Show simple item record

Molekularbiologische und Röntgenmikroskopische Charakterisierung der Heterochromatinproteine des Nematoden Caenorhabditis elegans

dc.contributor.advisorSchulze, Ekkehard Prof. Dr.de
dc.contributor.authorBahrami, Masoudde
dc.date.accessioned2012-04-16T14:57:03Zde
dc.date.available2013-01-30T23:50:40Zde
dc.date.issued2001-12-12de
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-0006-AC7E-9de
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-669
dc.description.abstractDer Nematode C. elegans ist der erste höhere Eukaryont, dessen Genom vollständig sequenziert wurde (The C. elegans Sequencing Consortium, 1998). Dieser Modelorganismus eignet sich für entwicklungsbiologische, genetische sowie biochemische Studien. In dieser Arbeit wurde C. elegans für die Erforschung der Heterochromatinproteine verwendet. Durch Sequenzvergleich mit Genom von C. elegans ergaben sich zwei homologe Gene, welche für drei Proteine HP1.1, HP1.2 und HP1.3 kodieren. Das Ziel dieser Arbeit war eine vergleichende Analyse dieser Proteine durch Röntgenmikroskopie und Lichtmikroskopie. Durch das Green Fluorescence Protein (GFP) Reportersystem war es möglich, HP1.1 als Fusionsprotein im Organismus darzustellen. Mit Hilfe konfokaler Mikroskopie wurde das Expressionsmuster und die subzelluläre Verteilung des Proteins im Embryo analysiert. Prominente fluoreszierende Chromatinstrukturen fanden sich in den Kernen der meisten Gewebe nahe peripherie des Zellkern. Transienter Knock! -out durch dsRNA vermittelte Interferenz mit hp1.1 mRNA zeigte variable Defekte in larvaler Entwicklung. Weiterhin konnte gezeigt werden, daß HP1.1 somaspezifisch exprimiert wird und nicht am Chromatin silencing in den Keimbahnzellen beteiligt ist. Die Beobachtungen in dieser Arbeit deuten daraufhin, daß HP1.1 in C. elegans eine ähnliche Funktion einnimmt wie bei den Organismen, die einen größeren Anteil an Heterochromatin besitzen.de
dc.format.mimetypeapplication/pdfde
dc.language.isoengde
dc.rights.urihttp://webdoc.sub.gwdg.de/diss/copyrdiss.htmde
dc.titleMolekularbiologische und Röntgenmikroskopische Charakterisierung der Heterochromatinproteine des Nematoden Caenorhabditis elegansde
dc.typedoctoralThesis
dc.contributor.refereeGrossbach, Ulrich Prof. Dr.de
dc.date.examination2001-10-30de
dc.subject.dnb570 Biowissenschaften, Biologiede
dc.description.abstractengThe nematode C. elegans was the first multicellular organisms whose genome was completely sequenced (The C. elegans Sequencing Consortium, 1998). This model organism is highly eligible for a variety of approaches in developmental biology, genetics and biochemistry. For the first time, in this study the nematode C. elegans was used to investigate heterochromatin binding protein 1 (HP1) homologs in order to analyze their molecular functions. A sequence alignment revealed three HP1 homologs, HP1.1, HP1.2, and HP1.3 in the genome of C. elegans. HP1.3 was detected with an anti mouse-HP1 antibody. The nematode C. elegans is a model organism which can be used for genetical and biochemical approaches. The aim of this work was to explore the role of heterochromatin protein homologs in C. elegans. Extrachromosomal and integrated arrays expressions fluorescent fusion proteins of living specimen by HP1.1::GFP were created, which allowed the cytological observation with a confocal laser scanning microscope. The dynamics of the HP1.1 distribution throughout the cell cycle has been documented. The expression of HP1.1::GFP begins with the 60-cell stage in embryogenesis. HP1.1 is present in a very high number of cells in most of the tissues. Furthermore, there are mostly six spot-like subnuclear structures in the chromatin near to the nuclear envelope of interphase nuclei. These subnuclear structures disappear dynamically with the onset of mitosis, when the nuclear envelope breaks down. HP1.1 separates from chromosomes in the prometaphase completely, and relocates to chromosomes at late metaphase. At the anaphase HP1.1 occupies binding sites of the spindle-fibers as a layer. For concomitant visualization of HP1 and DNA a double-label experiment was used, in which H1.1::CFP serves as a indirect marker for DNA. I show that HP1.1 is part of the outer kinetochore of C. elegans holocentric chromosomes using images recorded with a confocal laser scanning microscope. dsRNA with HP1.1 expression showed multiple and variable defects including embryonic death, slow growth, dumpy-like animals, and larval arrest. The molecular mechanisms of HP1.1 localization to the spot-like structures in interphase were analyzed by dsRNA with established HP1 interacting proteins in the hp1.1::gfp reporter strain. dsRNA with the SET domain proteins SU(VAR)3-9 (C41G7.4), and another SET domain (C15H11.5) relocated HP1.1::GFP to the cytoplasm. dsRNA with the lamin B receptor (B0250.7) led to a diffuse intranuclear distribution of HP1.1::GFP in the nuclei. dsRNA with ORC2 (F59E10.1) expression resulted in misregulation of the quantity of HP1.1 expression and a modified intranuclear distribution. These results have given rise to interpretations that HP1.1 is involved in a number of different chromatin protein complexes. HP1.1 is not involved in the chromatin silencing in the germ line of C. elegans as shown by HP1.1 dsRNA experiments in a germline silenced reporter strain. This is additional and confirmed by the analysis of HP1.1 expression patterns. HP1.1 is involved in a number of different chromatin complexes which show that HP1.1 is a strictly somatic protein. The observations in this work indicate that at the molecular level HP1.1 in C. elegans functions in contrast to organisms which contain visible amounts of heterochromatin.de
dc.contributor.coRefereeEngel, Wolfgang Prof. Dr. Dr.de
dc.subject.topicMathematics and Computer Sciencede
dc.subject.gerHeterochromatinde
dc.subject.gerGFPde
dc.subject.gerZellzyklusde
dc.subject.gerRNAide
dc.subject.engHeterochromatinde
dc.subject.engGFPde
dc.subject.engcell cyclede
dc.subject.engRNAide
dc.subject.bk42.23de
dc.identifier.urnurn:nbn:de:gbv:7-webdoc-966-6de
dc.identifier.purlwebdoc-966de
dc.affiliation.instituteBiologische Fakultät inkl. Psychologiede
dc.subject.gokfullWKde
dc.identifier.ppn341039683


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record