem vergleiche ich diese Ergebnisse mit denen von adulten Fruchtfliegen und schlage ein Schaltkreis-Modell vor, das erklärt, wie eine solche Kodierung der Intensität zustande kommen kann. Eine solche Spezifität für Intensitäten beim Lernen erweitert die bisher bekannte Fülle des ‚Inhalts’ von olfaktorischen Gedächtnisspuren und die Anforderungen an Computermodelle über Riechen und Geruchslernen. In Kapitel I.2 untersuche ich Ähnlichkeitsbeziehungen zwischen Duftpaaren anhand der Wahrnehmung von Larven. Ich verwende dazu vier verschiedene Typen von Lernexperimenten. Durch Kombination der Ergebnisse dieser vier Experimente erhalte ich eine aufgabenunabhängige Abschätzung der vom Tier wahrgenommenen Ähnlichkeiten zwischen Paaren von Duftstoffen. Ein Vergleich dieser wahrgenommenen Ähnlichkeiten mit veröffentlichten Messungen von physikalischen und chemischen Ähnlichkeiten ergibt eine schwache Korrelation. Eine erwähnenswerte Ausnahme zu dieser Korrelation ist das Duftpaar 3-Octanol und Benzaldehyd. Kapitel I.3 zeigt für zwei Duftstoffe (3-Octanol und 1-Octen-3-ol), dass die wahrgenommene Ähnlichkeit zwischen diesen beiden Duftstoffen abhängig ist von der Art des Trainings. Wenn die Tiere nicht-diskriminativ trainiert werden, werden die Düfte vom Tier generalisiert, während diskriminatives Training die wahrgenommene Unterschiede zwischen den Düften erhöht. Anosmische Or83b1-Mutanten haben diese Fähigkeiten verloren, was darauf hindeutet, das diese adaptive Anpassung in Nervenzellen stattfindet, die den Or83b-exprimierenden sensorischen Neuronen nachgeschaltet sind. In Kapitel II.1 untersuche ich die Auswirkung von Zugabe getrockneter Wurzeln der Pflanze Rhodiola rosea zum Fliegenfutter. Ich finde heraus, dass Rhodiola rosea dosisabhängig die olfaktorische Konditionierung von Drosophila-Larven verbessert. Die Zugabe von kommerziell verfügbaren Tabletten oder Extrakten zum Fliegenfutter hat keinen positiven Effekt auf solche „kognitiven“ Fähigkeiten, was uns möglicherweise erlaubt, zwischen den effektiven Substanzen der Wurzel und diesen Präparaten zu differenzieren. Drosophila als genetisch manipulierbarer Modellorganismus sollte uns nun weiterführende Analysen der molekularen Mechanismen erlauben, die dieser „kognitiven Verbesserung“ durch Rhodiola rosea zugrunde liegen. Kapitel II.2 beschreibe ich die Funktion von Synapsin, einem evolutionär konservierten präsynaptischen Phosphoprotein. Ich verwende dazu einen kombinierten verhaltensbasierten und genetischen Ansatz. Untersucht wird, wo und wie dieses Protein assoziative Plastizität im Gehirn von Drosophila-Larven beeinflusst. Diese Studie zeigt, dass eine Synapsin-abhängige Gedächtnisspur im Pilzkörper, einer „kortikalen“ Gehirnregion der Insekten, lokalisiert werden kann. Auf der molekularen Ebene zeigen die Ergebnisse dieser Studie Synapsin als einen im Verhalten wichtigen Effektor der AC-cAMP-Kaskade. * Many thanks to M. Schlayer, T. Niewalda and T. Saumweber for their help in this translation. KW - Drosophila KW - Insektenlarve KW - Geruchssinn KW - Lernen KW - Drosophila melanogaster KW - Olfaktion KW - Neurogenetik KW - Speicher KW - Neurogenetics KW - Drosophila melanogaster KW - Olfaction KW - Learning KW - Memory KW - Reinforcement Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-66316 ER - TY - THES A1 - Schmitt, Kathrin T1 - Identification and Characterization of GAS2L3 as a Novel Mitotic Regulator in Human Cells T1 - Die Identifizierung und Charakterisierung von GAS2L3 als neuer Regulator der Mitose in humanen Zellen N2 - Precise control of mitotic progression is vital for the maintenance of genomic integrity. Since the loss of genomic integrity is known to promote tumorigenesis, the identification of knew G2/M regulatory genes attracts great attention. LINC, a human multiprotein complex, is a transcriptional activator of a set of G2/M specific genes. By depleting LIN9 in MEFs, a core subunit of LINC, Gas2l3 was identified as a novel LINC target gene. The so far uncharacterized Gas2l3 gene encodes for a member of the family of growth arrest specific 2 (GAS2) proteins, which share a highly conserved putative actin binding CH and a putative microtubule binding GAS2 domain. In the present study GAS2L3 was identified as a LINC target gene also in human cells. Gene