A thermal denaturation study of chromatin and nuclease-produced chromatin fragments

Lewis, Peter N.

doi:10.1139/o77-106

Cited by 17 publications

(20 citation statements)

References 34 publications

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“…The mobility of this synthetic nucleosome on 3.5% Tris/borate/EDTA gels was 0.61 k 0.04 relative to a bromophenol blue marker and agrees exactly with that found by us for an 11-S native calf thymus 140-base-pair core nucleosome on the same gel system [19]. The thermal denaturation derivative profile at 260 nm for this synthetic nucleosome was quite similar to that obtained for a native particle (not shown).…”

Section: Preparation and Properties Of Synthetic Chromatin And Nucleosupporting

confidence: 88%

“…These gels were prerun with 25 pl of 1 M cysteamine hydrochloride to reduce the oxidizing effect of residual ammonium persulfate [9] on histone H3. Synthetic nucleosomes and the constituent DNA were characterized by gel electrophoresis using the 3.5 % Tris/ borate/EDTA gels of Maniatis et al [IS] as described previously [19]. The histone composition was determined by the discontinuous 18% sodium dodecylsulfate gels of Laemmli [20] as modified by Weintraub et al [21].…”

Section: Electrophoresismentioning

confidence: 99%

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Fluorescently Labelled Histones as Probes of Nucleosome Structure

Lewis

1979

European Journal of Biochemistry

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A fluorescent derivative of calf thymus histone H4 has been prepared by the reaction of methionine-84 with N-(iodoacetylaminoethyl)8-naphthylamine-l -sulfonic acid at pH 2.4 in 8 M urea. The preparation and characterization of this labelled histone is described. Fluorescence emission measurements indicate that the label on H4 undergoes a 3 -5-fold increase in emission intensity when H4 self-interacts or binds to DNA alone or is incorporated in a synthetic nucleosome. The changes observed are consistent with the formation of varied apolar environments around methionine-84, due most likely to histone-histone rather than histone-DNA interactions. Preliminary experiments indicate that the precise emission intensity of labelled H4 in the nucleosome is quite sensitive to conditions of ionic strength and histone integrity.There is now considerable interest in the structure of the fundamental chromosomal unit, the nucleosome (for a review see [l]). While X-ray and neutron diffraction studies [2,3] reveal details of the folding of this entity what is also needed is the ability to examine directly the possibility of conformational isomerism. It may be that the basic nucleosome folding scheme can adopt one of a number of discrete structures in response to variables such as ionic strength, degree of histone modification, and non-histone composition.One approach to studying this possibility involves the attachment of environment-sensitive probes to unique sites in the nucleosome [4-61. These probes could then be used to monitor structural changes, both equilibrium and dynamic, in response to variations in the parameters mentioned above. In this connection we wish to report the preparation and general properties of histone H4 labelled with N-(iodoacetylarninoethy1)-8-naphthylamine-l-sulfonic acid at the single methionine-84. This paper will deal mainly with the qualitative behaviour of this covalently bound fluorescent label in free solution and when complexed with DNA and the other histones in a nucleosome. Subsequent communications will be concerned with the quantitative aspects of these interactions and the use of this and other probes covalently attached to the other histones in the nucleosome to elucidate conformational transitions.Abbreviution. PhMeS02F, phenylmethylsulphonyl fluoride. Enzyme. Micrococcal nuclease (EC 3.1.4.7). MATERIALS AND METHODS Preparation of Histone H4 and H4 Peptide 69-102Electrophoretically homogeneous H4 was isolated by gel filtration on Bio-Gel P-10 [7] from a roughly equimolar mixture of histones H2A and H4 prepared by the method of Hooper and Smith [8]. To ensure the absence of methionine oxidation, the H4 samples were treated overnight with 50 % thioglycolic acid [91 and were recovered by acetone precipitation. A crude fraction of H4 peptide 69-102 was prepared by gel filtration on Sephadex G-25 of a partial digest (0.25 M acetic acid) of histone H4 [7]. Contaminating peptide 1-23 was removed by bringing the peptide mixture to 12% perchloric acid and spinning down the precipitate. The pelle...

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Section: Preparation and Properties Of Synthetic Chromatin And Nucleosupporting

confidence: 88%

Section: Electrophoresismentioning

confidence: 99%

Fluorescently Labelled Histones as Probes of Nucleosome Structure

Lewis

1979

European Journal of Biochemistry

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“…This analysis used a combination of circular dichroism and calorimetry, and the samples were analyzed at low salt concentrations to maintain optical clarity. It is unfortunate that these and many other studies (13)(14)(15)(16)(17)(18)(19)(20)(21) of nucleosome and chromatin melting cannot be strictly compared with our results because of the substantial differences in salt concentrations. Weischet et al (12) found the melting of core particles to be quite sensitive to salts in the range 0.1-10 mM.…”

Section: Discussionmentioning

confidence: 64%

Differential scanning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment.

