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BY-NC-ND 3.0 license Open Access Published by De Gruyter June 2, 2014

Physico-Chemical Studies of Isolated Chromatin Compared with in situ Chromatin after Partial Hepatectomy in the Rat

  • Paul Miller , Walfried A. Linden and Claudio Nicolini

Abstract

Chromatin was isolated from rat liver cells at 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, there was an increase in chromatin molar ellipticity measured at 276 nm, and a decrease in thermal stability 3 to 8 h after surgery. These events occured prior to the onset of DNA synthesis. These early changes be­ tween non-proliferating (G0) and proliferating (G1) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic changes in template activity.

Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by even the slightest shearing during chromatin preparation, suggesting the loss of native chromatin organization. To eliminate this problem, experiments were also conducted using chromatin in situ. A flow cytometer (FCM) was used to study unfixed liver cell suspensions stained with ethidium bromide (EB). Fluorescence was mea­ sured in the green spectral range after addition of increasing amounts of EB. Experimental evidence is provided that the same alteration in chromatin conformation can be best detected using low molar ratios of EB per unit DNA due to greater fluorescence emission in G1 respect to G0 cells.

These correlated studies demonstrate that the same changes controlling chromatin organization in situ are detected also in the tertiary-quaternary structure of “isolated” chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at the G0 -G1 and G1 -S transitions.

Received: 1978-12-15
Revised: 1979-2-19
Published Online: 2014-6-2
Published in Print: 1979-6-1

© 1946 – 2014: Verlag der Zeitschrift für Naturforschung

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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