Changes in the circular dichroic spectrum of calf thymus solubilized chromatin caused by ultraviolet irradiation

Summary UV irradiation of the chromatin caused an increase of the positive circular dichroic band in the vicinity of 275 nm (corresponding to DNA) and a deepening of the negative band of proteins at about 225 nm. These changes in the circular dichroic spectrum are monotonous in the range of doses studied (< 6 × 10⁴ J.m^–2). The increase of the positive circular dichroic band probably reflects the occurrence of local conformational changes in DNA, which include changes in base position (tilting, distance from helix axis) in the close neighbourhood of photoproducts. The presence of photoproducts in chromatin reduces changes in its circular dichroic spectra with temperature.     

The effect of various conditions of chromatin isolation on the nucleosomal structure of the isolated chromatin

Chromatin from calf thymus isolated under hypotonic conditions in the presence of various agents was investigated by methods of electron microscopy prior to and after EDTA treatment. It is shown that the presence of chelating agents and, especially, the application of considerable mechanical forces in the course of isolation may cause damage to the nucleosomal structure of the chromatin. Moreover, sufficiently great mechanical forces are liable to destroy the structure of the chromatin nucleosomal fibres even when they are packed in structures of a higher order of organization of the chromatin.     

The effect of the ionic strength on G-bands

Summary The most important factors, for direct demonstration of G-bands, are the ionic strength and the dye concentration of the staining solution. Sörensen buffer, ionic strength 0.10 and 0.50 vol% Giemsa produce G-banding of human metaphase chromosomes.     

Effect of heparin polyanion on chromatin preparations obtained in solutions of low ionic strength]

DNP obtained in low ionic strength solutions (0.7 mM Na-phosphate buffer, pH 7.0) was found to be dissociated under the effect of heparin. The dissociation order of the three histone fractions was established: H2a, H1, H4. The following order of histones is assumed: H2a, H2b, H1, H3, H4. Activation of the DNA and RNA synthesis in the eucaryotic cells, their nuclei and chromatin under the effect of low heparin doses should be associated not with the H1 histone dissociation, but with the dissociation of histones moderately rich in lysine--H2a, and, probably, H2b.     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.     

The effect of low ionic strength extracellular solutions on the resting potential in skeletal muscle fibers

Intracellular measurements of the resting potential were made in fibers of the frog sartorius muscle in solutions of varying salt composition and concentration to determine the effects of low ionic strength extracellular solutions on the resting potential. Changes in the glass microelectrode tip potential in low ionic strength solutions were minimized by adding ThCl₄ to the extracellular solution. These experimental conditions allowed measurement of the relationship of the resting potential to the concentration of the salt in the extracellular solution by replacing it with the nonionic substance, sucrose. Substitution of sucrose for the extracellular NaCl produced a stable depolarization which was logarithmically related to the NaCl concentration. Substitution of sucrose for choline Cl, instead of NaCl, produced the same degree of depolarization. When Na salts of anions less permeable than chloride (Br, I, NO₃) were used, the resting potentials in 116 mM solutions were close to those with chloride (±3mv). The depolarizations produced in low ionic strength solutions of these salts were significantly less than those with chloride.     

Polyamine addition to preparation media induces chromatin condensation, irreversibly at low ionic strength

Polyamines (spermine, spermidine) are commonly used as stabilizing cations in the chromatin preparation media. Their residual binding to chromatin is not easily reversed at low ionic strength, even after extensive dialysis, as evidenced by the use of labelled spermidine. Electric dichroism measurements show that their presence interferes with the physico-chemical characterization of chromatin by maintaining a condensed structure. These results give a definite answer to the controversy about the sign of optical anisotropy of chromatin determined by electric and flow dichroism techniques.     

On the origin of the positive band in the far-ultraviolet circular dichroic spectrum of fibronectin

The genesis of the positive bands in the far-ultraviolet circular dichroic spectra of human plasma fibronectin and its 31-kDa NH2-terminal heparin-binding fragment was studied. Spectra of ester derivatives of tyrosine, tryptophan, and phenylalanine and of model mixtures of these derivatives in which they are present in the same ratios as in the proteins indicate that all the aromatic side chains make substantial contributions to composite positive bands with maxima several nanometers below those of the proteins. In the presence of solvent perturbants such as polyethylene glycol and ethylene glycol, the bands of the model mixtures are red-shifted to the approximate positions they have in the spectra of the proteins. No additional red shift is seen with solvent perturbation of the proteins, suggesting that the conditions leading to this effect are satisfied within the proteins. A separate effect of solvent perturbation, an increase in amplitude of the positive band, occurs equally in solutions of free aromatic amino acid derivatives and in the proteins. This effect is used to estimate the relative accessibility of the chromophores of fibronectin and its fragment to different perturbants. The possible influence of protein secondary structure on the amplitude of the positive circular dichroic band is discussed.     

