Figure 1.
(A–E) Metaphase chromosomes released from mitotic CHO cells in a solution containing a crowding macromolecule in 100 µM K-Hepes buffer.
Representative fields of chromosomes cytocentrifuged and fixed in the same medium as that used for cell lysis. (A, B, F) phase-contrast images; (C–E) DNA labeled with YOYO-1. Chromosomes were released in (A) 12% PEG (Mr 8 kD); (B) 25% PEG; (C) 20% PEG; (D) 40% Ficoll (Mr 70 kD); (E) 12% dextran (Mr 10.5 kD). (F) Chromosomes isolated by a conventional method [29] from a sample of the mitotic cells used in panel A. Magnification is the same in all panels; scale bar in A, 5 µm.
Figure 2.
Images by transmission electron microscopy of chromosomes released in 12% PEG.
Sections are approximately longitudinal or transversal in (A) and (B), respectively. (C) Chromatin fibres in regions of lower density at the periphery of chromosomes; white arrows illustrate regions where fibres of ∼30 nm diameter are seen. Scale bars (A, B), 1 µm; (C), 30 nm.
Figure 3.
Influence of the concentration of crowding agent on chromosome dimensions.
Chromosomes released in 12% PEG were deposited on slides and incubated for 1 h with PEG at the concentration shown in 100 µm K-Hepes buffer, fixed in the same solution, and DNA was labeled with YOYO-1. (A) 3-D volume of the largest chromosome of CHO cells reconstructed from serial confocal sections; scale bar, 1 µm. (B) Length of the largest chromosome, diameter of randomly selected chromosomes, and these values expressed as the % of those in 12% PEG; error bars show SEM from measurements of ≥15 chromosomes. (C) Transverse linescans of fluorescence intensity across representative chomosomes labeled with YOYO-1. (D) Representative images of chromosomes incubated in 100 µm K-Hepes buffer with no PEG for 1 h and labeled with YOYO-1. Scale bar, 1 µm.
Figure 4.
Nucleosomal structure and nonhistone proteins of chromosomes released in 12% PEG.
(A) DNA fragments from chromosomes incubated with micrococcal nuclease, separated on a 2% agarose gel; M, length markers. (B) Proteins extracted from chromosomes in 0.2 N H2SO4 and separated in a 4–20% denaturing SDS-PAGE gel; markers (M) were purified histones from calf thymus. (C) Topoisomerase IIα and (D) SMC2 visualised by immunofluorescence (red); DNA was labeled with YOYO-1 (green). Scale bars, 1 µm.