Publikation: Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts
Dateien
Datum
Autor:innen
Herausgeber:innen
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
URI (zitierfähiger Link)
Internationale Patentnummer
Link zur Lizenz
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Sammlungen
Core Facility der Universität Konstanz
Titel in einer weiteren Sprache
Publikationstyp
Publikationsstatus
Erschienen in
Zusammenfassung
In the work presented, I was able to characterise several aspects of an in vitro DNA replication system with human cell extracts.
I could confirm that plasmids without special sequence characteristics are replicated by the system and that the replication of each template takes place only once, resembling the way genomic DNA is replicated in vivo. The occurrence of different kinds of replication intermediates was shown by electron microscopy, and the fate of the template DNA during the reaction was clarified. I also demonstrated that only a specific form of DNA can serve as substrate and that the products of the reaction can be separated by differential digest in a reasonable manner. Furthermore I was able to proof that the factors responsible for replication in vitro are the the same ones driving the reaction in the cell. It could also be demonstrated that the efficiency of the reaction depends on the cell cycle stage of the cells the protein extracts were prepared from. At last the reaction could be inhibited by depletion of ORC proteins, although the inhibition could not be reverted by the addition of recombinantly expressed ORC complexes.
In conclusion this work is a contribution towards the complete characterisation of an in vitro replication assay as a model for the replication of the genome in human cells.
Zusammenfassung in einer weiteren Sprache
In der vorliegenden Arbeit konnte ich eine Reihe von Eigenschaften eines In vitro-DNA-Replikations-Systems mit menschlichen Zellextrakten beschreiben.
Es konnte bestätigt werden, dass Plasmide unabhängig von deren Sequenz von dem System repliziert werden und dass jedes Plasmid, analog zur genomischen Replikation in vivo, genau einmal repliziert wird. Mit Hilfe von Elektronenmikroskopie konnte gezeigt werden, dass verschiedene Arten von Replikations-Intermediaten auftreten. Auch konnte das Schicksal der DNA während der Reaktion aufgeklärt werden. Es zeigte sich, dass nur eine spezielle Topoisoform von DNA dabei als Substrat der Reaktion dienen kann. Die Produkte der Reaktion wurden durch differentiellen Verdau voneinander getrennt. Desweiteren konnte ich beweisen, dass die Faktoren, die für die Replikation in vitro verantwortlich sind, die selben sind, die in der Zelle diese Aufgabe erfüllen. Ebenso wurde gezeigt, dass die Effizienz der Reaktion davon abhängt, in welcher Phase des Zellzyklus sich die Zellen zum Zeitpunkt der Präparation der Proteinextrakte befinden. Abschließend konnte die Reaktion durch die Depletion von ORC-Proteinen aus den Zellextrakten inhibiert werden, wohingegen das Aufheben der Inhibition durch die Zugabe rekombinant exprimierter ORC-Komplexe nicht gelang.
Die Arbeit hat so einen Beitrag zu der Charakterisierung eines in vitro-DNA-Replikations-Systems als Modell für die Genomreplikation in menschlichen Zellen geleistet.
Fachgebiet (DDC)
Schlagwörter
Konferenz
Rezension
Zitieren
ISO 690
ODRONITZ, Florian, 2004. Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts [Master thesis]BibTex
@mastersthesis{Odronitz2004Estab-8134, year={2004}, title={Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts}, author={Odronitz, Florian} }
RDF
<rdf:RDF xmlns:dcterms="http://purl.org/dc/terms/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#" xmlns:foaf="http://xmlns.com/foaf/0.1/" xmlns:void="http://rdfs.org/ns/void#" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/8134"> <foaf:homepage rdf:resource="http://localhost:8080/"/> <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/8134/1/F_Odronitz_Diplomarbeit.pdf"/> <dc:format>application/pdf</dc:format> <dcterms:abstract xml:lang="eng">In the work presented, I was able to characterise several aspects of an in vitro DNA replication system with human cell extracts.<br />I could confirm that plasmids without special sequence characteristics are replicated by the system and that the replication of each template takes place only once, resembling the way genomic DNA is replicated in vivo. The occurrence of different kinds of replication intermediates was shown by electron microscopy, and the fate of the template DNA during the reaction was clarified. I also demonstrated that only a specific form of DNA can serve as substrate and that the products of the reaction can be separated by differential digest in a reasonable manner. Furthermore I was able to proof that the factors responsible for replication in vitro are the the same ones driving the reaction in the cell. It could also be demonstrated that the efficiency of the reaction depends on the cell cycle stage of the cells the protein extracts were prepared from. At last the reaction could be inhibited by depletion of ORC proteins, although the inhibition could not be reverted by the addition of recombinantly expressed ORC complexes.<br />In conclusion this work is a contribution towards the complete characterisation of an in vitro replication assay as a model for the replication of the genome in human cells.</dcterms:abstract> <dc:rights>terms-of-use</dc:rights> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:40:52Z</dc:date> <dc:creator>Odronitz, Florian</dc:creator> <dcterms:issued>2004</dcterms:issued> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T17:40:52Z</dcterms:available> <dcterms:title>Establishment and Characterisation of an in vitro Replication System with Human Cell Extracts</dcterms:title> <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/8134/1/F_Odronitz_Diplomarbeit.pdf"/> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/8134"/> <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/> <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/> <dc:contributor>Odronitz, Florian</dc:contributor> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> <dc:language>eng</dc:language> </rdf:Description> </rdf:RDF>