Fractionation of mechanically sheared chromatin on ECTHAM-cellulose

Abstract

Chromatography of chromatin on the weak ion-exchange resin ECTHAM-cellulose was re-examined using the combined salt-pH elution conditions of Stratling, W.H., Van, N.T. and O'Malley, B.W. (1976) Eur. J. Biochem. 66, 423–433. When mechanically sheared rat liver chromatin was chromatographed on ECTHAM-cellulose the histone composition of eluted fractions was very similar, whereas early eluting fractions were enriched in non-histone proteins, including certain high mobility group proteins, and in hnRNP particles, containing newly synthesised RNA. Later eluting fractions were depleted in all of these components. The majority of hnRNP particles in early eluting chromatin were shown to be physically associated with chromatin by centrifugation in metrizamide. Hen erythrocyte chromatin contained no early eluting material. Size of DNA fragments was not a significant factor in determining the elution position of chromatin fragments. Early eluting material was not generated by endogenous nuclease and protease action. The conditions of chromatin preparation, and of elution of early chromatin fractions caused no gross disruption of chromatin structure, or dissociation of chromatin proteins, although some nucleosome sliding may have occurred. The conditions required for elution of some of the later fractions are sufficient to cause dissociation of protein, and alteration of chromatin conformation.