Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.

Abstract

Dark field scanning electron microscopy of unstained, unfixed samples of chromatin, histone-l-depleted chromatin, and nucleohistone has been used to identify an apparent subunit of chromatin, namely a disk-shaped structure we term the unit particle, which is probably about 135 A wide and 50 A thick in the hydrated state. The unit particles are found at rather uniform intervals along thin DNA-like fibers. Histone 1 depletion leads to a bimodal distribution of these spacings. Our observations suggest that the unit particle consists of a loop of nucleoprotein, perhaps around a histone core.After many attempts, with limited success, to describe the structure of the eukaryotic genetic material as a continuous structure, investigators are now turning their attention to models of a more discontinuous structure, involving subunits which might be "assembled" into chromatin. (20).The chemical composition and stoichiometry of the samples were characterized using the procedures of Bonner et al. (20). Intact chromatin possessed a total histone/DNA weight ratio of 1.12 I 0.04, an H1/DNA ratio of 0.29 + 0.02, and a non-histone protein/DNA ratio of 0.55 I 0.03. The histone/DNA ratio of HI-depleted chromatin was 0.83 + 0.02; no non-histone proteins were found. The nucleohistone had a ratio of histone/DNA of 1.02 I 0.02 and an Hi/ DNA ratio of 0.14 + 0.02; no non-histone proteins were found.All specimen solutions were "desalted" immediately before preparation, by exclusion on Sephadex G-100 equilibrated with 1 mM NaPO4, pH 7.0. About 2 gl of this Mg/ml solution of chromatin were then placed on a 20-30 A thick hydrophilic carbon film, blotted, and air dried.Electron Microscopy. All specimens were examined at 30 keV in dark field using the high-resolution scanning transmission electron microscope developed in the laboratory of . Microscope operating conditions were as described before, except that we chose not to outgas the specimen by baking (17). The elastically and inelastically scattered electron signals were usually simultaneously stored in digital form directly on magnetic tape, using a 512 X 512 picture element format (16,17). At low instrumental magnification each picture element represented 15.4 A at the specimen; at high magnification each picture element represented 4.96 A. The magnification was determined from

Self-assembly of single and closely spaced nucleosome core particles

“…One turn was measured to consist of 83 ± 4 base pairs (Fig. 7) which is in accord with models proposed previously (22,23,43,44). Hence, these results are consistent with those obtained by DNase II digestion.…”

Nucleosome Arcs and Helices

“…Thus, the shape of the nucleosome core is more nearly a short cylinder than a sphere. This conclusion is in agreement with recent reports (27)(28)(29) …”

“…3, a and b). In the projections observed most frequently, the characteristic concentration of the stain in the middle of the circular nucleosomes (25,28,31) was clearly visible (Fig. 3b), an indication that in this projection one is looking through rows of superimposed nucleosomes.…”

Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher‐order structures by laser‐plasma X‐ray contact microscopy

“…Eukaryotic chromosomes are composed of chromatin fibres which themselves consist of unit structures (Watanabe et d., 1990), nucleosomes of 10-15nm diameter (Langmore & Wooley, 1975;Oudet et al, 1975) and superbeads of 15-5Onm diameter (Hozier et d., 1977;Azorin et al, 1982;Zentgraf & Franke, 1984). Bordas et d. (1986) have shown that the native uncondensed chromatin fibre in solution has an outer diameter of c. 30 nm using synchrotron radiation scattering analysis.…”