Studies on the structure of isolated chromatin in three different solvents

Hollandt, H.; Nothohrn, Holger; Riedel, Frank; Harbers, Eberhard

doi:10.1093/nar/6.5.2017

Cited by 26 publications

(12 citation statements)

References 23 publications

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“…As shown in Fig. 5, and has been recognized formerly [53], there are indications for the existence of a higher structural order also in 0.2 mM EDTA solvent, either residues of quaternary structure or another type of superstructure in addition to the tertiary structure. However, the amount of quaternary structure is very low or the assumed super-structure is a fairly loose and wide-spread one.…”

Section: For Chromatin From F K T I O T Zsupporting

confidence: 67%

“…3 shows a Guinier plot of the desmeared scattering data of isolated chromatin. By approximating a straight line to the experimental points one can deduce from the slope the radius of gyration of the cross-section [53]. In Fig.3 two straight lines with different slopes are approximated by linear regression.…”

Section: Resultsmentioning

confidence: 99%

“…The scattering intensity curves were registered with a Kratky camera as described previously [53] Glatter [54] which permits smoothing of data without altering the radius of gyration. For some of the experimental data the indirect transformation into real space [55] was applied, resulting in the distance distribution functions.…”

Section: Small-angle X-ray Scatteringmentioning

confidence: 99%

“…Subsequent increase of the ionic strength up to physiological level causes the formation of higher-order structures. In general it is assumed that these superstructures are identical with the original ones within the cell nuclei [53]. However, the sedimentation and X-ray data indicate that reconstitution of chromatin is incomplete and apparently not perfect.…”

Section: For Chromatin From F K T I O T Zmentioning

confidence: 99%

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Structural Investigations on Isolated Chromatin of Higher‐Order Organisation

Brust

Harbers

1981

European Journal of Biochemistry

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Chromatin of a high structural order was prepared by a method avoiding drastic mechanical stress, as well as strong changes of the ionic strength, and was investigated by small-angle X-ray scattering. The results obtained support the idea that the quaternary structure of completely hydrated chromatin in solution is a solenoid with a diameter of about 32-34 nm. Gel chromatography of this chromatin yields solutions containing mainly quaternary structures with differing portions of tertiary structure. Decrease of the ionic strength results in an increase of loosening and unfolding of quaternary structures. Reconstitution by readjustment of the physiological ionic strength indicates slight differences between reconstituted and original higher-order structures.Chromatin structure appears to be a hierarchical one. The elementary subunit is the nucleosome [I -61 of which the over all shape is well known now. Some details about the arrangement of D N A and histones within the nucleosome are established [7-111 and several models of primary organization of nucleosomes are discussed [8,12 -The next level of organization is the tertiary structure of chromatin which appears to be an arrangement of nucleosomes like 'beads on a string' or tightly packed like a rod with a diameter of about 10-11 nm in solvents of low ionic strength [1,16]. In solvents of higher ionic strength part of the chromatin is organized in a higher-order conformation [ 16 -321 forming a quaternary structure. In electron micrographs this sometimes appears as a 'knobby' fiber of about 25 -30 nm in diameter. However, whether the quaternary structure is organized in the form of 'superbeads' [26,27, 32-34] containing 6-12 nucleosomes or as a solenoid [17,[29][30][31] is still an open question [16].In order to investigate chromatin in solution it has first to be isolated. Even with mild methods avoiding shear and mechanical stress, one can obtain isolated chromatin only as a fragmentary material. A procedure often applied is the lysis of the cell nuclei in 0.2 mM EDTA [38]. In this solvent chromatin appears as a string of nucleosomes, i.e. the tertiary structure. Increasing the ionic strength of the solvent leads to reconstitution of higher-order structures. These quaternary structures appear to be very similar to the original ones in intact cell nuclei; however, it is not known how far they are really identical. By avoiding drastic changes of ionic strength during lysis of the nuclei it is possible to obtain solutions of chromatin having a highly preserved structural order [39,40]. For our structural studies applying small-angle X-ray scattering higher-order chromatin was obtained by a modification of the method of Rees et al. [39,41]. By enzymatic autolysing of the cell nuclei chromatin with a very high sedimentation coefficient can be isolated. Purification of this chromatin by gel chromatography results in solutions containing native quaternary structure and still a certain amount of tertiary structure. This accumulation of quaternary structure material ...

