Abstract
A method for electron microscopic demonstration of supranucleosomal (20-30 nm chromatin) fiber loops was developed. Chicken erythrocytes were treated with varying concentrations of detergents, such as Joy, sodium N-lauroyl sarcosinate, and sodium laurylsulfate, and then fixed with a formalin solution. The fixed cells were centrifuged onto an electron microscope grid, followed by staining and metal shadowing. Thin-sectioned specimens of the fixed cells were prepared routinely. Although supranucleosomal fiber loops could be observed when any one of these detergents was used, Joy gave the best result. Electron micrographs of rotary-shadowed specimens of erythrocyte ghosts formed by treatment with a low concentration (0.07-0.11 w/w%) of Joy showed a halolike, radial arrangement of supranucleosomal fiber loops around the ghost cells. The width of the halo was about 3 micron. By increasing the detergent concentration (approximately 8% Joy), nucleosome fibers and naked DNA appeared and increased in number, indicating that the supranucleosomal fibers were disassembled by the action of the detergent. Thin-sectioned specimens of cells treated with 0.09% Joy showed granulofibrillar chromatin radially dispersed from the nuclear cage. The fibers were thought to be identical with the supranucleosomal fibers observed in the rotary-shadowed specimens.
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