expression analysis revealed that GAS2L3 transcription, in contrast to all other GAS2 family members, is highly regulated during the cell cycle with highest expression in G2/M. The GAS2L3 protein showed a specific localization pattern during the M phase: In metaphase, GAS2L3 localized to the mitotic spindle, relocated to the spindle midzone microtubules in late anaphase and concentrated at the midbody in telophase where it persisted until the end of cytokinesis. Overexpression of a set of different GAS2L3 deletion mutants demonstrated that the localization to the mitotic microtubule network is dependent on the C-terminus, whereas the midbody localization is dependent on full length GAS2L3 protein. Additionally, exclusive overexpression of the CH domain induced the formation of actin stress fibers, suggesting that the CH domain is an actin binding domain. In contrast, the GAS2 domain was neither needed nor sufficient for microtubule binding, indicating that there must be an additional so far unknown microtubule binding domain in the C-terminus. Interestingly, immunoblot analysis also identified the C-terminus as the domain responsible for GAS2L3 protein instability, partially dependent on proteasomal degradation. Consistent with its specific localization pattern, GAS2L3 depletion by RNAi demonstrated its responsibility for proper mitosis and cytokinesis. GAS2L3 depletion in HeLa cells resulted in the accumulation of multinucleated cells, an indicator for chromosome mis-segregation during mitosis. Also the amount of cells in cytokinesis was enriched, indicating failures in completing the last step of cytokinesis, the abscission. Strikingly, treatment with microtubule poisons that lead to the activation of the spindle assembly checkpoint (SAC) indicated that the SAC was weakened in GAS2L3 depleted cells. Although the exact molecular mechanism is still unknown, fist experiments support the hypothesis that GAS2L3 might be a regulator of the SAC master kinase BUBR1. In conclusion, this study provides first evidence for GAS2L3 as a novel regulator of mitosis and cytokinesis and it might therefore be an important guardian against tumorigenesis. N2 - Der korrekte Verlauf durch die Mitose des Zellzyklus trägt entscheidend zur Aufrechterhaltung der genomischen Integrität bei. Da ein Verlust der genomischen Integrität die Tumorentstehung begünstigt, ist die Identifizierung neuer G2/M regulatorischer Gene ein Forschungsbereich, der großes Interesse weckt. Der humane Multiproteinkomplex LINC ist für die transkriptionelle Aktivierung einer Vielzahl G2/M spezifischer Gene verantwortlich. Durch die Depletion von LIN9 in MEFs, einer Kernkomponente von LINC, wurde Gas2l3 als ein neues Zielgen von LINC identifiziert. Das bisher uncharakterisierte Gas2l3 Gen codiert für ein der GAS2 (growth arrest specific 2) Familie zugehöriges Protein, deren Mitglieder sich durch eine hoch konservierte putative Aktin-bindende Domäne (CH) und eine putative Mikrotubuli-bindende Domäne (GAS2) auszeichnen. In der vorliegenden Arbeit konnte gezeigt werden, dass GAS2L3 auch in humanen Zellen ein Zielgen von LINC ist. Die Transkription von GAS2L3 wies, im Gegensatz zu allen anderen GAS2 Familienmitgliedern, eine starke Regulation während des Zellzyklus auf, wobei die höchste Genexpression in der G2/M Phase vorlag. Das GAS2L3 Protein zeigte eine spezifische Lokalisation während der M Phase: In der Metaphase findet sich GAS2L3 an der mitotischen Spindel, wandert von dort an die Mikrotubuli der zentralen Spindel der Anaphase und konzentriert sich in der Telophase am Midbody, wo es bis zum Ende der Zytokinese verweilt. Der Einsatz unterschiedlicher Deletionsmutanten demonstrierte, dass die Lokalisation an die mitotischen Mikrotubuli vom C-Terminus abhängig ist, wohingegen die Lokalisation am Midbody von der gesamten Proteinsequenz abhängt. Die Ausbildung von Aktin-Streß-Filamenten nach alleiniger Überexpression der CH Domäne deutete darauf hin, dass die CH Domäne eine Aktin-bindende Domäne ist. Die GAS2 Domäne hingegen wurde weder für die Interaktion mit Mikrotubuli gebraucht, noch war sie alleine für diese ausreichend. Alle Daten weisen darauf hin, dass GAS2L3 eine bisher unbekannte Mikrotubuli-bindende Domäne im C-Terminus trägt. Interessanterweise ist der C-Terminus auch