Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes.

Abstract

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60-75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.