Higher-Order Structure Of Chromatin From Resting Cells : I. Electron Microscopy Of Chromatin From Calf Thymus

Abstract

Extremely large domains of the genome of resting cells (calf thymus) have been visualized in the electron microscope by combining mild extraction procedures with a non-artifactual method of mounting the sample (the phospholipid monolayer technique). The observed chromatin strands, free from distortion, reach contour lengths up to 60 micrometers. After lysis of the nuclei, four classes of fibres may be identified on the basis of their diameters (30, 24, 18 and 11 nm, respectively). The morphology of giant chromatin strands is strikingly regular; long trains of equally sized, arc-shaped segments are observed, their length being, in many cases, multiples of a fixed value. The inflection points delimiting contiguous segments are often associated with laminar fragments of the nuclear envelope or, less frequently, linked to fibrillar elements. It appears that higher-order structures of chromatin in resting cells conform, to a large extent, to a so called ‘drapery-like’ mode, according to which a continuous strand runs between contiguous anchorage sites placed on the nuclear envelope. Because of the presence of regularly spaced inflection points, this organization is much more ordered than expected. Spontaneous unwinding of the fibres at low ionic strength, limited nuclease digestion, and relaxation in the presence of ethidium bromide, have been used as probes of the conformation. All these experiments rule out its identification with a single-strand helix. The final ordered state is attained by folding the basic 11 nm strand and by winding up this configuration on itself. This leads to a coiled-coil or ‘rope-like’ model. The 11 nm strand is ‘punctuated’ by sharp kinks. Roughly, it may be assimilated to a chain of semirigid, freely joined elements. As a consequence, local flexibility is greatly enhanced, so allowing the assembly mode described.

Thermodynamics of condensation of nuclear chromatin. A differential scanning calorimetry study of the salt-dependent structural transitions

“…This correspondence has been verified by inspection in the electron microscope of large chromatin fragments released from disrupted nuclei. In the case of native material (Figure 2C), we observe thick fibers, which show local uncoiling, conforming to the coiled-coil or ropelike model for the higher order structure (Cavazza et al, 1983). Thirty minutes after equilibration in 10 mM Na+, chromatin is found in the extended form (D), which after dialysis takes on the morphology characteristic of the native state (E).…”

“…Preparation and Characterization of Nuclei from Calf Thymus. Nuclei were isolated from calf thymus by a modification of the method described previously (Cavazza et al, 1983). During preliminary experiments, we noticed that the activity of endogenous proteases and nucleases could be suppressed almost completely by performing both the grinding and the washing steps in dissociation medium (DM) (75 mM NaCl, 24 mM Na2EDTA, 5 mM NaHS03, and 1 mM PMSF, pH 7.8) which was therefore used instead of the low-salt buffer (0.25 M sucrose, 10 mM Tris, and 5 mM MgCl2, pH 8).…”

“…A droplet of chromatin suspension was applied to a carbon-coated grid for 15 min, the excess removed by filter paper, and the adsorbed material negatively stained with 1% uranyl acetate. Thermally denatured chromatin was also visualized by the phospholipid monolayer technique, as already described (Cavazza et al, 1983;Balbi et al, 1989). A Siemens 102 electron microscope operating at 80 kV was used.…”

Structural domains and conformational changes in nuclear chromatin: a quantitative thermodynamic approach by differential scanning calorimetry

“…Electron Microscopy. Nuclei and chromatin fragments obtained by hypotonic swelling were adsorbed on a phospholipid monolayer as already described (Cavazza et al, 1983) and shadowed with platinum at an angle of 16°from two directions 90°a part. For routine observations on the morphology of nuclei, a droplet of the suspension was applied to a carbon-coated grid, the excess solution removed by filter paper, and the sample stained with 1% uranyl acetate.…”