Intact chromatin, chromatin minus histone H1, and nuclease digestion fragments have been studied by very small angle neutron scattering. The results are not consistent with a straight chain of nucleosomes and require the presence of a higher order coiling in monovalent salt solutions. The data are interpretable by a structure having a cross section radius of gryration of 8.5 +/- 1 nm, which suggests an outer diameter for a coil of nucleosomes of 27 +/- 3 nm.

Chromatin fibers were studied in solutions of mM monovalent salt by small angle neutron scattering. The variation of the cross section radius of gyration with H2O/D2O contrast shows that DNA is at much larger average radial distances from the fiber axis than histone. Consequently, the coils of DNA in a core particle must be approximately parallel to the fiber direction. The radii of gyration suggest that the maximum diameter of chromatin and nucleosomes is approximately 14 nm and that the DNA id distributed in two radial layers. The concentration dependence of the scattering maxima near 14 nm spacings furnishes independent support for a 14 nm external diameter and can be interpreted by a double DNA layer configuration.

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...

Very small angle neutron scattering studies have been made on intact nuclei under a variety of solution conditions. Scattering maxima are observed at 30 to 40 nm and at 18 nm in most environments. Although the spacing, intensity and presence of the maximum near 40 nm varies considerably with environment the 18 nm is rather constant. The 30 to 40 nm maximum appears to be best interpreted by the presence of 35 to 50 nm diameter fibers in nuclei. An important result is that no scattering maximum was observed near 11 nm, suggesting that a tightly super coiled nucleofilament with such a pitch is not present.

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