Cell membrane‐dependent chromatin condensation

Abstract

It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non-ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNAspecific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed. 0 1995 wiley-Liss, IIIC.

Genome size and chromatin condensation in vertebrates

“…The inverse ratio (DMS/IMS) was taken because there is an inverse relation between the OM fluorescence intensity (i.e., accessibility of DNA to fluorochrome) and the degree of chromatin condensation. Olivomycin was chosen as a fluorescent probe because it is the largest of the DNA-specific fluorochromes, which makes it most sensitive to the chromatin condensation state (Vinogradov 1995b). Possible GC content-related effects of OM binding, formaldehyde fixation and genome size were controlled using partial correlation analysis.…”

“…The degree of cell membrane-dependent chromatin condensation was determined as described earlier (Vinogradov 1995b(Vinogradov , 1996. Briefly, red blood cells were suspended in phosphate-buffered saline (PBS) supplemented with 0.7 mM EDTA, pH 7.5 at a concentration of approximately 10 6 cells/ml.…”

“…In addition to optical problems, there is a large error caused by the species specificity of acid hydrolysis used for the Feulgen reaction, especially when nuclei with a high DNA content are compared with smaller nuclei used as a standard, because the curves of acid hydrolysis greatly differ between them (Bachmann 1970;James 1972;Kato et al 1980). Fixation used in absorption photometry may involve additional problems related to differences in chromatin condensation (Vinogradov 1995b). The results of flow cytometry are much more consistent (especially, if base pair specificity of fluorochromes is taken into account), even when different staining techniques are used (Fig.…”

“…However, it is more difficult to estimate the degree of chromatin condensation as compared with genome size. After cell membrane damage, there is a drastic decondensation of chromatin, which is reflected in a sharp increase of DNA accessibility to DNAtropic fluorochromes and DNase I digestion (Vinogradov 1995b(Vinogradov , 1996Loborg and Rundquist 1997). Therefore, this level of chromatin structure cannot be studied by biochemical methods, which require cell disruption.…”

“…Therefore, this level of chromatin structure cannot be studied by biochemical methods, which require cell disruption. A special method for quantitative monitoring of cell membranedependent chromatin condensation with flow cytometry was proposed (Vinogradov 1995b). In the present work, the relationship between genome size and the degree of chromatin condensation was studied in the erythrocytes of 36 vertebrate species from six classes.…”