Low-angle neutron scattering from chromatin subunit particles

Pardon, J. F.; Worcester, David L.; Wooley, John; Tatchell, Kelly; Holde, Kensal E. van; Richards, B. M.

doi:10.1093/nar/2.11.2163

Cited by 208 publications

(57 citation statements)

References 23 publications

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“…As predicted in our model, the DNA was found to be on the outside (see Pardon et al, 1975). The ubiquity of nucleosomal structure among eukaryotes was clearly established by Dennis Lohr's demonstration of nucleosomes in yeast (Lohr & van Holde, 1975).…”

supporting

confidence: 73%

A random walk amid the macromolecules

Holde

1996

Protein Science

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supporting

confidence: 73%

A random walk amid the macromolecules

Holde

1996

Protein Science

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“…Subunits from higher eukaryotes contain two copies each of histones H2A, H2B, H3, and H4 associated with about 140 base pairs of DNA (12,14). Recent neutron diffraction studies (15) have confirmed earlier proposals (10,11) that these histones form a "core" about which the unit length of DNA is wrapped. Lysine-rich histones HI (and H5 in erythrocytes) appear to be associated primarily with the "spacer" DNA, 30-60 base pairs long, between globular subunits (14,16,17).…”

mentioning

confidence: 82%

Chromatin subunits from baker's yeast: isolation and partial characterization.

Nelson¹,

Beltz²,

Rill³

1977

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

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The organization of proteins along DNA in chromatin of Saccharomyces cerevisiae (baker's yeast) was examined by analyzing the DNA and nucleoprotein products obtained after digestion of yeast nuclei with staphylococcal nuclease. Yeast DNA is digested in situ at regularly spaced cleavage sites about 160 Before the transcriptional apparatus of yeast can be related with certainty to that of higher cells, similarities in genome organization must be established. Interphase chromatin fibrils of higher eukaryotes have a "beaded string" structure consisting of repeated arrays of globular particles (about 100 A diameter) connected by thin nucleoprotein filaments (9-12). Nucleases cleave these interconnecting regions, producing individual "subunits" and larger oligomeric nucleoprotein units that can be readily isolated (12, 13). Subunits from higher eukaryotes contain two copies each of histones H2A, H2B, H3, and H4 associated with about 140 base pairs of DNA (12,14). Recent neutron diffraction studies (15) have confirmed earlier proposals (10, 11) that these histones form a "core" about which the unit length of DNA is wrapped. Lysine-rich histones HI (and H5 in erythrocytes) appear to be associated primarily with the "spacer" DNA, 30-60 base pairs long, between globular subunits (14,16,17).Yeast chromatin potentially may have a subunit structure since yeast nuclei contain several major basic proteins that electrophorese similarly to histones (18,19). In fact, staphylococcal nuclease digests yeast chromatin at discrete intervals, indicating that proteins are arranged along the DNA in-a periodic manner (18,20,21). However, some caution is warranted in interpreting these results because of reports that yeast lacks histones Hi and H3 (22) and that yeast ribosomal proteins may be coisolated with or otherwise mistaken for histones (23).As described below, we have confirmed the periodic structure of yeast chromatin and have isolated and partially characterized monomeric subunits and oligomer units from yeast that appear to be nearly identical to subunits from higher eukaryotes.MATERIALS AND METHODS Isolation and Digestion of Yeast Nuclei. Saccharomyces cerevisiae strain A8209B was obtained from Dr. Gerald Fink (Cornell University). Yeast cells and protoplasts were prepared as described by deKloet et al. (24). Prior to isolation of nuclei, protoplasts (OD6Wj nm = 1-1.5) were incubated for 45 min at 300 in 20 mM KH2PO4 (pH 6.2)/2 mM MgCl2/2% glucose/ 0.3% casamino acids/20% sorbitol to permit return to a metabolically active state (24). The protoplasts were then rapidly cooled to 40 and washed with cold 20% sorbitol. Nuclei were isolated (at 0-40) by a method modified slightly from that of Blobel and Potter (25). Protoplasts were washed once in TKMC buffer (50 mM Tris-HCI, pH 7.5/25 mM KCI/5 mM MgCI2/1 mM CaCI2) containing 0.25 M sucrose, then twice in the same solution containing 0.5% Triton X-100. The crude nuclear pellet was purified by centrifugation through dense sucrose in TKMC buffer plus 0.1 mM phenylmethylsulfo...

