Electron Tomography of Metaphase Nucleolar Organizer Regions:
Evidence for a Twisted-Loop Organization |
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Authors: | Laurent Heliot Herv Kaplan Laurent Lucas Christophe Klein Adrien Beorchia Martine Doco-Fenzy Monique Menager Marc Thiry Marie-Franoise ODonohue and Dominique Ploton |
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Institution: | Laurent Heliot, Hervé Kaplan, Laurent Lucas, Christophe Klein, Adrien Beorchia, Martine Doco-Fenzy, Monique Menager, Marc Thiry, Marie-Françoise O’Donohue, and Dominique Ploton |
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Abstract: | Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil. |
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