Condensation of Interphase Chromatin in Nuclei of Synchronized Chinese Hamster Ovary (CHO-K1) Cells

Abstract

Reversibly permeabilized cells have been used to visualize interphase chromatin structures in the presence and absence of biotinylated nucleotides. By reversing permeabilization, it was possible to confirm the existence of a flexible chromatin folding pattern through a series of transient geometric forms such as supercoiled, circular forms, chromatin bodies, thin and thick fibers, and elongated chromosomes. Our results show that the incorporation of biotin-11-dUTP interferes with chromatin condensation, leading to the accumulation of decondensed chromatin structures. Chromatin condensation without nucleotide incorporation was also studied in cell populations synchronized by centrifugal elutriation. After reversal of permeabilization, nuclei were isolated and chromatin structures were visualized after DAPI staining by fluorescent microscopy. Decondensed veil-like structures were observed in the early S phase (at an average C-value of 2.21), supercoiled chromatin later in the early S (2, 55 C), fibrous structures in the early mid S phase (2, 76 C), ribboned structures in the mid-S phase (2, 98 C), continuous chromatin strings later in the mid-S phase (3,28), elongated prechromosomes in the late S-phase (3, 72 C), precondensed chromosomes at the end and after the S phase (3, 99 C). Fluorescent microscopy revealed that neither interphase nor metaphase chromosomes are separate entities but form a linear array arranged in a semicircle. Linear arrangement was confirmed by computer image analysis.