Physical Studies of Isolated Eucaryotic Nuclei

Abstract

A B S T R A C TThe degree of chromatin condensation in isolated rat liver nuclei and chicken erythrocyte nuclei was studied by phase-contrast microscopy as a function of solvent pH, K + and Mg ++ concentrations Data were represented as "phase" maps, and standard solvent conditions selected that reproducibly yield granular, slightly granular, and homogeneous nuclei Nuclei in these various states w e r e examined by ultraviolet absorption and circular dichroism (CD) spectroscopy, low-angle X-rav diffraction, electron microscopy, and binding capacity for ethidium bromide Homogeneous nuclei exhibited absorption and CD spectra resembling those of isolated nucleohistone. Suspensions of granular nuclei showed marked turbidity and absorption flattening, and a characteristic blue-shift of a crossover wavelength in the CD spectra. In all solvent conditions studied, except pFI < 2 3, low-angle X-ray reflections characteristic of the native, presumably superhelical, nucleohistone were observed from pellets of intact nuclei. Threads (100-200 A diameter) were present in the condensed and dispersed phases of nuclei fixed under the standard solvent conditions, and examined in the electron microscope after thin sectioning and staining Nuclei at neutral pH, with different degrees of chromatin condensation, exhibited similar binding capacitacs for ethidium bromide. These data suggest a model that views chromatin condensation as a close packing of superhelical nucleohistone threads but still permits condensed chromatin to respond rapidly to alterations in solvent environment. I N T R O D U C T I O NIsolated eucaryofic nuclei undergo rapid morphological changes in response to alterations of solvent pH and electrolyte concentration (Ris and Mirsky, 1949; Anderson and Wilbur, I952;Philpot and Stanier, 1956). As examined with the phase-light microscope, nuclei appear to swell and become optically homogeneous with removal of divalent cations at neutral pH, whereas in the presence of sufficient monovalent or divalent cations, or at acid pH, nuclei are not swollen and appear characteristically "granular," due to differential refractility of ehromatin condensations These nuclear states furnish convenient material for comparing morphologic and other physical properties, in order to understand the mechanisms of chromatin condensation. These states gain further interest by analogy with similar morphologic changes seen during nuclear "activation" (Harris, 1967;Ringertz, 1969).In the present studies, nuclei have been isolated from two tissues, rat liver and chicken erythrocyte. Rat liver nuclei contain considerable amounts of dispersed chromatin, chicken erythrocyte nuclear chromatin is principally condensed. Employing phase-contrast microscopy, morphologic "phase" maps relating nuclear appearance to solvent composition have been constructed Standard solvent conditions have been defined, and the properties THE JOURNAL OF CELL BIOLOGY • VOLIY~E 53~ 1972 • pages 715-736 715 on

X-Ray Diffraction Studies of Nucleohistone: A Polyhelical Model of Chromosome Organization

“…This discrepancy is well beyond the limits of -experimental error, so that it does not appear possible to use a single coil to take into account the results obtained. The position of the peaks has also been found to vary significantly in different samples (6,7). Furthermore, there are some features of the scattering curves which are best interpreted with a model based on a double coil.…”

“…1. A more detailed discussion will be presented elsewhere (11 6, the 0°and 1200 curves cannot be continued because the peaks become superimposed and cannot be distinguished in the continuously decreasing scattering curve. For 1800, a peak is detected in this area, although it is due to an equatorial reflection which gradually dominates the second order peak.…”

“…In more concentrated systems it has been known for some time (2,3) that nucleohistone acquires a more ordered conforma--tion, characterized by a series-of diffraction rings which have been interpreted to arise either from intermolecular packing (3) or from the intramolecular nucleohistone conformation (4,5). More recently new x-ray diffraction studies have been published (6,7) which show that the position and relative intensity of the diffraction rings may vary depending on the conditions used to prepare the samples. In this paper we show that these results can be interpreted on the basis of a polyhelical model of nucleohistone structure.…”

Low-angle X-ray scattering by disordered and partially ordered helical systems

“…This leads to the conclusion that a mixture of coils of different dimensions is required to interpret the experimental pattern. Furthermore, a double-coil model appears to be more appropriate, since the variations in the intensity of the peaks experimentally found under different conditions (Olins & Olins, 1972;Bradbury, Molgaard, Stephens, Bolund & Johns, 1972) A, ct=0 °. In both cases the thickness of the coils was r= 10 A.…”

Association of cyclic GMP with gene expression of polytene chromosomes of Drosophila melanogaster.

“…Several studies (29,30) of the role of cations in chromatin condensation suggest that under appropriate ionic conditions (i.e., high cation), condensation of the chromatin occurs because the negative charge on the DNA phosphates is neutralized by the binding of cations. In this scheme, chromatin dispersion occurs due to the repulsion of negative charges on the DNA brought about by mechanisms that decrease the concentration of bound cation or add negative charges to the DNA.…”