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Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of high mobility group (HMG) binding are preserved in 1 M urea. Specificity is lost in 4 M urea. Thermal denaturation and circular dichroism show a dramatic reversal of the effects of urea on nucleosomes when HMG 14 or 17 is bound, indicating stabilization of the nucleosome by HMG proteins. Complexes formed between reconstructed nucleosomes containing purified inner histones plus poly(dA-dT) and HMG 14 or 17 demonstrate that the HMG binding site requires only DNA and histones. Electron microscopy reveals no major structural alterations in the nucleosome upon binding of HMG 14 or 17. Cross-linking the nucleosome extensively with formaldehyde under cooperative HMG binding conditions does not prevent the ionic strength-dependent shift to noncooperative binding. This suggests mechanisms other than internal nucleosome conformational changes may be involved in cooperative HMG binding.

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.

Thin sections (0.1-0.25 micron) of isolated chicken erythrocyte nuclei were examined at various tilt angles. Stereo pairs of electron micrographs document the parallel alignment of 25-nm chromatin fibers adjacent to the nuclear envelope, and demonstrate a fiber substructure consistent with close-packed arrays of nucleosomes.

Considerable evidence relates the nonhistone proteins high mobility group (HMG) 14 and HMG 17 with the structure of active or potentially active chromatin. In this study, bulk nucleosome core particles prepared from chicken erythrocytes and the complex formed by binding two HMG 14 molecules per nucleosome core were studied by use of small-angle neutron scattering techniques. By varying the H2O/2H2O ratio, and hence the contrast between the solvent and the particles, it was possible to determine the radius of gyration of the protein and of the DNA independently and as a function of HMG 14 binding. The results show an increase of 0.9 +/- 0.6 A (mean +/- SEM) in the protein radius of gyration and of 2.7 +/- 0.6 A in the DNA radius of gyration upon binding of HMG 14 to the nucleosome. These changes are considered in the light of several postulated modes for the unfolding or perturbation of the nucleosome structure. Modeling calculations demonstrate that the observed changes in radius of gyration for the DNA and for the protein are too small to be consistent with an overall unfolding or opening of the core particle upon HMG 14 binding. However, the observed changes are consistent with several models that involve only minor changes in the structure. It is postulated that the differences observed may be an indication of the type of conformational change occurring in active nucleosomes.

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.

Spread chromatin fibers, from isolated eucaryotic nuclei, reveal linear arrays of spherical particles (upsilon 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, upsilon 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 fro electron microscopy. These results suggest that the particulate (upsilon body) structures observed by electron microscopy may be closely related to the native configuration of chromatin.

Spread chromatin fibers and isolated chromatin fragments prepared from chicken erythrocyte nuclei were stained with dilute aqueous uranyl acetate. High-resolution electron micrographs reveal two new morphological features exhibited by many of the chromatin nu bodies: (a) lateral association of the nu body with the connecting strand, and (b) a centrally stained spot approximately 15 A wide, possibly corresponding to a hole or crevice within the nu body.

Human autoimmune sera specific for proliferating cell nuclear antigen (PCNA)/cyclin (auxiliary protein for DNA polymerase delta) demonstrated the presence of epitopes within the macro- and micronuclei of the hypotrichous ciliated protozoa Euplotes eurystomus. Tightly bound PCNA/cyclin was localized at the site of DNA synthesis in macronuclei, the rear zone of the replication band. Starvation or heat shock, conditions that reduce macronuclear replication, resulted in a decrease of PCNA/cyclin in replication bands. Micronuclei also exhibited PCNA/cyclin localization which persisted for a large proportion of the vegetative cell cycle and exhibited significant resistance to adverse culture conditions. Immunoprecipitation of 35S-labeled soluble Euplotes proteins with PCNA/cyclin autoimmune sera revealed a spectrum of low molecular mass proteins. PCNA/cyclin-like proteins have now been observed in the widely divergent species: human, rat, amphibian, yeast, and ciliated protozoa.

The antigenic properties of the polypeptide chains of human 7S γ-globulin have been related to two major non-cross-reacting antigenic determinants of the whole molecule. These determinants, called S and F, were obtained by hydrolysis of γ-globulin with papain. Antisera against whole γ-globulin and against S and F fragments were used in techniques of immune diffusion. Light (L) chains of γ-globulin showed reactions of partial identity with S fragments, and thus are antigenically deficient with respect to these fragments. Antisera directed against F determinants did not react with L chains but did react with heavy (H) polypeptide chain preparations. In addition, the major component of H chain fractions did not appear to contain determinants in common with the S fragments. L chains of a γ-myeloma protein were shown to be antigenically deficient with respect to the whole myeloma molecule, and antigenically identical with the Bence-Jones protein of the same patient. Correlation of these results with those of previous investigations have led to the conclusions that the S fragment which is known to contain the combining region of antibody molecules, consists in part of L chains or portions of L chains, and that the F fragment, which mediates several other functions of the whole molecule, is composed in part of portions of H chains.

Reconstitution of phage-neutralizing activity was observed after admixture of separated L and H polypeptide chains of purified antibodies obtained from single sheep or guinea pigs and directed against f1 and f2 phages. Hybrid mixtures of complementary chains of antiphage antibodies and γ-globulins of the same animal showed less activity than the homologous mixtures of chains from antibodies of the same specificity. 7S interspecies molecular hybrids could be formed between L or H chains of sheep γ-globulins and the complementary chains of guinea pig γ-globulins. In contrast to the results obtained within a single animal species, mixtures of L or H chains of antiphage antibodies from one species with the complementary chains of antibodies to the same phage from the other species showed slight or negligible potentiation of neutralizing activity.

Admixture of separated L and H polypeptide chains of 7S γ-globulins under appropriate conditions has been found to result in the reconstitution of 7S molecules. The chains were mixed in 0.5 N propionic acid and when dialyzed into neutral aqueous buffers interacted to form reconstituted material in greater than 30 per cent yield. This material had sedimentation coefficients of 6S to 7S, a weight average molecular weight of 160,000, and its antigenic structure and electrophoretic properties were the same as those of 7S γ-globulin. By the use of I131 and I125 labels on the different types of chains, combined with ultracentrifugation of chain mixtures in sucrose density gradients, the 7S product was found to contain both isotopes in ratios consistent with the presence of two L and two H chains. After hydrolysis with papain, the reconstituted material yielded products resembling S and F fragments. All of the isotope corresponding to L chains was found in the counterpart of the S fragment; the isotope corresponding to the H chain fraction was present in both fragments. The activity reconstituted from chains of a purified guinea pig antibody to f1 phage was found to be associated mainly with the 7S material. Hybrid molecules containing rabbit L chains and human H chains and of human L chains and rabbit H chains were formed by the same techniques of reconstitution. It was found that the interchain disulfide bonds of native 7S γ-globulins could be broken and reoxidized, as could those of reconstituted 7S material. Reduced L and H chains mixed in propionic acid, dialyzed against neutral buffers containing mercaptan, then against neutral buffers in the absence of mercaptan, formed stable 7S molecules of molecular weight 160,000, which were dissociable only after reduction.