Dynamic organization of chromosomes in the mammalian cell nucleus
type: CreativeWork
description: Uncovering the motifs of a higher order nuclear architecture and its implications on nuclear function has raised increasing interest in the past decade. The nucleus of higher eukaryotes is considered to display a highly dynamic interaction of DNA and protein factors. There is an emerging view that there are hierarchical levels of gene regulation, reaching from epigenetic modifications at the DNA- and histone level to a higher order functional nuclear topology, in the context of which gene-activating and -repressing processes influence the gene expression profile of an individual cell beyond the sequence information of the DNA. The present work focuses on the analysis of the dynamic aspects of higher order nuclear architecture in living cells. As a prerequisite, an in vivo replication labeling strategy was developed, that enabled the simultaneous visualization of early and mid-to-late replicating chromatin as well as single chromosome territories on the basis of a labeling/segregation approach. The presented scratch replication labeling protocol combines a high labeling efficiency with reduced “damaging” effects and can be successfully applied to a number of adherently growing cell lines, including primary human fibroblasts. In addition, a live cell observation system was developed that facilitates time-lapse confocal (4D) microscopy over elongated time periods which made it possible to follow a complete cell cycle or more. To address possible long-range movements of chromosome territories (CTs) during an entire interphase, fluorescence labeling of a small number of CTs was performed in living HeLa cells stably expressing histone H2B-GFP. This was achieved by in vivo scratch replication labeling with fluorescent nucleotides. Labeled cells were cultivated for several cell cycles until labeled chromatids had segregated. Such cells were followed by time-lapse confocal microscopy over time-scales of up to 20 hours covering major parts or the complete cell cycle. Position…
abstract: Uncovering the motifs of a higher order nuclear architecture and its implications on nuclear function has raised increasing interest in the past decade. The nucleus of higher eukaryotes is considered to display a highly dynamic interaction of DNA and protein factors. There is an emerging view that there are hierarchical levels of gene regulation, reaching from epigenetic modifications at the DNA- and histone level to a higher order functional nuclear topology, in the context of which gene-activating and -repressing processes influence the gene expression profile of an individual cell beyond the sequence information of the DNA. The present work focuses on the analysis of the dynamic aspects of higher order nuclear architecture in living cells. As a prerequisite, an in vivo replication labeling strategy was developed, that enabled the simultaneous visualization of early and mid-to-late replicating chromatin as well as single chromosome territories on the basis of a labeling/segregation approach. The presented scratch replication labeling protocol combines a high labeling efficiency with reduced “damaging” effects and can be successfully applied to a number of adherently growing cell lines, including primary human fibroblasts. In addition, a live cell observation system was developed that facilitates time-lapse confocal (4D) microscopy over elongated time periods which made it possible to follow a complete cell cycle or more. To address possible long-range movements of chromosome territories (CTs) during an entire interphase, fluorescence labeling of a small number of CTs was performed in living HeLa cells stably expressing histone H2B-GFP. This was achieved by in vivo scratch replication labeling with fluorescent nucleotides. Labeled cells were cultivated for several cell cycles until labeled chromatids had segregated. Such cells were followed by time-lapse confocal microscopy over time-scales of up to 20 hours covering major parts or the complete cell cycle. Position…
author: Schermelleh, Lothar
date published: 2003-07-15
date modified: 2026-07-24T02:51:41Z
provider: Ludwig Maxmilians Universität München
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Ludwig-Maximilians-Universität