A study of histones in amoeba nuclei by indirect immunofluorescence method.

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

A study on histones in amoeba nuclei by indirect immunofluorescence method

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

Comparative study of the myoid cells of human embryonal and adult thymus by the indirect immunofluorescence method]

The indirect immunofluorescence method showed that decreased (in comparison with myoid cells of adult human thymus) content of muscle antigens in the myoid elements of the embryonic organ caused a greater secretory activity of these elements at the early embryogenesis. Due to increased secretory activity of the myoid cells internal medium of the embryonic thymus contained more antigens common to the muscle tissue than the adult human thymus. The fact that during the ontogenesis the functional activity of the myoid cells correlated with the rate of lymphoid tissue formation favours a suggestion that heteroorganic antigens provide the thymus lymphocytes with information concerning the autoantigen structure necessary to induce natural immunological tolerance.     

Use of the indirect immunofluorescence method for the comparative analysis of histones

With the help of indirect immunofluorescence on the model systems--a ploid line of wheat, haploid and diploid cells of Chlamydomonas reinhardii, glass beads with adsorbed histones--a study was made of the dependence of ultimate dilutions (UD) of antihistone sera on the quantity, density and immunochemical properties of histones--antigens. The UD value in the artificial model system (glass beads) increased with the rise in the quantity and density of histones on bead to some definite limits, and then the UD remains constant to be determined only by the titre of antiserum. In natural model system (wheat, Chlamydomonas reinhardii), with the rise in quantities of DNA and histones in the nucleus their densities remain constant, with no changes of UD values being observed. The results obtained are discussed in terms of establishing the dependence of UD on immunochemical properties of histones-antigens. Thus, the method of indirect immunofluorescence may be used for comparative analysis of immunochemical properties of histones in various objects.     

Preparation and characteristics of antisera to individual histone fractions]

A method is described of preparation and characterization of antisera to pure individual histone fractions not conjugated with other proteins or haptens. Rabbits were given two injections of the antigen and the whole immunization schedule took only three weeks. The antisera were characterized by immunofluorescent technique using mouse liver sections and smears of rat liver nuclei.     

Modification of histone binding in calf thymus chromatin by protamine.

When calf thymus chromatin is incubated with protamine, the protein binds to DNA, forming a chromatin-protamine complex. The binding reaches a saturating level at the weight ratio of protamine to DNA of approximately 0.5. Although the saturated binding of protamine to DNA does not cause major displacement of histones from calf thymus chromatin, examination of the dissociation profiles by salt in combination with urea of protamine-treated chromatin shows that the histone-DNA interactions are markedly altered by such binding. The dissociation of histones from the chromatin-protamine complex requires less NaCl but the same concentration of urea as that for untreated chromatin, suggesting that the electorstatic interactions between the histones and DNA are decreased as a result of protamine binding. When protamine concentration is increased beyond that required for saturated binding to DNA during in vitro exposure of calf thymus chromatin to protamine, lysine-rich histone is completely displaced.     

Carbon magnetic resonance studies of the self-aggregation of calf thymus histones.

Self-aggregation of calf thymus histones H2A and H4 was studied by means of 13C magnetic resonance. Analyses of the changes in the intensities of several side-chain resonances, induced by added NaCl, confirm that carbon magnetic resonance can be used to monitor aggregation phenomena more accurately than other physicochemical methods. In particular the validity of an original computer-based method to treat 13C intensities is confirmed. The details of these aggregation phenomena are critically discussed in the light of a simple experiment on a 13C-enriched sample of H2B.     

A study of ratios between histone fractions]

The amount of individual fractions in the whole histone isolated from the blood of hen, frog (Rana ridibunda), bream (Abramis brama), from rat thymus and from the locust (Schistocerca gregaris) was studied quantitatively. It is shown that the ratios between fractions F2a1, F2b, F2a2 and F3 are fixed. The share of each fraction in the sum of fractions F2a1, F2b, F2a2, F3 was found to be approximately 22.5, 30.5, 21.0 and 26.0 per cent, respectively. The share of the fraction F1 can variate within a rather wide range.     

A study of calf thymus histone fraction F2(b) by gel filtration

The gel-filtration behaviour of calf thymus histone fraction F2(b) was studied at three different salt concentrations (0.01m-, 0.10m- and 1.00m-sodium chloride) and two different pH ranges (pH3–4 and pH6.7–7.1). Other histone fractions [F1, F2(a) and F3] were also utilized to assist interpretation of the data. It was found that the Stokes radius of histone fraction F2(b) was not significantly changed when the salt concentration was increased, implying that the aggregation of the individual histone molecules (Edwards & Shooter, 1969) resulted in only relatively minor changes in the hydrodynamic volume. Aggregation would appear to be due to the salting out of hydrophobic regions giving rise, in the aggregate, to a compact core of hydrophobic groups from which protrude the remaining basic parts of the molecule. Repulsion between charged groups on the basic regions of individual histone molecules would give the aggregate approximately spherical symmetry, the diameter of the aggregate approximating to the length of a single histone molecule.