Chromatin organization in detergent‐lysed chicken erythrocyte nuclei

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.

Scanning force microscopy of chromatin fibers in air and in liquid

“…Supranucleosomal fibers were obtained from chicken erythrocytes by Procedure 1 based on cell lysis by treatment with 100 mM NaCl, 5 mM Tris-HCl, pH 7.0 (Zentgraf et al 1980); Procedure 2, according to Seki et al (1987) in which cells are treated with 0.12% sarcosyl (sodium N-lauroyl sarcosinate, Sigma, Deisenhofen, Germany) at pH 8.7, and Procedure 3, in which extended nucleosomal chains were obtained after hypotonic lysis (Miller and Bakken 1972) according to Trendelenburg and Puvion-Dutilleul (1987).…”

“…The initial steps in the histone H1-induced condensation of nucleosomes have been described (Sogo and Thoma 1989). Other groups have reported the salt-dependent condensation of isolated unfolded chromatin to a 30 nm supranucleosomal fiber (Christiansen and Griffith 1977, Finch and Klug 1976, McGhee et al 1983, Seki et al 1987, Thoma and Koller 1981, Widom 1986, Williams et al 1986, Zentgraf et al 1980. Proposed models for the arrangement of the 30 nm fiber are the superbead model (Renz et al 1977, Zentgraf et al 1980) and a plethora of helical models based on solenoidal (Finch andKlug 1976, Thoma et al 1979), crossed-linker (Bordas et al 1986, Williams et al 1986, and twisted-ribbon (Woodcock et al 1984, Worcel et al 1981 topologies.…”