Electron microscopy of chromatin subunit particles

Holde, Kensal E. van; Sahasrabuddhe, Chintaman G.; Shaw, Barbara Ramsay; Bruggen, E.F.J. Van; Arnberg, Annika C.

doi:10.1016/0006-291x(74)90348-9

Cited by 106 publications

(42 citation statements)

References 6 publications

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“…7. for HI-depleted chromatin to under these conditions both nucleases cleave the internucleosomal linker region between chromatin subunits [21,56]. The finding of Altenburger et ul.…”

Section: Efrhct Of the Removal Of Histone Hi On The Digestion Of Chromentioning

confidence: 83%

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

1978

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It has been demonstrated by digestion studies with micrococcal nuclease that reconstitution of complexes from DNA and a mixture of the four small calf thymus histones H2A, H2B, H3, and H4 leads to subunits closely spaced in a 137 -I 7-nucleotide-pair register. Subunits isolated from the reconstituted complex contain nearly equimolar amounts of the four histones and sediment at 1 1.6 S. On DNase I digestion both the reconstituted complex and the separated subunits gave rise to series of single-stranded DNA fragments with a 1 0-nucleotide periodicity. This indicates that the reconstitution leads to subunits very similar to nucleosome cores. Nucleosome cores closely spaced in a 140-nucleotide-pair register were also obtained upon removal of histone H1 from chromatin by dissociatiop with 0.63 M NaCl and subsequent ultracentrifugation. In reconstitution experiments with all five histones (including histone H1) our procedure did not lead to tandemly arranged nucleosomes containing about 200 nucleotide pairs of DNA.In the presence of EDTA, DNase I1 cleaved calf thymus nuclei and chromatin at about 200-nucleotide-pair intervals whereas in the presence of Mg2 + cleavage at intervals of approximately half this size was observed. The change in the nature of the cleavage pattern, however, was no longer found after removal of histone H1 from chromatin. This indicates that H1 influences the accessibility of DNase I1 cleavage sites in chromatin. This finding is discussed with respect to the influence of histone HI on chromatin superstructure.

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“…7. for HI-depleted chromatin to under these conditions both nucleases cleave the internucleosomal linker region between chromatin subunits [21,56]. The finding of Altenburger et ul.…”

Section: Efrhct Of the Removal Of Histone Hi On The Digestion Of Chromentioning

confidence: 83%

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

1978

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“…4, bE), suggesting that the salt treatment had destroyed most of the nucleosomes. In fact electron microscopy revealed that only 6 Replicating SV40 DNA molecules appear supercoiled after extraction (24). In the cell they are most likely complexed with proteins in the same ratio as completed circular molecules (25).…”

Section: Experiments and Resultsmentioning

confidence: 99%

“…In these particles, termed nucleosomes (2), about 200 base pairs of DNA are associated with the four histonies F2a,, F2a2, F2b, and F3 (2)(3)(4)(5)(6)(7). Such a repeating unit structure can be formed in vitro by association of the four histonies and linear l)NAs (2).…”

mentioning

confidence: 99%

Folding of the DNA double helix in chromatin-like structures from simian virus 40.

Germond¹,

Hirt²,

Oudet³

et al. 1975

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

578

341

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Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions. In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA. Incubation with an untwisting extract converts the twisted complexes to relaxed structures. Extraction of the DNA from the relaxed complexes yields supercoiled molecules. The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.In eukaryotic nuclei, the fundamental structure of chromatin fibers appears to be a flexible chain composed of globular particles connected by DNA filaments (1, 2). In these particles, termed nucleosomes (2), about 200 base pairs of DNA are associated with the four histonies F2a,, F2a2, F2b, and F3 (2-7). Such a repeating unit structure can be formed in vitro by association of the four histonies and linear l)NAs (2).In the nucleosomes the DNA is under constraint, since it is compacted about 5-fold compared to its length in the extended double helical form (2). The nature of this constraint can be studied by the association of histones to covalently closed circular DNA molecules, since supercoiling or unwinding of the DNA within the nucleosome (luring its formation would alter the supercoiling of the rest of the molecule (8). In addition, from the known thermodynamic prolerties of superhelical DNAs (8-10), the influence of the degree of superhelicity on the formation of nucleosome structures can provide information on whether the formation of a nucleosome is equivalent to an unwinding or winding of the double helix. Simian virus 40 (SV40) D)NA is particularly attractive for such a study for two reasons. First, two circular covalently closed allomorphic forms of this DNA are available, the superhelical DNA I and the relaxed circular DNA Ir which results from the incubation of DNA I with an untwisting extract (FE) (l1). This extract is thought to introduce a single-strand nick into superhelical DNA and to reseal the nick after the torsional tension in the double helix has been relieved. Second, SV40 DNA and the four histones are associated in vivo anld the complexes can be isolated from virions (12, 13) and from infected cells. In the latter case, the complex appears as a compactecl structure with about 20 nueleosomes (14).We

