Quaternary structure of chromatin.

Abstract

Neutron scattering experiments on chro- This evidence has come mainly from nuclease digestion studies and electron microscopy of chromatin and nuclei (4-6). However,, except for the work df Ris, which implies that the 100 A unit thread doubles up in the presence of calcium ions (7), the highei-order folding of the unit thread has been somewhat neglected. Previous x-ray scattering studies on chromatin, although directed at tertiary structure of the order of 100 A, often showed weak inflections in the scattering profile at spacings corresponding to about 400 and 200 A (see, for example, Fig. 2 of ref. 2),. It now seems that the weakness of these low angle x-ray reflections was due to slit smearing from the x-ray diffractometer.This paper presbnts the results obtained from neutron investigations of the higher-order arrangement of the 100 A unit thread. Very low angle reflections are indeed found to be quite prominent in the neutron scattering at spacings of up to 460 A. It is shown that a quaternary chromatin structure does exist, and it is suggested that a coiling of the 110 A unit thread with an average pitch of about 500 A and a radius of 130 A is consistent with these results. METHODSCalf thymus chromatin was isolated by the method of Panyim et al. (8), which is based on the isolation of nuclei, their purification, and their subsequent gentle disruption. Chromatin samples were concentrated by 'centrifugation, and all samples with the exception of a few controls were dialyzed against three changes of the chosen H20/D20 mixture, with a known amount of salt.Neutron scattering experiments were done with the NILS diffractometer built by K. Ibel (9). Exposure times were on the order of 20 min using a AX/X of 8%. With this diffractom-1043 eter and monochromatization, instrumental smearing of the scattering patterns are considerably less than with the x-ray diffractometers used for previous small angle x-ray scattering (2). Even so, the' maxima in the scattering profile are somewhat smeared out.For the discussion that follows,, it is worth discussing some of the features of neutron scattering. The neutron scattering amplitude of each atom or isotope is dependent only on the nuclear structure and not oh the atomic number, as is the case for x-rays. Thus, the scattering from a molecule in solution at zero angle is a function of the sum of the scattering lengths of its atoms or of the atoms in its compl8hent molecular parts, minus that in the same volume of solvent. H20 has a .scattering length density of -0.6 X 1010/cm3, while pure D20 has a value that is 10 times larger and positive. Because the density and atomic abundances of DNA and protein are dissimilar, the neutron scattering from each will vary rather differently with the solvent scattering length density, which is only a function of the H20/D20 content of the solvent. At a D20 content of 38%o by volume, the scattering from histone will be almost zero, whereas DNA will scatter very weakly in the neighborhood of 65% D20. For the results of this paper, it s...

Chromosome, DNA and Plant Evolution

“…Five different investigations (Olins and Olins, 1974;Kornberg, 1974;Bram et at., 1975;Lacy and Axel, 1975;Gottesfeld et at., 1975) have shown that the DNA of chromatin in metabolic cells such as those of calf thymus consists of spheres or pellets of a DNA-histone complex, in which the helix of DNA is tightly bound to histone and of the most constant kind condensed into supercoils, representing a tertiary structure. Such condensed DNA is inaccessible to RNA polymerase enzyme and must be regarded as inactive in transcription.…”

“…These spheres are separated by intervals of the DNA helix that are less tightly condensed, the spacing of which may be determined by other, more varied kinds of histone (Kornberg, 1974). The diameter of the spheres is estimated at 60-80 A (Olins and Olins, 1974) or 100 A (Kornberg, 1974;Bram et at., 1975). These spheres may contain unicate sequences, repetitive sequences, or even noncoding DNA (Lacy and Axel, 1975;Gottesfeld et at., 1975).…”

Chromatin architecture: investigation of a subunit of chromatin by dark field electron microscopy.

“…However, lateral stacking of the particles in the oriented gels might lead to equatorial orientation of the 55 A reflection. (3) The interparticle spacings might provide the basis for the recent observations by neutron diffraction of 400 and 200 A spacings which seem to be concentration independent (36). (4) The significant amount of free fiber(s) which can be associated with the unit particles might provide binding sites for non-histone proteins without requiring extensive restructuring of the histone-histone, histone-DNA or particle-particle interactions.…”

Presence of protein A24 in rat liver nucleosomes.

“…Several lines of evidence have suggested that nucleosomes are packaged into superhelical arrays of chromatin fibers (39)(40)(41)(42). It is possible that proteins A24 and Bu are involved in the supercoiling of chromatin and analysis of the amounts of these proteins in nucleosome multimers will aid in determining their distribution along the chromatin fiber.…”