Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study.

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.

Chicken-erythrocyte inner histone tetramer has been complexed with several natural and synthetic DNA duplexes by salt-gradient dialysis at various protein/DNA ratios. The resulting complexes, in low-ionic-strength buffer, have been examined by electron microscopy, circular dichroism, and thermal denaturation. Electron microscopy reveals nucleosomes (v bodies) randomly arranged along DNA fibers, including poly(dA-dT)*poly(dA-dT), poly(dI-dC)*poly(dI-dC), but not poly(dA)*poly(dT).Circular dichroism studies showed prominent histone or-helix and "suppression" of nucleic acid ellipticity (X>240 nm). Thermal denaturation experiments revealed Tm behavior comparable to that of HI-(or H5-) depleted chromatin. Tm III and Tm IV increased linearly with G+C% (natural DNAs), but were virtually independent of the histone/DNA ratio; therefore, the melting of nucleosomes along a DNA chain is insensitive to adjacent "spacer" DNA lengths. This suggests that Tm III and Tm IV arise from the melting of different domains of DNA associated with the core v body.

Isolated chicken erythrocyte nuclei have been incubated with dilute concentrations of the bifunctional cross-linking agent glutaraldehyde (0–20 mM) in order to stabilize histone-histone interactions within the native nucleus. The kinetics of the disappearance of acid-soluble histones, free amino groups, and of individual histones have been observed to be pseudo first-order. Apparent first-order rate constants for the disappearance of individual histones correlate with the lysine mole percent of that fraction and follow the ranking, kapp: F1 > F2C > F2B ≥ F2A2, F2A1, F3. Histone polymers were observed to form very rapidly during the fixation reaction. Partial fractionation and amino acid analyses of these polymers support the view that they are composed principally of cross-linked (F2C)n molecules (where n = 2 to ∼8). The rate of glutaraldehyde reaction with free amino groups in histones is drastically reduced in solvents that promote chromatin decondensation (i.e., low ionic strengths in the absence of divalent cations) whereas the formation of cross-linked F2C polymers is less severely reduced. It is proposed that some F2C histones exist in close proximity within the isolated erythrocyte nucleus.