Antibody labelling and flow cytometric analysis of metaphase chromosomes reveals two discrete structural forms

Metaphase chromosomes from cultured Chinese Hamster Ovary cells were labelled in suspension with a monoclonal antibody to histone 2B, counterstained with propidium iodide (PI) and analysed by flow cytometry. Contour plots of antibody binding (FITC fluorescence) against DNA content (PI fluorescence) revealed two discrete forms of each individual chromosome, showing high and low levels of antibody binding respectively. The two types of chromosome were easily distinguishable by immunofluorescence microscopy. The distribution of individual chromosomes between the two populations was related to chromosome size, with larger chromosomes predominating in the low-labelling population and vice versa. Variations in ionic strength, pH, divalent cation concentration or preparation procedure influenced the absolute level of antibody binding but did not eliminate the two populations. In contrast, preincubation with intercalating dyes, such as ethidium and propidium, inhibited antibody binding and generated a single, low-labelling population. Preliminary evidence suggests that in vivo changes in chromosome structure can affect the distribution of chromosomes between the two populations. Prolonged exposure of cells to Colcemid prior to chromosome isolation, a procedure known to increase chromosome condensation, resulted in a progressive shift into the low-labelling population. Our results suggest that chromosomes undergo a relatively rapid transition from the high-labelling to the low-labelling form during the prometaphase-metaphase stage of mitosis. The timing of this transition appears to be size dependent, with the larger chromosomes preceding the smaller. The transition may represent a change in chromosome condensation.