Biochemical and Electron‐Microscopic Evidence that the Subunit Structure of Chinese‐Hamster‐Ovary Interphase Chromatin Is Conserved in Mitotic Chromosomes

Abstract

Biochemical and EM studies demonstrate that the subunit structure of Chinese hamster ovary cell interphase chromatin is conserved in mitotic chromosomes. Digestion of purified chromosomes or nuclei with micrococcal nuclease [EC 3.1.4.7] produces DNA in discrete size classes, as visualized by polyacrylamide gel electrophoresis, which are common to the 2 materials. Early in digestion the DNA fragments are integral multiples of a monomer approximately 177 base pairs in length, whereas after extensive digestion the remaining DNA fragments migrate ahead of the monomer position. The size of the repeating DNA unit was confirmed as being smaller than that produced by micrococcal nuclease digestion of rat liver nuclei by direct comparison. EM of partially unravelled chromosomes at low ionic strength shows tightly packed spheres (nucleosomes) approximately 12 nm in diameter which are often arranged as linear chains. Chromosomal material prepared for EM after varying extents of micrococcal nuclease digestion is composed of fragments containing progressively fewer nucleosomes, which parallels the loss of high DNA multimer bands in gel electrophoresis. Material unravelled from chromosomes in the presence of NaCl consists of nucleosomes packed in a different configuration which suggests the origin of higher order structures in chromosomes.