Analysis of subunit organization in chicken erythrocyte chromatin.

Abstract

Micrococcal nuclease digestion of intact chicken erythrocyte nuclei is shown to result in the formation of core nucleoprotein particles containing about 140 base pairs of DNA. These core particles, which are almost entirely devoid of histones fI and f2c, are derived from transient nucleoprotein particles containing an average of approximately 180 base pairs of DNA. Oligomers of these latter particles may be isolated after brief nuclease digestion. (2). The latter has been proposed by Kornberg (9) to be the basic DNA repeating unit associated with an eight-histone complex. We will show that the eight-histone complex is associated with 140 base pairs of DNA, and that the remaining DNA in the subunit is especially nuclease-sensitive. MATERIALS AND METHODSIsolation and Digestion of Erythrocyte Nuclei. Blood was obtained from adult White Leghorn chickens by cardiac puncture in the presence of heparin. After centrifugation at 3000 X g for 10 min, the plasma and buffy coat were removed. The erythrocytes were washed twrice with an isotonic saline solution and frozen at -600 until needed. The frozen erythrocytes were thawed at 370 in an equal volume of 0.15 M NaCl, 0.015 M Na citrate, pH 7.2 (saline/citrate) and centrifuged at 3000 X g for 10 min; the nuclear pellet was resuspended in 0.25% Nonidet P-40 in saline/citrate. In some experiments 1 mM phenylmethane sulfonyl fluoride was added to inhibit protease action (15). The nuclei were repelleted, washed with saline/citrate and resuspended in 0.3 M sucrose, 0.75 mM CaCl2, 10 mM Tris-HCl, pH 7.2 at a concentration of 2 X 108 nuclei per ml. Digestion of the nuclei by micrococcal nuclease (Worthington) was carried out at 370 with 125 units of nuclease per ml of nuclei suspension. The digestion reaction was terminated by making the solution 10 mM in EDTA, 0.15 M NaCl, and 1% sodium dodecyl sulfate (NaDodSO4). After pronase treatment (0.5 mg/ml) for 4 hr at 370 the DNA was extracted by an NaDodSO4-phenol procedure (16).When the digested chromatin was to be fractionated on an agarose A-Sm column, the reaction was terminated by the addition of EDTA to 10 mM and cooling on ice. After centrifugation at 12,000 X g for 15 min, the nuclei were resuspended in 10 ml of 10 mM Tris-HCI, pH 7.5, 0.7 mM EDTA and disrupted by homogenization for about 1 min at a medium setting on a Virtis homogenizer. The nuclear debris was pelleted at 10,000 X g for 15 min. The supernatant was made 7% in sucrose and applied to a Bio-Rad A-Sm column, 90 X 2.5 cm, equilibrated with 10 mM 0

Comparative subunit structure of HeLa, yeast, and chicken erythrocyte chromatin.

“…Nuclei were isolated and resuspended in digestion buffer as previously described for yeast (10), chicken erythrocyte (8), and HeLa (12) with the following exceptions: for all organisms, 0.1 mM phenylmethylsulfonyl fluoride was added to cell lysis buffer and again during resuspension in digestion buffer; for HeLa cells, lysis buffer contained 1 mM CaCl2, digestion buffer contained 0.1 mM CaC12, and the nuclei were washed one time in digestion buffer; for yeast, digestion buffer had 0.05 mM Ca++. Digestions were done at about 300 gg/ml of DNA and 120 units/ml (Worthington units) of The percent of DNA made acid soluble during the digestion was measured on aliquots of the stopped digestion mixture from the same times of digestion as extracted above.…”

“…It was suggested for chicken erythrocyte (8) that this behavior reflects the existence of two well defined DNA domains within the repeating unit: a relatively nuclease resistant "core" DNA fragment bound tightly to the eight histones, and a nuclease sensitive "spacer" domain which is distinguished by its relative accessibility to nuclease digestion.…”

“…Intranuclear digestion of eukaryotic chromatins by staphylococcal nuclease produces a series of discretely sized DNA fragments (oligomers) with sizes that are integral multiples of a basic size (monomer) (1). Evidence indicates that this pattern of fragments results from partial protection of the DNA by a repetitive organization of histones (2,3), which appear in the electron microscope (4, 5) as globular particles [termed nucleosomes (6)], spaced along the chromatin strands.The size of the DNA contained in monomeric and oligomeric nucleoprotein fragments decreases with digestion time; monomers are digested to relatively stable "core particles" containing about 140 base pairs (bp) of DNA and eight histone molecules (7,8). It was suggested for chicken erythrocyte (8) that this behavior reflects the existence of two well defined DNA domains within the repeating unit: a relatively nuclease resistant "core" DNA fragment bound tightly to the eight histones, and a nuclease sensitive "spacer" domain which is distinguished by its relative accessibility to nuclease digestion.…”

“…The size of the DNA contained in monomeric and oligomeric nucleoprotein fragments decreases with digestion time; monomers are digested to relatively stable "core particles" containing about 140 base pairs (bp) of DNA and eight histone molecules (7,8). It was suggested for chicken erythrocyte (8) that this behavior reflects the existence of two well defined DNA domains within the repeating unit: a relatively nuclease resistant "core" DNA fragment bound tightly to the eight histones, and a nuclease sensitive "spacer" domain which is distinguished by its relative accessibility to nuclease digestion.…”

Closely Spaced Nucleosome Cores in Reconstituted Histone . DNA Complexes and Histone-H1-Depleted Chromatin

“…This enzyme cleaves the DNA preferentially within the nucleosomes and generates a characteristic pattern of single-stranded DNA fragments which differ in size by 10 nucleotides [19]. A similar series of DNA fragments has been obtained on digestion of nucleosome core particles with DNase I [6,9,20]. Recently the digestion of chromatin by a different nuclease, DNase 11, was shown to lead to a novel cleavage pattern depending on the ionic conditions.…”