Dissociation and reconstitution of chromatin without appreciable degradation of the proteins

Ballal, N.R.; Goldberg, David; Busch, Harris

doi:10.1016/0006-291x(75)90418-0

Cited by 46 publications

(12 citation statements)

References 19 publications

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“…In the first modification, the extraction buffer contained 0.05 M NaHSC>3 at pH 5.0 (Panyim et al, 1968;Bartley and Chalkley, 1970). In the second modification, the extraction buffer was 0.14 M NaCl, 0.05 M acetate, pH 5.0, containing 0.1 mM phenylmethanesulfonyl fluoride (PMSF)2 (Ballal et al, 1975). In using the latter procedure, a stock solution of 0.1 M PMSF in 2propanol was diluted 1000-fold into fresh homogenizing buffer solution prior to each wash, as PMSF is known to eventually hydrolyze in aqueous solution (Gold, 1967).…”

Section: Methodsmentioning

confidence: 99%

Conformational changes in subfractions of calf thymus histone H1

Smerdon¹,

Isenberg²

1976

Biochemistry

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This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.

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Section: Methodsmentioning

confidence: 99%

Conformational changes in subfractions of calf thymus histone H1

Smerdon¹,

Isenberg²

1976

Biochemistry

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“…Rat liver nuclei were isolated by the method of Hewish and Burgoyne (1). PMSF: was added to 0.5 mM in all extraction buffers to inhibit proteolytic activity (23).…”

Section: A) -Preparation Of Nuclei and Chromatinmentioning

confidence: 99%

The distribution of histone H1 subfractions in chromatin subunits

Gorka¹,

Lawrence²

1979

Nucl Acids Res

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Rat liver chromatin was digested with micrococcal nuclease to various extents and fractionated into nucleosomes, di and trimers of nucleosomes on an isokinetic sucrose gradient. In conditions under which degradation of linker DNA within the particles was limited, the electrophoretic analysis of the histone content showed that the overall content of H1 histone increased from nucleosomes to higher order oligomers. Moreover, the histone H1 subfractions were found unevenly distributed among the chromatin subunits, one of them, H1--3 showing most variation. A more regular distribution of these subfractions was found in subunits obtained from a more extended digestion level of chromatin. It is suggested that the H1 subfractions differ in the protection they confer upon DNA.

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“…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.…”

Section: Methodsmentioning

confidence: 99%

Analysis of subunit organization in chicken erythrocyte chromatin.

Shaw¹,

Herman²,

Kovacic³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

258

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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

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Dissociation and reconstitution of chromatin without appreciable degradation of the proteins

Cited by 46 publications

References 19 publications

Conformational changes in subfractions of calf thymus histone H1

Conformational changes in subfractions of calf thymus histone H1

The distribution of histone H1 subfractions in chromatin subunits

Analysis of subunit organization in chicken erythrocyte chromatin.

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