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

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Abstract
Chromatin was isolated from rat liver cells for 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, chromatin molar ellipticity measured at 276 nm increased, and thermal stability decreased 3-8 h after surgery. This occured prior to DNA synthesis onset. These early changes between non-proliferating (G0) and proliferating (G1) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic template activity change. Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by slight shearing during chromatin preparation, suggesting native chromatin organization loss. 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 measured in the green spectral range after addition of increasing EB amounts. Experimental evidence is provided that the same chromatin conformation alteration 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 in the tertiary-quaternary structure of isolated chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at G0-G1 and G1-S transitions.

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