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.

Spread chromatin fibers, from isolated eucaryotic nuclei, reveal linear arrays of spherical particles (o bodies), about 70 A in diameter, connected by thin filaments about 15 A wide. These particles have been observed in freshly isolated nuclei from rat thymus, rat liver, and chicken erythrocytes. In addition, v bodies can be visualized in preparations of isolated sheared chromatin, and in chromatin reconstructed from dissociating solvent conditions (i.e., high urea-NaCl concentration). As a criterion for perturbation of native chromatin structure low-angle X-ray diffraction patterns were obtained from nuclear pellets at different stages in the preparation of nuclei for electron microscopy. These results suggest that the particulate (v body) structures observed by electron microscopy may be closely related to the native configuration of chromatin.The DNA of eucaryotic chromosomes exists in a highly folded condition largely as a consequence of its interactions with the histone proteins (Huberman, 1973;Pardon and Richards, 1973;Hnilica, 1972;DuPraw, 1970). Numerous models of this folded state have been presented based upon data from low-angle X-ray diffraction and from electron microscopy. The models which appear to be favored, at present, consist of superhelical coiling of a fundamental nucleohistone strand (Pardon and Wilkins, 1972;Bram and Ris, 1971). Previous studies from our laboratory (Olins and Olins, 1974) have presented evidence that chromatin fibers consist of chains of spheroid particles about 70 A in diameter (v bodies) which could contain DNA folded by association with a small number of histone molecules. This paper extends these observations to soluble chromatin and to reconstructed chromatin. In addition, parallel, low-angle X-ray diffraction studies are included which indicate that the particulate structures observed by electron microscopy are closely related to the native state of chromatin. MATERIALS AND METHODS ReagentsFormaldehyde (10%) was prepared fresh by dilution from a stock of 37% analytical reagent grade formaldehyde. Chemicals were reagent grade or better. Buffers and solutions were made from glass-distilled water. Preparation of NucleiRat thymus, rat liver, and chicken erythrocyte nuclei were prepared as described previously Olins, 1974, 1972;Olins and Wright, 1973). Nuclei, recovered after pelleting from high sucrose concentrations, were washed and centrifuged twice in 0.05 M sodium cacodylate buffer (pH 7.5), 0.025 M KCI, 5 mM MgCI2 (CKM), and washed and centrifuged once with 0.2 M KCI before the swelling procedures.Swelling of the nuclei was generally accomplished by suspending the KCI-washed nuclear pellet in 0.2 M KC1 to a concentration of approximately 108 nuclei/ml, followed by a dilution of about 200-fold with distilled water. Nuclei were allowed to swell for 10-15 rain, then the suspension was made approximately I% in formaldehyde by the addition of 0.1 vol of 10% formaldehyde (pH 57,8

We have performed low-angle neutron scattering studies on reconstituted complexes of fd DNA and the gene 5 protein that is produced during infection of Escherichia coli by filamentous fd phage. Essentially identical helical complexes have been made with normal protonated DNA or DNA in which at least 87% of the nonexchangeable protons are replaced by deuterium. From neutron scattering profiles of both complexes over a range of D2O/H2O solvent mixtures, the DNA deuteration is shown to have a dramatic influence on the measured cross-sectional radius of gyration. Most importantly, data for the complex containing deuterated DNA lead to a more negative slope in a plot of the square of the cross-sectional radius of gyration vs. the inverse of the solute-solvent contrast, compared with the slope of a plot of data for the complex containing protonated DNA. This means that, in a cross-sectional view of the complex, the DNA is near the center of the structure. By our analysis, the DNA has a cross-sectional radius of gyration of 17.6 +/- 3 A, while the protein has a cross-sectional radius of gyration of about 33.5 A. Therefore, the model for the structure of the helical complex that has been proposed from X-ray diffraction studies on gene 5 protein crystallized with oligodeoxynucleotides [McPherson, A., Jurnak, F., Wang, A., Kolpak, F., Rich, A., Molineux, I., & Fitzgerald, P. (1980) Biophys. J. 32, 155-170] is not valid for the complex in solution. From our neutron diffraction data we have also obtained values for the solvent-excluded volume and mass per unit length. The relation of our findings to the solution structure of the complex is discussed.

The ability of the immune system to precisely target and eliminate aberrant or infected cells has long been studied in the field of infectious diseases. Attempts to define and exploit these potent immunological processes in the fight against cancer has been a longstanding effort dating back over 100 years to when Dr. William Coley purposefully infected cancer patients with a cocktail of heat-killed bacteria to stimulate anti-cancer immune processes. Although the field of cancer immunotherapy has been dotted with skepticism at times, the success of immune checkpoint inhibitors and recent FDA approvals of autologous cell therapies have pivoted immunotherapy to center stage as one of the most promising strategies to treat cancer. This review aims to summarize historic milestones throughout the field of cancer immunotherapy as well as highlight current and promising immunotherapies in development.

Chromatin fibers consists of globular nucleohistone particles (designated nu bodies) along the length of the chromatin DNA with approximately 6-to7-fold compaction of the DNA within the nu bodies. We have calculated theoretical small-angle x-ray scattering curves and have compared these with experimental data in the literature. Several models predict maxima at the correct angles. The first maximum (approximately 110 degrees A) results from interparticle interference, while both the spatial arrangement and the structure factor the nu bodies can contribute to the additional small-angle maxima. These calculations suggest models which can account for the electron microscopic observation that chromatin is seen as either approximately 100-or approximately 200-to 250 degrees A-diameter fibers, depending on the solvent conditions. They also account for the limited orientability of the x-ray pattern from pulled chromatin fibers.