The organization of supercoiled DNA from human chromosomes

Nucleoids obtained from metaphase-arrested HeLa cells by gentle lysis in non-ionic detergent and 2 M salt were prepared for electron microscopy by Kleinschmidt spreading techniques. From biophysical studies nucleoids have been shown to contain high molecular weight DNA which is supercoiled and topologically constrained. The contents of nucleoids disperse and collapse into 2 dimensions on the water surface as discrete particles (spreads), which are provisionally equated with metaphase chromosomes. In some cases separate spreads are linked. The structure of the spreads is complex. A preliminary description is presented, although we believe that some of the structures reported may be products of preparation and spreading rather than significant features of the organization of intact nucleoids. There is considerable variation in the appearance of different spreads which may be related to the degree of unfolding, spreading or damage of the different preparations. Each spread consists of one or more core areas surrounded by a network of fibres. Cores are composed, at least in part, of compressed fibres and superficially appear to constrain fibres of the network. In the network the inter-twining of pairs of fibres and the occurrence of flat fibre spirals (disks) are interpreted as evidence of DNA supercoiling, but other fibres of similar thickness are not visibly supercoiled. High-order structures including groupings of disks are observed: these may be the structural correlates of the folded domains of supercoiled DNA established biophysically. The relative proportion of disks and other network structures varies between spreads.