Viscosity of chromatin solutions increases with increasing ionic strength

Increasing the ionic strength of rat liver chromatin solutions above 0.4 M causes increasing viscosity. This indicates transformation of the compact chromatin molecules to more elongated forms. In the range of 0.4–0.5 M ionic strength histone H1 is dissociating continuously from the chromatin and the quaternary structure chromatin unravels. At ionic strength higher than 0.5 M the viscosities of chromatin solutions are furthermore increasing due to structural deformation. Near 0.7 M ionic strength the core histones H2A and H2B begin to dissociate from the chromatin, and the opening of the nucleosome cores leads to increasing elongation of the chromatin molecules.     

Interactions between core histones and chromatin at physiological ionic strength

Addition of core histones to chromatin or chromatin core particles at physiological ionic strength results in soluble nucleohistone complexes when polyglutamic acid is included in the sample. The interaction between nucleosomes and added core histones is strong enough to inhibit nucleosome formation on a closed circular DNA in the same solution. Complexes consisting of core particles and core histones run as discrete nucleoprotein particles on polyacrylamide gels. Consistent with the electrophoretic properties of these particles, protein cross-linking with dimethyl suberimidate indicates that added core histones are bound as excess octamers. Histones in the excess octamers do not exchange with nucleosomal core histones at an ionic strength of 0.1 M and can be selectively removed from core particles by incubating the complexes in a solution containing sufficient DNA. Under conditions where added histones are confined to the surface of chromatin, the excess histones are mobile and can migrate onto a contiguous extension of naked DNA and form nucleosomes.     

Effects of ionic strength on endogenous nuclease activity in chelated and nonchelated chromatin

Calf thymus chromatin, isolated using a standard (low ionic strength, but nonchelating) isolation protocol, dialyzed against either Tris-PMSF or Tris-EDTA, was reconstituted in a high salt compacting buffer (COM) or a low salt dispersing buffer (DIS) prior to digestion with endogenous nucleases. A greater level of enzyme activity occurred when chromatin was in a condensed state (COM buffer) and not chelated prior to digestion. In contrast, chromatin chelated by dialysis against Tris-EDTA prior to digestion showed higher levels of enzyme activity in the dispersed state (DIS buffer). Nonchelated undigested chromatin contained 0.280 +/- 0.16 ug copper/mg DNA and and 0.305 +/+- 0.09 ug zinc/mg DNA. Chelation removed about 78% of copper per mg DNA and approximately 65% of zinc per mg DNA. In COM buffer after a 20 min digestion, the solubilized fraction was enriched in copper showing about 20 X more metal per mg DNA than nonchelated chromatin. Approximately the same amount of zinc was found in both chelated and nonchelated chromatin while there was less zinc in chelated chromatin solubilized in DIS buffer. Thus, chelation has important effects on the digestibility of chromatin and on the type of ionic environment that provides the most favorable conditions for endogenous nuclease activity.     

Absorption and circular dichroic spectral studies on the self-association of daunorubicin

The self-association of daunorubicin in aqueous solution has been examined using visible absorption, fluorescence, and CD measurements. Spectral changes in the concentration range 10^?6 to 1.5 × 10^?3M have been interpreted in terms of a monomer–dimer equilibrium for daunorubicin. The data have been analyzed using a nonlinear curve-fitting technique. The results obtained in this study differ markedly from previously published results, and possible reasons for this difference are discussed. The effect of solvent, temperature, and ionic strength on the dimerization process is investigated.     

Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength

When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer [1]. At this ionic concentration, chromatin maintains its native structure [2]. In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM. The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin. These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins. The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.     

pH-induced conformational changes in chromatin subunits measured by circular dichroism and immunochemical reactivity

Changes in the conformational state of chromatin core particles from chicken erythrocytes were studied by both immunochemical and biophysical methods as a function of pH and ionic strength. When the pH of core particles in a solution of ionic strength 3, 60 or 220 mM was lowered from pH 7.5, a sharp transition in the circular dichroism spectrum of DNA monitored between 320 and 260 nm was observed at pH 6.65. This change in DNA ellipticity was totally reversible. Binding to core particles of antibodies specific for histones H2B, H2A, H3 and for the IRGERA (synthetic C-terminal) peptide of H3 was used to follow changes in histone antigenicity. Binding was studied in the pH range 7.5-5.35, and at ionic strength of 60 and 220 mM. A change in reactivity of some histone epitopes was observed around pH 6.2–6.5. However, the changes observed by circular dichroism and antibody binding pertain to different components of chromatin subunits and they probably reflect independent phenomena. The alteration in accessibility of these determinants at the surface of core particles was completely reversible and was dependent on ionic strength. The conformation changes in core particles occurring near physiological ionic strength and pH may reflect dynamic changes in chromatin structure that possess functional significance.