The quantitative protein composition of calf thymus chromatin.

The proteins of calf thymus chromatin were analysed quantitatively using a combination of polyacrylamide and cellogel electrophoreses. Quantification was achieved by determining the staining values of each purified histone with amido-black. The results indicate that, on average, 700 molecules histone F1, 850 of F2al, 1440 of F2a2, 1 890 of F2b, 2380 of F3 and 500-1000 non-histone molecules are bound per 10(5) base pairs of DNA. This suggests a moderately dense protein covering of the DNA.     

The solubility of calf thymus chromatin in sodium chloride

The solubility of calf thymus chromatin and chromatin depleted of F1-histone has been examined under various conditions in sodium chloride. F1-depleted DNH was more soluble than native DNH at low concentrations but this difference became small at high concentrations (1mg/ml). Both exhibited minimum solubility in 0.15M -NaCl. The effect of pH and of maleylation of the mino acid side chains on the solubility implied that electrostatic interactions dominated the precipitation reaction. Urea had no effect on the solubility of either complex. N.m.r. studies showed that the chromatin behaved as a rigid complex at all salt concentrations less than 0.6 molar.     

Laser Raman spectra of calf thymus chromatin and its constituents.

Extensive Raman measurements have been made on calf thymus chromatin, core chromatin, the (H3,H4)/DNA complex, and isolated DNA. The results indicate that the alpha-helical content of the nucleosomal histones gradually increases as they form the heterocomplexes that lead to the formation of the octameric nucleosome core. The secondary structure of the latter is not modified as it binds to DNA. The spectra indicate that the DNA essentially retains its B conformation in nucleosomes, although slight changes probably occur in the ribose-phosphate backbone. No specific interactions between the nucleosomal histones and DNA can be established from the spectra, but histone H1 possibly interacts selectively with the thymine bases.     

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.     

Reactive properties of the amino groups of histones in calf thymus chromatin

The reactivity of the five main histones in calf thymus chromatin towards acetic anhydride has been determined using the technique of competitive labelling (Kaplan et al., 1971; Kaplan, 1972). Under trace labelling conditions all the histones incorporate radioactive label from tritiated acetic anhydride, however, only histone IIb2 incorporates the label at a rate which is consistent with a substantial portion of its amino groups being fully exposed. Identification of the reactive functional groups of histone IIb2, revealed that the reactivity could be accounted for entirely by the reactivity of its N-terminal proline. It is concluded that the vast majority if not all the ?-amino groups of histones in calf thymus chromatin have abnormally high pK_a values and thus appear to be buried, while the N-terminus of histone IIb2 is exposed. This conclusion supports the hypothesis that the ?-amino groups of histones form salt linkages with the phosphates of DNA.     

Circular dichroism of micrococcal nuclease-treated calf thymus chromatin (soluble chromatin) in presence of CH3HgOH

Exposing (soluble) calf thymus chromatin and, as reference, protein-free native calf thymus DNA (both in 0.01 M Na+, pH 6.8, 25 degrees C) to increasing concentrations of CH3HgOh produces cooperative transitions in their CD spectra. In the case of chromatin, and there especially at low concentrations of methylmercury, they are due to reactions affecting the relative orientation of the bases in the constituent DNA, without disrupting base-pairing. In the case of protein-free DNA, and with chromatin at higher methylmercury concentrations, the CD changes signal collapse of the DNA secondary structure. Primary data (molar ellipticities [theta], zero-ellipticity points, and rotational strengths R) are presented as a function of methylmercury concentration and wavelength. The results are discussed in relation to previous findings of this laboratory regarding methylmercury-DNA and methylmercury-chromatin interactions, and it is pointed out that the structural alterations observed with chromatin at low levels of methylmercury may very well be the primary events in a chain that is responsible for the teratogenic and clastogenic damages caused by organic mercury.     

A study of the interaction between ethidium bromide and rye chromatin: comparison with calf thymus chromatin.

We studied the interaction of ethidium bromide with rye and calf thymus chromatin. Both types of chromatin have the same dye accessibility, which is about 50% of that of DNA. From this result we conclude that the molecular structure of these two chromatins is similar. For rye, the extraction of H1 produces no change in the binding of ethidium bromide. The subsequent extraction of H2A and H2B produces a 14% increase in the binding, and the removal of H3 and H4, another 54% increase. At this stage, the number of binding sites is still less than that of DNA. This is presumably due to the presence of some tightly bound non-histones. Thus, the arginine-rich histones and the tightly bound non-histones are most responsible for limiting the binding of ethidium bromide to rye chromatin.     

Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.     

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.