The use of fluorescence correlations spectroscopy to probe chromatin in the cell nucleus.
All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small system, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well-characterized DNA preparations, including calif thymus DNa, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and it decreases as the cells leave the resting state and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000648:ultrastructure*; D055598:Chemical Phenomena; D002621:Chemistry; D002843:Chromatin / Q000648:ultrastructure*; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D004996:Ethidium; D008433:Mathematics; D013539:Simian virus 40 / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland / Q000032:analysis
D002843:Chromatin; D004279:DNA, Viral; D004247:DNA; D004996:Ethidium