Scatter analysis of discrete-sized chromatin fragments favours a cylindrical organization.

Fragments of chromatin containing 23 +/- 2.5 nucleosomes have been fractionated after light nuclease treatment of chicken erythrocyte nuclei. Low-angle scattering measures the total z-average radius of gyration of the already well-defined particles and the shape of scatter curves can be compared with three-dimensional analysis as opposed to cross-section analysis of long chromatin fragments. The data show that the particles are not spherical, have no detectable hole in the center of the structure and are best represented by a solid rod-like shape such as that generated by a coil of nucleosomes with the centre perhaps filled with linker DNA and histone H1/H5. 23 nucleosome fragments, where the DNA is partially fragmented, have near-identical scatter curves to the above-defined intact particles, indicating the primary importance of histone proteins in maintaining the integrity of the chromatin higher-order structure. Neutron scattering shows the radii of gyration to be contrast-independent, which fits in with the model calculations for solenoids. Particles with fragmented DNA and the intact particles, therefore, behave as sections of a solenoidal higher-order structure and possibly are observed as "superbeads' only during the folding and unfolding pathways of nucleosome multimers.

Histone H5 promotes the association of condensed chromatin fragments to give pseudo-higher-order structures.

We describe two distinct situations in which chicken erythrocyte chromatin fragments associate in solution. The erythrocyte-specific histone H5 is implicated since chromatins that do not contain H5 do not show this behaviour. Well-defined oligomers of between approximately 6 and approximately 18 nucleosomes prepared at low ionic strength condense and associate when the ionic strength is raised to 75 mM, forming pseudo-higher-order structures. The associated forms, probably predominantly dimers, are stabilized by migration of about 10% of the H5, and of the minor lysine-rich histone H1, from the non-associated forms, probably reflecting the preference of H5 for higher-order structures observed previously [Thomas, J. O. and Rees, C. (1983) Eur. J. Biochem. 134, 109-115]. Since the final (H1 + H5) content of the aggregate at 75 mM is never higher than that of the fragment prepared at low ionic strength, migration is probably to a small proportion of sites that have inevitably become vacant due to handling losses at the higher (but not at low) ionic strength. H5 thus maximizes its interactions in the condensed state of chromatin and even maintains the association of two or more fragments without continuity of the DNA. Aggregates of oligomers larger than about 18 nucleosomes may be too long to withstand hydrodynamic shear forces in the absence of such continuity. During nuclease digestion of nuclear chromatin, H5 and, to a lesser extent, H1, are released from the ends of very short fragments and bind to larger oligomers of various sizes leading to heterogeneous aggregates that survive exposure to low ionic strength. These aggregates, in contrast to those described above, have up to 60% more H5 and 20% more H1 than chromatin prepared at low ionic strength. Whether the excess H5 and H1 bind non-specifically or to a second low-affinity binding site on each nucleosome is not known. The associated forms described above (1) are well defined and potentially useful for structural studies, whereas the other aggregates (2) seem less likely to be directly relevant to the native structure of chromatin.

Solubility and structure of domains of chicken erythrocyte chromatin containing transcriptionally competent and inactive genes.

Chromatin generated by micrococcal nuclease digestion of erythrocyte nuclei can be fractionated into two pools of differing solubility in solvents containing 0.15-0.25 M NaCl. A fixed percentage of the chromatin is soluble under these conditions, independent of the average size of the DNA in the unfractionated chromatin. Chromatin containing particular gene sequences is also distributed between soluble and insoluble fractions in a way that is independent of the average size of the starting material. However, the actual percentage of gene copies present in each fraction is not necessarily the same as for bulk chromatin. The transcriptionally active chicken erythrocyte adult beta-globin gene is more soluble than the bulk, while the ovalbumin gene in the same tissue is less soluble. These differences do not appear to be related to variations in content of RNA, core histones, or two classes of non-histone proteins. Instead, we find that the soluble chromatin pool is somewhat depleted in histones H1 and H5 and contains lower molecular weight DNA than precipitable chromatin. The soluble fraction can be made insoluble by addition of H1. If the precipitable chromatin fraction is redigested to reduce its size and then recombined with the soluble fraction and reprecipitated, the distribution of globin gene is randomized. The results suggest that the partitioning of chromatin into soluble and insoluble pools in 0.15-0.25 M NaCl arises from redistribution of a limiting amount of histones H1 and H5 to the chromatin fractions containing the longest DNA.

Chicken erythrocyte chromatin and nuclear envelope antigens.

Chromatin and inner layer nuclear envelope were isolated from chicken erythrocyte nuclei. Two antisera against dehistonized chromatin and nuclear envelope of chicken erythrocytes were obtained. Using the antiserum against dehistonized chromatin of erythrocytes we found: the presence of the antigens at approximate mol. wts of 56,000 and 77,000 tightly bound with DNA and characteristic of only erythrocyte chromatin; localized antigens at approximate mol. wts of 63,000, 68,000 and 92,000 tightly bound with DNA and common only for chromatin and nuclear envelope of chicken erythrocytes; heterogeneity of the antigens tightly bound with DNA. Using the antiserum against inner layer nuclear envelope we did not find antigens specific only for nuclear envelope and absent in erythrocyte chromatin. Some of the antigens were present in the control preparations of chicken liver chromatin and may be regarded as being species specific.

Effects of chromatin protein fractions on transcriptional activity of chicken thrombocyte and erythrocyte chromatin.

1. Chromatin proteins of chicken thrombocytes and erythrocytes were separated into three fractions by successive extraction with 5 M urea containing various salt concentrations and pH values. Molecular composition of protein fractions was determined by SDS-polyacrylamide gel electrophoresis. 2. The efficiences of the chromatin residues after sequential protein extractions as well as those of reconstituted DNA-protein fraction complexes, in serving as a template for the in vitro RNA synthesis were measured in order to identify the effect of each fraction. 3. The different involvement of chromatin protein fractions of template properties of thrombocyte and erythrocyte chromatin was stated.

Occurrence of H1o-like protein and protein A24 in the chromatin of bullfrog erythrocytes lacking histone 5.

Electrophoretic analysis of acid-soluble chromosomal protein isolated from the erythrocytes of the bullfrog Rana catesbeiana reveals that the nucleated erythrocytes contain five major histones (H1A, H2A, H2B, H3, and H4) and three minor histone-like proteins (H1B, R1, and R2). Histone 5, found as an additional major histone of avian erythrocytes, is not detected in the frog erythrocytes. Three minor components of the bullfrog erythrocytes, which are not present in the avian erythrocytes, have been purified to electrophoretic homogeneity and characterized by amino acid analysis, NH2-terminal analysis, tryptic peptide mapping, and immunological techniques. H1B extracted with 5% HClO4 along with H1A has a very similar amino acid composition and tryptic peptide map to H1o, a subfraction of lysine-rich histones found in nondividing mammalian cells. Microcomplement fixation also shows that H1B and bovine liver H1o share some common antigenic determinants. R1, a basic protein having a ratio of basic/acidic amino acids of 2.0 and 20 mol % lysine, is distinguished from any chromosomal proteins characterized so far on the basis of electrophoretic mobility and amino acid composition. On the other hand, R2 is identified as protein A24 on the basis of its electrophoretic mobility, amino acid composition, and tryptic peptide map. Since H1o and protein A24 are considered to be involved in the inhibition of DNA replication and RNA synthesis, respectively, H1o-like protein and protein A24 in the frog erythrocyte lacking H5 may have central roles in genetic inactivation during erythrocyte maturation.