Large-scale isolation of a native deoxyribonucleohistone complex from baker's yeast

Abstract

A method for large scale isolation of a native deoxyribonucleohistone complex from yeast is described. Crude chromatin, obtained after disrupting yeast cells at low ionic strength, contains a large amount of lipids, partially due to contaminating membranes. Most of them are removed by a Triton X-100 treatment, followed by step-gradient centrifugation. About 90% of the pellet may be solubilized by mild procedures, the composition of the soluble material being: histone/DNA = 1.0;nonhistone proteins/DNA = 0.55; RNA/DNA = 0.18. Histones can be obtained with high purity. Micrococcal nuclease digests DNA to yield a series of oligomeric fragments, with an average repeat length of about 160 base pairs. Circular dichroism spectra show that (theta) 270 is reduced by about 30% when compared to pure DNA and that chromosomal proteins are not denatured. These results indicate that the components of the complex conserve the native state.