Heterogeneity of chromatin subunits in vitro and location of histone H1.

Varshavsky, Alexander; Bakayev, Valery V.; Georgiev, G.P.

doi:10.1093/nar/3.2.477

Cited by 323 publications

(145 citation statements)

References 20 publications

Supporting

Mentioning

133

Contrasting

Order By: Relevance

“…The core particles were prepared from Hl-depleted chromatin of mouse Ehrlich ascites tumor cells as described (3,11). The core particles (0.5 mg/ml) were methylated ( (1978) 4185 was denatured at 1000 for 1 min.…”

Section: Methodsmentioning

confidence: 99%

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

Mirzabekov¹,

Shick²,

Belyavsky³

et al. 1978

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

119

View full text Add to dashboard Cite

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particle has been determined by a sequencing procedure based on crosslinking histones to the 5'-terminal DNA fragments produced by scission of one DNA strand at the point of crosslinking. The position of histones on DNA has been identified by measuring the length of crosslinked DNA fragments. The results demonstrate that each of the histones is arranged within several adjacent or dispersed DNA segments of a little less than 10 nucleotides in length. Histone-free intervals are located between these segments at the regular distances of about (1O). nucleotides from the 5' end of the DNA and are likely to face one side of the DNA helix. Histones appear to be arranged in a similar manner on both DNA strands and do not form "locks" around DNA. A linearized model of the core particle is proposed.The periodic structure of chromatin now appears to be well established (for review, see refs. 1 and 2). A repeat unit of chromatin, termed the nucleosome, is made up of an octamer aggregate of histones containing pairs of each of the four main types, H2A, H2B, H3, and H4, which together with histone HI (3) is complexed with DNA of a variable length (150-240 base pairs). The basic structural element of the nucleosome in all cell types seems to be a core particle containing the histone octamer and DNA of about 140 base pairs in length (4, 5). Recent studies of the core particle crystals suggest that its structure is a flat disc of dimensions 110 X 110 X 57 A; about 1.75 turns of DNA in the B form are wound around the protein core (6). The proximity of histones in chromatin has been revealed by histone-histone crosslinking experiments (for review, see ref. 1). Histones H3 and H4 appear to play a central part in nucleosome formation (7) as first suggested by Kornberg (8). In the nucleosome the DNA sites separated by 10-nucleotide intervals are exposed to the action of different nucleases (7, 9). As determined by methylation experiments (10, 11), histones are partly buried in the major groove and leave the minor groove of DNA well exposed.Here we report a high-resolution map for the arrangement of histones along the core DNA determined by a sequencing procedure (11) in which histones were crosslinked to DNA within the core (12). Crosslinking causes the specific scission of one DNA strand at the point of crosslinking and the attachment of only the 5'-terminal DNA fragment to histones. The size of the single-stranded DNA fragments crosslinked to each histone species has been measured to identify the position of histones on one strand of the core DNA from its 5' end. A fairly clear picture of the detailed organization of histones has emerged from this map. Preliminary results of this work have been published elsewhere (11). MATERIALS AND METHODSCrosslinking Histones to DNA. The core particles were prepared from Hl-depleted chromatin of mouse Ehrlich ascites tumor cells as described (3, 11). The core particles (0.5 mg/ml) were methylated (abou...

show abstract

Section: Methodsmentioning

confidence: 99%

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

Mirzabekov¹,

Shick²,

Belyavsky³

et al. 1978

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

119

View full text Add to dashboard Cite

show abstract

“…1). The current, widely-used assay differs little from that originally described by Fried and Crothers 7 and Garner and Revzin 8 , although precursors to the technique can be found in the earlier literature [9][10][11] . Mobility-shift assays are often used for qualitative purposes, although under appropriate conditions they can provide quantitative data for the determination of binding stoichiometries, affinities and kinetics 3,6,12 .…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

View full text Add to dashboard Cite

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

show abstract

“…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).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Nelson¹,

Beltz²,

Rill³

1977

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

View full text Add to dashboard Cite

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...

show abstract

Heterogeneity of chromatin subunits in vitro and location of histone H1.

Cited by 323 publications

References 20 publications

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

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

Contact Info

Product

Resources

About