Chromatin substructure: an electron microscopic study of thin-sectioned chromatin subjected to sequential protein extraction and water swelling procedures.

Electron microscopic observations and measurements were made on thin-sectioned chromatin fibers and fibrils obtained from nuclei of mature chicken erythrocytes. The nuclei were isolated in low ionic strength gum arabic and octanol then extracted sequentially with (1) 0.14 M NaCl, (2) 0.25 N HCl, (3) buffer saturated phenol, (4) hot 5% SDS and 0.14 M 2-mercaptoethanol and, (5) 0.4 N NaOH. The amount of nuclear protein removed at each of the first four extraction steps was 1, 86, 3 and 11% of the total, respectively. Each extract was characterized by electrophoretic profiles. At each extraction the chromatin was fixed by adding large quantities of a mixture of equal volumes of sodium cacodylate buffered 8% (w/v) glutaraldehyde (pH 6.8) and 2% OsO4 (w/v), directly into (1) an aliquot of the chromatin in extraction fluid, and (2) an aliquot of the chromatin after water washing and swelling. Three size classes of chromatin structure were seen in thin sections prepared for high resolution transmission electron microscopy and stained with uranyl acetate and lead citrate. A thick fiber of about 25 + nm diameter was the predominant large fiber seen in freshly isolated nuclei or in nuclei after salt extraction. This 25 + nm fiber has a substructure consisting of 3.2-5.2 nm diameter fibrils. After water swelling of such freshly isolated or salt extracted nuclei a fiber of about 10 nm diameter was the predominant large fiber instead of the 25 nm diameter fiber. The HCl extraction step which is known to remove histones, caused the disappearance of both the 25 nm and the 10 nm fibers. High magnification (600,000 x) micrographs of the chromatin at all procedural steps, except the last NaOH step, reveal the fibril to be omnipresent. This fibril tends to decrease somewhat in diameter during the protein extraction steps to a 2.5 nm diameter fibril after the hot SDS extraction. A fibril of 2.5 nm diameter is expected of naked double helical DNA stained with a positive stain. The NaOH, which is known to denature DNA, completely destroyed the remaining fibril. We inerpret our results to indicate that the larger chromatin fiber seen in micrographs of thin-sectioned chromatin has a fibrillar substructure which probably represents a double coil of native DNA which may have a thin protein coating of its own. The latter fibril may in turn be wrapped around a hydrophobic histone domain, perhaps reflected in the 10 nm diameter fiber which is seen upon swelling of the chromatin. This 10 nm diameter fiber is thought to be further packaged by folding into the 25 + nm diameter chromatin fiber most frequently reported in thin sections of eukaryotic cell nuclei in situ.