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“…A well-characterized entity within the basic subunit of chromatin, the nucleosome, is the "core particle," which has a molecular weight of 200,000 and contains approximately 140 base pairs of DNA and two molecules each of the histones H2A, H2B, H3, and H4 (1,2). Electron microscopy (3,4), neutron scattering (5,6), and x-ray diffraction (7) studies indicate that the "core particle" is a disk 110 A wide by 55 A high with two turns of DNA localized around the compact histone center. The arrangement of histones in the center and along the DNA within a nucleosome is not known.…”

mentioning

confidence: 99%

Characterization of the histone core complex.

Chung¹,

Hill²,

Doty³

1978

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

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A stable histone core complex containing equimolar ratios of H2A, H2B, H3, and H4 has been isolated from chicken erythrocyte chromatin in high salt. This complex has been characterized by sedimentation and chemical crosslinking studies. In velocity ultracentrifugation, only one single sharp sedimentation boundary has been observed with sedimentation coefficient S = 3.8 L 0.1. The molecular weight of the histone core com has been investigated by low-speed sedimentation equilibrium studies over a wide range of protein concentration. Analysis of the apparent weight-average molecular weight as a function of concentration indicates that the histone core complex in 2 M NaCl, pH 9.0, is in equilibrium between a tetramer (H2AXH2BXH3XH4) and an octamer I(H2AXH2BXH3XH4)12 species with a tetramer molecular weight of 55000. The equilibrium constant K is approximately 1.2 X 10-5 liter per mol at 100. Evidence of such a tetramer-octamer equilibrium in solution is also supported by the results of our chemical crosslinking experiments on the histone core complex. A well-characterized entity within the basic subunit of chromatin, the nucleosome, is the "core particle," which has a molecular weight of 200,000 and contains approximately 140 base pairs of DNA and two molecules each of the histones H2A, H2B, H3, and H4 (1, 2). Electron microscopy (3, 4), neutron scattering (5, 6), and x-ray diffraction (7) studies indicate that the "core particle" is a disk 110 A wide by 55 A high with two turns of DNA localized around the compact histone center. The arrangement of histones in the center and along the DNA within a nucleosome is not known. Proximity of certain histones within the nucleosome has been established by protein crosslinking studies; in particular, the use of zero-length crosslinking agents, ultraviolet light, tetranitromethane, and carbodiimide, has demonstrated close contact between H2B and H4, H2B and H2A, and H3 and H4, respectively (8, 9); furthermore, H2B is probably bound simultaneously to both H2A and H4 in vvo (8). Solution studies on mixtures of isolated histones have shown that histones H2A, H2B, H3, and H4 interact pairwise and that each possesses at least two distinct domains for interaction with other histones (9). Their interactions can be roughly divided into two categories: strong interactions between the pairs H2A-H2B, H2B-H4, and H3-H4, and weak interactions between the pairs H2A-H3, H2B-H3, and H2A-H4 (9). Along with dimeric complexes found in mixtures of histone pairs, the arginine-rich histones form a tetramer [(H3)(H4)]2 (10-13). In addition, more complex oligomeric structures are found in solutions of high ionic strength (2 M NaCl) at neutral to alkaline pH (pH 7-10) after gentle extraction of the chromatin with high salt, although the precise nature of the oligomers is not clear (6,(14)(15)(16)(17). The elevated ionic strength and pH reduce the repulsions between the highly charged histones, possibly providing an environment similar to that provided by DNA in the nucleosome. In fact, the c...

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Low-angle neutron scattering from chromatin subunit particles

Cited by 208 publications

References 23 publications

A random walk amid the macromolecules

A random walk amid the macromolecules

Chromatin subunits from baker's yeast: isolation and partial characterization.

Characterization of the histone core complex.

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