Study of chromatin organization with trypsin immobilized on collagen membranes |
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Authors: | Christian Marion Bernard Roux Laurence Pallotta Pierre R Coulet |
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Institution: | 1. Laboratoire de Physico Chimie Biologique Université Claude Bernard, Lyon I, 43, Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France;2. Laboratoire de Biologie et Technologie des Membranes du CNRS, Université Claude Bernard, Lyon I, 43, Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France |
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Abstract: | Trypsin immobilized on collagen membranes has been used to digest chromatin polynucleosomes. With this method, the use of protease inhibitor is avoided and the digestion time easily controlled simply by taking the membrane out of the chromatin solution. Its most fundamental advantage is however to allow the mild removing of the most accessible histone fragments without addition of salt then without perturbation of their ionic environment. Degradation of histone fractions were correlated with conformational changes using circular dichroism and electric birefringence measurements. On digestion, the sign of birefringence reversed, becoming negative, and an increase of molar ellipticity was observed. These changes reflecting the unfolding of DNA correspond to the digestion of H1 and also of fragments of H3. This would indicate that H3 and particularly its basic terminal regions, play a fundamental role in the maintenance of chromatin in a compact structure. |
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