für die hohe Instabilität des GAS2L3 Proteins, die teilweise durch den Abbau im Proteasom verursacht wird, verantwortlich. Entsprechend der spezifischen Lokalisation zeigte die Depletion von GAS2L3 durch siRNA Transfektion dessen Wichtigkeit für den korrekten Verlauf der M Phase. GAS2L3 depletierte HeLa Zellen zeigten eine Anreicherung von multinukleären Zellen, welche ein Indikator für die fehlerhafte Verteilung der Chromosomen in der Mitose sind. Ein Hinweis auf Probleme im Beenden der Zytokinese stellte die erhöhte Anzahl von Zellen dar, die sich in der Zytokinese befanden. Eines der auffallendsten Merkmale war ein geschwächter mitotischer Spindelkontrollpunkt, den GAS2L3 depletierte Zellen nach der Behandlung mit den Kontrollpunkt aktivierenden Mikrotubuli-Giften aufwiesen. Auch wenn der exakte molekulare Mechanismus hierbei noch unbekannt ist, deuten erste Experimente darauf hin, dass GAS2L3 die Aktivität von BUBR1, einer essentiellen Kinase des mitotischen Spindelkontrollpunkts, beeinflusst. Alle Daten dieser Arbeit verdeutlichen die Wichtigkeit von GAS2L3 als einen neuen Regulator der Mitose und Zytokinese. Somit ist anzunehmen, dass die korrekte Funktion von GAS2L3 entscheidend zum Schutz vor Tumorentstehung beiträgt. KW - Mensch KW - Zelle KW - Mitose KW - Kernspindel KW - Kontrolle KW - Genregulation KW - Spindelkontrollpunkt KW - Zytokinese KW - Midbody KW - GAS2L3 KW - LIN9 KW - Zellzyklus KW - LIN9 KW - GAS2L3 KW - mitosis KW - cytokinesis KW - spindle assembly checkpoint Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-52704 ER - TY - JOUR A1 - Clauss, Gerd A1 - Winkler, Christoph A1 - Lohmeyer, Jürgen A1 - Anders, Fritz A1 - Schartl, Manfred T1 - Oncofetal antigen in Xiphophorus detected by monoclonal antibodies directed against melanoma-associated antigens N2 - Monoclonal antlbodies (MAbs) directed against Xiphophorus melanoma cells were deve(oped and tested by lndirect immunofluorescence and Immunoperoxidase staining for reactivity with a panel of I 5 allogeneic tissues and 12 allogeneic cell llnes. The reactivity of such MAbs was restricted to melanoma cells from tumor biopsies and melanoma-derived cell lines. ln addition, all embryonie cells of all histiotypes from developmental stages later than mld·organogenesis and from corresponding short term in vitro cultures reacted with these MAbs. ln contrast, normal tissues and organs from adult fish dlsplayed no reactivity, thus implying that the melanoma-associated antigens detected by the MAbs described are oncofetal antigens. KW - Physiologische Chemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61784 ER - TY - JOUR A1 - Friedenreich, Hildegard A1 - Schartl, Manfred T1 - Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells N2 - ln order to construct fish specific expression vectors for studies on gene regulation in vitro and in vivo a variety of heterologous enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial chloramphenicol acetyl transferase reporter gene in three teleost fish cell lines. Several viral enhancers were found to be constitutively active at high Ieveis. The human metallothionein promoter showed inducible expression in the presence of heavy metal Ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient splicing and Iack of translation were observed, pointing to limitations in the use of heterologous genes in gene transfer experiments. On the contrary, some heterologous promoters and enhancers functioned in fish c as weil as in their cell type of origin, indicating t at corresponding transcription factors are sufficient conserved between fish and human over a period of 900 million years of Independent evolution. KW - Physiologische Chemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61774 ER - TY - JOUR A1 - Dracopoli, Nicholas C. A1 - Feltquate, David M. A1 - Sam, Brigitta A1 - Schartl, Manfred T1 - Taql and Mspl RFLPs are detected by the human 2,3-biphosphoglycerate mutase (BPGM) cDNA N2 - No abstract available KW - Physiologische Chemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61763 ER - TY - JOUR A1 - Schartl, Manfred T1 - Homology of melanoma-inducing loci in the genus Xiphophorus N2 - Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Same of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern Iod from