Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry.

摘要: Abstract Thermal denaturation of nuclear DNA is studied in situ individual cells or isolated cell nuclei by employing the property fluorochrome acridine orange (AO) to differentially stain native and denatured using an automated flow-through cytofluorimeter for measurement fluorescence. RNAse-treated cells, nuclei, are heated, stained measured while suspension AO-DNA interaction under equilibrium conditions. Measurements made rapidly (200 cells/sec); subpopulations from a sample can be chosen on basis differences their staining light-scattering properties analysed separately. rapid; it approaches maximum during first 5 min heating. Divalent cations stabilize against denaturation. At low pH transition occurs at lower temperature width curves (‘melting profiles’) increased. Decrease ionic strength lowers melting temperature. This effect much more pronounced pretreated with acids conditions known remove histones. Histones thus appear providing counterions. least four separate phases distinguished profiles situ; they believed indicate different points complexes particular A decrease (nuclear) ability scatter light coincides situ, possibly representing altered refractive and/or reflective nuclei. Formaldehyde, commonly used prevent renaturation, not present method. The heat-induced alterations chromatin adequately stabilized after cooling absence this agent. Cells heated 60–85 °C exhibit increased total fluorescence AO-staining, which due unmasking new sites DNA. increase neither correlated melting, nor presence Possibly, reflects destruction superstructure maintained temperatures associations other than histone macromolecules (nuclear membrane).