Investigations have been carried out into the salt-induced higher-order folding in the transcriptionally active chromatin region of rat liver nuclei by nuclease digestion, sedimentation and CD. The sensitivity of active chromatin in nuclei to micrococcal nuclease was suppressed by raising the ionic strength from 25 to 90 mM, indicating the occurrence of salt-induced condensation. The rate of sedimentation of fractionated inactive chromatin fragments of both moderate (∼3.5 kbp) and large (∼8.8 kbp) size increased maximally to the same extent, while that of active chromatin fragments was dependent on their size. The rate of sedimentation of moderately sized active chromatin fragments (∼5.5 kbp) showed a maximal 15% increase at 90 mM ionic strength. In contrast, a large increase (at least 60%) in the sedimentation rate of large active chromatin fragments (∼21 kbp) was observed at 65 mM ionic strength. A reasonable degree of higher-order folding was observed in large active chromatin fragments even at 25 mM ionic strength. On considering the percentage increase in sedimentation rate as a measure of the higher-order folding of chromatin, a different type of higher-order folding was observed in active chromatin fragments. Although the percentage increase in sedimentation decreased from 40 to 24% with an increase in the size of active chromatin from ∼3 to ∼9 kbp, a further increase in size up to 16 kbp brought the percentage increase back to 40%. CD studies agreed with the conclusions drawn from sedimentation studies. Active chromatin from hypothyroid rats showed similar folding behaviour, but the order of folding was slightly lower than for control active chromatin, at all sizes.

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