the platyfish Xiphophorus maculatus the melanoma-inducing gene has been doned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanomapredisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a geneduplication event, thereby acquiring the oncogenic potential. The homology of the melanomainducing Iod points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus. KW - Physiologische Chemie KW - Xiphophorus Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61757 ER - TY - JOUR A1 - Winkler, Christoph A1 - Vielkind, Jürgen R. A1 - Schartl, Manfred T1 - Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes) N2 - Species of small fish are becoming useful tools for studies on vertebrate development. Wehave investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporaland spatial expression patterns ofbacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as weil as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function. KW - Physiologische Chemie KW - Medaka - Genetransfer - Transient expression - DNA fate - Fish developmental biology Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61743 ER - TY - JOUR A1 - Schartl, Manfred A1 - Schlupp, Ingo A1 - Schartl, Angelika A1 - Meyer, Manfred K. A1 - Nanda, Indrajit A1 - Schmid, Michael A1 - Epplen, Jörg T. A1 - Parzefall, Jakob T1 - On the stability of dispensable constituents of the eukaryotic genome: Stability of coding sequences versus truly hypervariable sequences in a clonal vertebrate, the amazon molly, Poecilia formosa N2 - In dooal unisexual vertebrales, the genes specifying the males become dispensable. To study tbe rate of such geoes the gynogeoetic all-female fisb Poecilillfonnolll was treated with androgens. Phenotypic males were obtained that exbibited the complete set of male cbaracteristics of dosely related gooocboristic species, induding body proportions, pigmentation, the extremely complex insemination apparatus of poecilüd fish, sexual bebavior, and spermatogeoesls. Tbe apparent stabllity of such genic structures, induding those involved in androgen regulation, is contrasted by high instability of noncoding sequeaces. Frequent mutations, thelr donal transmission, and at least two truly hypervariable Iod leading to individual difl'ereaces between these othenrise donal organisms were detected by DNA fingerprinting. These observations substantiate the concept that also in "ameiotic" vertebrates certain compartments of the genome are more prooe to mutatiooal alterations than others. KW - Physiologische Chemie KW - DNA fingerprinting KW - androgen-induced masculinization KW - gynogeaesls KW - simple repeat sequences Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61731 ER - TY - JOUR A1 - Förnzler, Dorothee A1 - Wittbrodt, Joachim A1 - Schartl, M.anfred T1 - Analysis of an esterase linked to a locus involved in the regulation of the melanoma oncogene and isolation of polymorphic marker sequences in Xiphophorus N2 - Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecu/ar identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confinn that ES 1 is a distant marker for R ( approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity Ievels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markersthat bridge the large distance between ESl and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding Iandmarks on a physical map of the R-containing chromosomal region. KW - Physiologische Chemie KW - Xiphophorus KW - melanoma ; oncogene regulation ; esterase ; molecular marker sequences Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61726 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Schartl, Manfred A1 - Feichtinger, Wolfgang A1 - Epplen, Jörg T. A1 - Schmid, Michael T1 - Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences N2 - Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically dernonstrahle sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)\(_n\)/(gaca)\(_m\) are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)\(_4\) probe. The (GATA)\(_4\) oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In cantrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)\(_4\). Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)\(_4\) probe. KW - Physiologische Chemie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61715 ER -