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Cell cycle control of higher-order chromatin assembly around naked DNA in vitro
We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with...
Formato: | Texto |
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Lenguaje: | English |
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The Rockefeller University Press
1991
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2289196/ https://www.ncbi.nlm.nih.gov/pubmed/1661728 |
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collection | PubMed |
description | We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis- specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM- 2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle. |
format | Text |
id | pubmed-2289196 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1991 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22891962008-05-01 Cell cycle control of higher-order chromatin assembly around naked DNA in vitro J Cell Biol Articles We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis- specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM- 2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle. The Rockefeller University Press 1991-12-02 /pmc/articles/PMC2289196/ /pubmed/1661728 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Articles Cell cycle control of higher-order chromatin assembly around naked DNA in vitro |
title | Cell cycle control of higher-order chromatin assembly around naked DNA in vitro |
title_full | Cell cycle control of higher-order chromatin assembly around naked DNA in vitro |
title_fullStr | Cell cycle control of higher-order chromatin assembly around naked DNA in vitro |
title_full_unstemmed | Cell cycle control of higher-order chromatin assembly around naked DNA in vitro |
title_short | Cell cycle control of higher-order chromatin assembly around naked DNA in vitro |
title_sort | cell cycle control of higher-order chromatin assembly around naked dna in vitro |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2289196/ https://www.ncbi.nlm.nih.gov/pubmed/1661728 |