Fractionation of Unfixed Chromatin by Buoyant-Density Centrifugation in Gradients Containing 3-Iodo-1, 2-Propanediol and Metrizamide

Abstract

Buoyant-density centrifugation of unfixed chromatin has been performed in a newly devised medium containing 3-iodo-l, 2-propanediol and metrizamide. Chromatins were obtained from isotopically labeled mouse hepatoma cells in suspension culture, either grown normally or density labeled in a medium containing bromodeoxyuridine, by mild digestion of isolated nuclei with micrococcal nuclease. When a mixture of normal and density labeled chromatin, marked with [¹⁴C]thymidine and [³H]bromodeoxyuridine, respectively, was centrifuged in the medium, chromatin peaks represented by labeled DNA were resolved to the extent expected from their separate banding profiles. Centrifugation of an equivalent chromatin mixture labeled with [¹⁴4C] and [³H]lysine, respectively, also yielded resolution of chromatin peaks represented by labeled proteins. Only small amounts of labeled proteins were dissociated from chromatin in the gradient medium. Labeled proteins recovered from the gradient fractions were analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The results suggested that most of the histones remained associated with the original stretches of DNA during the centrifugal fractionation period. Essentially all of the dissociated proteins were found to be non-histone proteins.