Touchette¹,

Cole²

1985

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

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Differential scanning calorimetry revealed that chromatin melts in four distinct transitions in intact HeLa nuclei at 60°C, 76C, 88°C, and 105°C. Calorimetry of whole cells was characterized by the same four transitions along with another at 65°C, which was probably RNA. Isolated chromatin, however, melted in only two transitions at 72°C and 85°C. Very brief digestion of HeLa nuclei with either micrococcal nuclease or DNase I resulted in the conversion of a structure that melted at 105°C to one that melted at 880C. Further digestion with micrococcal nuclease to the level of the mononucleosome did not result in any further substantial changes in either enthalpy or melting temperatures. In contrast, further DNase I digestion that resulted in cleavage within the nucleosome produced a pronounced shift in melting temperatures to two broad transitions at 62C and 78°C.Eukaryotic DNA exists as long continuous molecules but is condensed on the order of 1000-fold to fit within interphase nuclei (1). The lowest level of folding is that of the nucleosome, a repeating subunit consisting of two each of the core histones H2A, H2B, H3, and H4, along with about 200 base pairs of DNA and one molecule of histone H1. Histone H1 is believed to effect further condensation to a 300-A fiber that has been postulated to be a solenoid (2).Studies of chromatin structure above the level of the solenoid have been hampered due to the tendency of chromatin fibers to form aggregates and turbid suspensions at physiological salt conditions. The use of differential scanning microcalorimetry in studies of chromatin structure has the advantage that samples need not be optically clear as is required for spectral studies (3,4 (7). These workers correlated the intensities of the endotherms observed with the relative amounts of DNA and RNA present in several samples.The results of our study suggest that differential scanning calorimetry can be used to characterize the native chromatin structures in nuclei and that nuclei are to be preferred over isolated chromatin and whole cells. The thermodynamically significant chromatin structures of whole cells were shown to be retained in the nucleus, but a major structure was destroyed by brief micrococcal nuclease treatment. MATERIALS AND METHODSCell Culture. HeLa cells, strain S3, were maintained in suspension culture at cell densities of 2-8 x 105 per ml in Joklik's modified spinner medium supplemented with 5% calf serum. Cells were harvested at cell densities of 5-6 x 105 per ml.Isolation of Nuclei. All steps were carried out at 0-40C. (pH 7.5). NaCl, MgCl2, and CaCl2 were added to final concentrations of 150 mM, 1 mM, -and 1 mM, respectively, and the chromatin was incubated for 1 hr at room temperature. Insoluble chromatin was centrifuged for 10 min at 8000 x g and the pellet was used for calorimetric scanning.Gel Electrophoresis of DNA Digestion Fragments. Following digestion by micrococcal nuclease or DNase I, nuclei were pelleted and dissolved in 5 ml of 1 M NaCl/0.6% NaDodSO4 and then extracted ...

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“…(6) or crude chromatin (21) by gel filtration on a Bio-Gel A-5m column (1.6cm x 90 cm) eluted with 10 mM Triscacodylate -0.7 mM EDTA (pH 8) (22). Mononucleosomes free of histone H1 were prepared by gel filtration of similar digests which had the HI-containing material previously precipitated by 0.12 M NaCl (23).…”

Section: Nucleosome Preparationmentioning

confidence: 99%

“…1 ) . One of the more sensitive methods for examining changes in nucleosome structure is thermal denaturation monitored at 260 nm (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Since there is now good evidence to suggest that mononucleosomes are heterogeneous ( 18,19) and that a factor for nucleosonle assembly exists (20), there is a strong possibility that a mononucleosome can adopt one of a number of potential conformations depending upon its protein (or RNA) complement and its past history.…”

Section: Introductionmentioning

confidence: 99%

Rearrangements and reconstitution of mononucleosomes as monitored by thermal denaturation measurements

Chiu¹,

Lee²,

Lewis³

1980

Can. J. Biochem.

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Derivative melting profiles of calf thymus mononucleosomes have been examined for changes resulting from variations in solvent pH and ionic strength, histone H1 content, and DNA size. Samples of mononucleosomes were found to rearrange during freeze-drying to form an altered monomer and a series of noncovalent multimers. The derivative melting profiles of these particles differ significantly from those for the untreated monomer and dimer. The noncovalent dimer exhibited a new melting transition at 66 degrees C involving approximately 18 base pairs of DNA normally associated with the highest melting transition. Mononucleosomes were reconstituted from 6 M guanidine hydrochloride to give particles with physical properties including melting profile which were virtually indistinguishable from those of the starting material. This result confirms the notion that no structural domains exist in the histone core that can be irreversible denatured by noncovalent perturbations.

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A thermal denaturation study of chromatin and nuclease-produced chromatin fragments

Cited by 17 publications

References 34 publications

Fluorescently Labelled Histones as Probes of Nucleosome Structure

Fluorescently Labelled Histones as Probes of Nucleosome Structure

Differential scanning calorimetry of nuclei reveals the loss of major structural features in chromatin by brief nuclease treatment.

Rearrangements and reconstitution of mononucleosomes as monitored by thermal denaturation measurements

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