[Effect of the supranucleosomal chromatin organization on histone-DNA interrelations]. / Vliianie nadnukleosomnoi organizatsii khromatina na vzaimootnosheniia gistonov i DNK.

It has been demonstrated by the method of competitive displacement of own chromatin histone by excess total histone that chromatin dispersity influence the strength of histone-DNA interactions in a medium of physiological ionic strength. Histone NI was removed from chromatin after the quantity of total histone added to chromatin was equivalent to that existing in chromatin. The proportion of histones H2A and H2B removed from chromatin was increased after mechanical of ultrasonic degradation of chromatin at 5-20-fold excess of total extra-histone. In some histone preparations, the removal of histones H2A and H2B was not detectable at even 200-fold excess of total histone. This may be explained by strengthening histone-DNA interactions in superhelical loops of chromatin.

Stability of nuclear thyroid-hormone receptor and its behavior on hydroxylapatite.

Rat-liver chromatin was kept under several conditions. The maximum binding capacity (Bmax) and the apparent association constant (Ka) for triiodothyronine (T3)-binding were determined. Binding activity (Bmax x Ka) was unstable at 0 and -20 degrees C in the reaction buffer. Maximum binding activity was found upon storage at -70 degrees C or in liquid nitrogen where about 70% of the original activity remained after 3 weeks. With storage, Ka decreased more rapidly than Bmax which showed little change. These temperature-dependent changes in the stored chromatin were specific for the receptor and not observed for other characteristics of the chromatin, i.e., template activity and the electrophoretic separation pattern of chromatin proteins. The elution profile of the receptor on a hydroxylapatite column was unchanged during storage of the chromatin, suggesting that the chemical nature of the binding protein(s) may not change during storage.

Chemically induced gene expression. Manipulation of the transforming ability of simian virus 40 minichromatin by specific chemical hyperacetylation of histones H3 and H4.

We have developed a non-enzymatic acetylating procedure, closely resembling the situation in vivo, utilizing acetyl adenylate, an acetylating agent in vivo, that mimics the enzymatic hyperacetylation of specific histone species. Analysis of the acetylated species of calf thymus histones produced from reaction with soluble chromatin yielded the same species generated in vivo and observed during active gene transcription. Four species of histone H4 and three of histone H3 occur with no alteration in histones H2A or H2B. This procedure has been utilized to hyperacetylate simian virus 40 (SV40) minichromatin in vitro in order to study the effect of acetylated compared to non-acetylated minichromatin in cellular transformation of cultured Balb/3T3 cells. Transformed cell foci appeared only in the cultures infected with hyperacetylated SV40 minichromatin. To select for cellular transformation, foci were transferred to agar-lined culture flasks and grown in the suspension of 1% methylcellulose. The selected cells were plated on slides and analyzed for the presence of T-antigen by indirect immunofluorescence. The hyperacetylated-minichromatin-infected cells exhibited T-antigen-specific fluorescence, while non-acetylated-minichromatin-treated cells and normal cells showed no specific fluorescence. These results suggest a major role for histone hyperacetylation in the mechanism of SV40 viral transformation.

Effects of a chromatin low molecular weight peptidic fraction on differentiation markers and virus production in Friend leukemia cells.

Low molecular weight chromatin peptides exert a dose-dependent inhibition of Dimethylsulfoxide (DMSO)-induced erythroid differentiation of murine Friend Leukemia Cells (FLC). This effect correlates with the degree of purification of the peptide fractions. Crot analysis of globin mRNA amounts in DMSO-treated FLC given the peptides showed a 4-5-fold decrease of messenger RNA in the cytoplasma with no nuclear storage of globin transcripts. Spectrin accumulation in "induced" FLC is inhibited as well. The effects of the peptides on erythroid markers are reversible upon removal of the compounds. They also appear to be specific for induced gene expression as (1) no effects are observed on cell growth and RNA synthesis in normal non-differentiating cell lines; and (2) no changes have been detected with regard to the expression of integrated viral genes coding for continuous shedding of viral particles.

Pyruvate kinase isozymes in various tissues of rat, and increase of spleen-type pyruvate kinase in liver by injecting chromatins from spleen and tumor.

Pyruvate kinase [EC 2.7.1.40] in various tissues of rats was separable into seven kinds of pI-isozymes by isoelectric separation with Ampholine carrier ampholytes; pI 5.4-isozyme, pI 5.6-isozyme, pI 6.2-isozyme (2 kinds), pI 6.6-isozyme, pI 7.4-isozyme, and pI 7.8-isozyme. Some of these pI-isozymes contained bound fructose 1,6-diphosphate (FDP). The bound FDP was completely dissociated when the pI-isozymes were salted out with ammonium sulfate. In the FDP-free form, pyruvate kinase was classified into three types, liver-type (type L) of pI 6.2, muscle-type (type M) of pI 7.4, and spleen-type (type M2) of pI 7.8. The liver-type isoenzyme had two kinds of FDP-binding sites; the pI 5.6-isozyme and pI 5.4-isozyme were obtained when one and two kinds of sites were bound with FDP, respectively. The association and dissociation of FDP at both sites were reversible in the presence and absence of 0.15 M KC1 (high ionic strength). The muscle-type isoenzyme had no FDP-binding site. The spleen-type isoenzyme had two kinds of FDP-binding sites, like the liver-type isoenzyme. When the ionic strength of solutions containing the enzyme and FDP was sufficiently low, one and two kinds of the sites could bind with FDP, converting the enzyme into pI 6.6-isozyme and pI 6.2-isozyme, respectively. FDP bound with one kind of site (the 2nd site) was easily dissociable, but FDP bound with the other kind of site (the 1st site) was not. Provided that the 1st site carried bound FDP, the 2nd site was associable at high ionic strength. The liver-type isoenzyme free of FDP and the spleen-type isoenzyme bound with FDP at both sites had similar pI values of 6.2 and were not separable by isoelectric separation. Some properties of these pI-isozymes were compared. When Rhodamine sarcoma was transplanted in rats, the content of spleen-type isoenzyme in the livers increased. When rats were injected with chromatin prepared from either Rhodamine sarcoma or spleen, the content of spleen-type isoenzyme in the livers again increased. This was not observed on the injection of chromatin prepared from liver, indicating that the factor capable of controlling the gene expression was present in chromatins of sarcoma and spleen but barely or not at all in chromatin of liver.

The effect of antiestrogens on egg yolk protein synthesis and estrogen-binding to chromatin in the rooster liver.

Nafoxidine and CI-628, two well known antiestrogenic compounds, reduce the stimulating effect of estradiol on the estrogen-binding capacity of the liver chromatin from roosters. In vitro both antiestrogens compete with [3H]estradiol for the binding sites on the liver chromatin. They inhibit the estrogen-induced synthesis of egg yolk proteins (vitellogenin) and fail to induce this estrogen-specific protein synthesis by themselves. They show the ability, however, to increase the estrogen-binding sites on the liver chromatin to some extent.

Increased survival of mouse ependymoblastoma hosts previously treated with dehistonized tumor chromatin.

Half of the mice treated with dehistonized chromatin (protein-DNA complex) from a transplantable mouse ependymoblastoma before subcutaneous challenge with the syngeneic tumor survived twice as long as untreated animals.