Separation by equilibrium centrifugation in CsCl gradients of density-labelled and normal deoxyribonucleoprotein from chromatin

Abstract

Deoxyribonucleoprotein containing labelled DNA and/or proteins was prepared from chromatin of mouse cells grown in vitro, and treated with formaldehyde so that the proteins were no longer dissociated from the DNA in caesium chloride solutions. DNP so treated forms a symmetrical band of density 1.384 g/ml. (23 °C) when centrifuged to equilibrium in CsCl gradients. DNP in which thymidine in one strand of the DNA has been replaced by 5-iododeoxyuridine (hybrid DNP) shows a higher density of 1.402 g/ml., and mixtures of normal and hybrid DNP can be partially separated in CsCl gradients. DNP prepared from a mixture of normal DNP- and hybrid DNP-containing cells is similarly resolved into partially overlapping bands; thus aggregation or exchange of proteins between the two DNP species does not appear to occur during preparation. These methods may be applicable to biochemical studies of the pattern of distribution of chromosomal proteins during chromosome replication.