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Section: For Chromatin From F K T I O T Zsupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Small-angle X-ray Scatteringmentioning

confidence: 99%

Section: For Chromatin From F K T I O T Zmentioning

confidence: 99%

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Structural Investigations on Isolated Chromatin of Higher‐Order Organisation

Brust

Harbers

1981

European Journal of Biochemistry

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“…The mass per unit length of chromatin has been measured by small-angle scattering (10)(11)(12)(13)(14). The parameter has also been measured by hydrodynamic methods (7,15) and light scattering (16).…”

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Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.

Gerchman

Ramakrishnan

1987

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

131

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Neutron scattering in solution and scanning transmission electron microscopy were simultaneously done on chicken erythrocyte chromatin at various salt and magnesium concentrations. We show that chromatin is organized into a higher-order structure even at low ionic strength and that the mass per unit length increases continuously as a function of salt concentration, reaching a limiting value of between six and seven nucleosomes per 11 nm. There is no evidence of a transition from a 10-nm to a 30-nm fiber. Fiber diameter is correlated with mass per unit length, showing that both increase during condensation. We also find that there is no essential difference between the mass per unit length measured by scanning transmission electron microscopy and neutron scattering in solution, showing that the ordered regions seen in micrographs are representative of chromatin in solution.Nucleosomes in chromatin interact to form a higher-order structure, referred to as the 30-nm filament. Despite a large number of structural studies on chromatin, there is no unanimity on its higher-order structure. The nature and sources ofconflicting views ofthe structure ofthe 30-nm fiber have been summarized (1).Several models of the 30-nm fiber propose a helical structure for chromatin. These include the solenoidal model (2), the helical-ribbon model (3,4), the crossed-linker doublehelical model (5), and the nonsequential single-helix model (6). All of these models share certain similarities; for example, they all satisfy constraints imposed by electric dichroism measurements on the angle of the nucleosomal disk with respect to the fiber axis (7,8). Most ofthe models have similar repeat dimensions that would show up as peaks in a diffraction pattern. The helical-ribbon model predicts certain additional peaks due to the width of the ribbon. These additional peaks have not been observed in an experiment on oriented chromatin (9), but it is possible that an adjustment in the parameters of the helical-ribbon model would eliminate the additional reflections that are predicted.Despite such similarities, the models differ considerably in detail. Even at a coarse level, the various models predict different values for the mass per unit length of condensed chromatin. Thus a measurement of the physical characteristics of chromatin condensation could help to substantiate or rule out some of the existing models for chromatin structure.The mass per unit length of chromatin has been measured by small-angle scattering (10-14). The parameter has also been measured by hydrodynamic methods (7, 15) and light scattering (16). These experiments generally yield a value of 6-7 nucleosomes per 11 nm for the mass per unit length of the condensed 30-nm fiber. However, scanning transmission electron microscopy (STEM) studies on chromatin (4, 5) have yielded much higher values, of up to 11.6 nucleosomes per nm. Felsenfeld and McGhee (1) have pointed out that the discrepancy may be due to the fact that in STEM studies one selects ordered regions of chromatin, wh...

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Chromatin structure studied by linear dichroism at different salt concentrations

1982

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SynopsisWe have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LDIA over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55", and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1M NaCl or in 0.1 mM MgC12. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na+, this can be explained as a screening effect due to the bulk concentration of Na+. With Mg2+ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaC1, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.

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Studies on the structure of isolated chromatin in three different solvents

Cited by 26 publications

References 23 publications

Structural Investigations on Isolated Chromatin of Higher‐Order Organisation

Structural Investigations on Isolated Chromatin of Higher‐Order Organisation

Chromatin higher-order structure studied by neutron scattering and scanning transmission electron microscopy.

Chromatin structure studied by linear dichroism at different salt concentrations

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