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“…Both endogenous (6)(7) and exogenous (8)(9)(10)(11)(12)(13)(14) nucleases appear to recognize a repeating nucleoprotein unit along the chromatin fiber. Furthermore, chromatin particles isolated from nuclease-treated or sonicated chromatin resemble v-bodies in the electron microscope (15)(16)(17). Thus many lines of evidence support the subunit or "beads-on-astring" model of chromatin structure (2,18).…”

mentioning

confidence: 87%

Structure of transcriptionally active chromatin.

Gottesfeld¹,

Murphy²,

Bonner³

1975

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

108

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Rat-liver chromatin has been fractionated into transcriptionally active and inactive regions [Gottesfeld eta. (1974) Proc. Nat. Acad. Sci. USA 71, 2193USA 71, -2197 and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by the endonuclease DNase IL The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nucleaseresistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition. A regular repeating unit in chromatin was first suggested from the x-ray diffraction studies of Pardon et al. (1) MATERIALS AND METHODSChromatin Fractionation. Chromatin was prepared from rat liver by the method of Marushige and Bonner (22) and treated with diisopropylfluorophosphate (DFP) to inhibit endogenous protease activity (23). Fractionation was carried out as diagrammed in Fig. 1; details of this method have been published previously (21).Preparation of Chromatin Subunits. Nuclease-resistant subunits of rat-liver chromatin were prepared as follows:DNase II was added to 10 units per A260 unit of chromatin in 25 mM sodium acetate (pH 6.6). Digestion was carried out at 240 and was terminated after 90 min by raising the pH to 7.5 with 0.1 M Tris-HCl (pH 11). Nuclease-resistant subunits from chromatin fraction P1 were prepared by homogenizing the pellet fraction in 25 mM sodium acetate (pH 6.6) and redigesting with DNase as described above for whole chromatin. Undigested chromatin (about 20% of the input DNA) was removed by centrifugation at 27,000 X g for 10-15 min. The supernatant was layered on isokinetic sucrose gradients in SW25. 1 cellulose nitrate tubes. The gradients were formed according to Noll (24); the parameters were CTOP = 15% (weight/volume), CRES = 34.2% (weight/ volume), and VMIX = 31.4 ml. All solutions contained 10 mM Tris-HCl (pH 8). Centrifugation was at 25,000 rpm for [36][37][38][39][40][41][42] hr. Gradients were analyzed with an ISCO UV Analyzer and chart recorder. Fractions from these gradients were rerun on 5-24% isokinetic sucrose gradients. The parameters were CTOP = 5.1% (weight/volume), CRES = 31.4% (weight/volume) and VMIX = 9.4 ml. Centrifugation was in the SW 41 rotor at 39,000 rpm at 40 for 16.5 hr.Subunits were also prepared from chromatin devoid of histone I. Removal of this histone was accomplished by extraction of Virtis-sheared chromatin (45 V, 90 sec) with 0.5 M NaCl at 4°. The resultant nucleohistone was pelleted by centrifugation in the Ti 50 rotor at 50,000 rpm for 18 hr.The pellet was digested with nuclease as described above.Redigestion of Chromatin Fraction S2. Chromatin of fraction S2 was redigested with nuclease in three different ways: the DNase II present...

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Electron microscopy of chromatin subunit particles

Cited by 106 publications

References 6 publications

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

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

Folding of the DNA double helix in chromatin-like structures from simian virus 40.

Structure of transcriptionally active chromatin.

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