The effect of polyene antibiotics on fractions of DNP both bound and not bound to dog kidney nuclear membranes]

Effects of amphotericine B and nistatine on nuclear membrane-bound DNP (DNPm) and free DNP (DNPf) from dog kidney are studied. Intravenous injection of amphotericine B resulted in the increase of binding of DNP particles with nuclear membrane: the content of DNA in DNPm fraction was 50-fold increased. The injection of nistatine did not affect DNP binding with nuclear membrane. Amphotericine B alone increased the protein-DNA ratio and decreased the RNA/DNA ratio in DNPf fraction. Both amphotericine B and nistatine sharply increased the protein/DNA ratio and practically did not change the RNA/DNA ratio in DNPm fraction. Amphotericine B produced considerable changes in temperature denaturation of DNA in DNPo, while nistatine produced no effect. Both antibiotics considerably changed the composition of acid soluble proteins in DNPm and DNPf, non-histone proteins in DNPf, and also they caused the changed and quantitative redistribution of separate lipid components in DNPm lipids. Polyene antibiotics are suggested to effect on animal cell nuclear structures.     

Conformational effects of histones H1 on DNA structure. Comparative study between H1-1, H1(0), H5 and sperm holothuria phi 0

Interactions of mammalian histones, H1-1 and H1(0), phi 0 from holothuria sperm and H5 with poly(dA-dT), poly(dG-dC) and poly(dG-me5dC) were measured by a nitrocellulose filter binding assay and circular dichroism. All of the proteins bound to every one of the polymers, but differed in the extent of binding, which depended on the polynucleotide/protein ratios and ionic strength. The order of retention of all polymers was phi 0 greater than H1-1 greater than H1(0). The binding of H1(0) to poly(dG-me5dC) was remarkably sensitive to ionic strength. The proteins caused changes in the spectral features of the polynucleotides, but differed in the type and extent of the change. Complexes prepared with H1-1 and H1(0) with all polymers showed a strongly negative psi spectrum. Complexes of poly(dA-dT) and phi 0, at a protein/polynucleotide ratio of 0.4, displayed a distinctive spectrum, giving the appearance of a Z-like DNA spectrum, at low ionic strength. At higher ionic strength the complexes showed a psi spectrum. Complexes of poly(dG-me5dC) in the Z or B conformation with phi 0 showed spectral features characteristic of a mixture of a Z-like and a psi spectrum. In contrast, H5 reduced the Z-DNA spectral features in the presence of Mg, and produced an inversion of the B spectrum up to a polynucleotide/protein ratio of 0.24. These findings demonstrate the ability of different proteins to produce changes in the conformation of DNA. This may reflect the ability of chromatin to undergo differential condensation, depending on both the base composition of DNA and the type of H1 histone bound to it.     

The comparative analysis of interaction between acridine orange and DNA-containing systems]

The interaction between acridine orange (AO) and diluted and concentrated solutions of DNA, DNP systems and chromatin suspension at the physiologic ionic strength was investigated. The effect of AO on DNP systems was also investigated. It was shown that highest possible number of AO molecules bound to DNA made up 70% of the total number of nucleotides. The model of AO binding to DNA is proposed and used for calculation of constants of stronger and weaker AO-binding capacities equal to 6-10(6) M-1 and 1,7-10(5) M-1, respectively. The AO-DNA binding constants in DNP-complex are five as low. The primary number of binding sites in chromatin suspension made up 10% of the corresponding sites in DNA and increased as AO was adsorbed. AO induced the supercontraction of oriented DNP systems at the physiologic ionic strength and the appearance of the low-temperature melting hump.     

Studies on deoxyribonucleoprotein structure. Small-angle X-ray scattering in solutions of various ionic strengths.

The cross-sectional radius of gyration of the deoxyribonucleoprotein (DNP) threads was measured by small-angle X-ray scattering in a wide range of ionic strengths (from 0.0005 to 2 M NaCl). For DNP in a solution of low ionic strength, this value is 30 Å. The increase of ionic strength results in partial deproteinization of DNP, while the cross-sectional radius of gyration varies from 25 Å for DNP in 0.7 M NaCl to 10 Å for DNP in 2 M NaCl. It is suggested that gradual deproteinization by the increase of NaCl concentration causes conformational changes, which are associated with the alteration of the DNP superstructure. The data are interpreted on the basis of the superhelical model of DNA packing in DNP; however, the coexistence of superhelical and unfolded regions in the DNP structure is also a possibility.     

Nonhistone proteins HMG1 and HMG2 suppress the nucleosome assembly at physiological ionic strength

The effect of nonhistone protein HMG1 and HMG2 from pig thymus on the in vitro nucleosome assembly has been examined with plasmid pSV2-gpt DNA and pig thymus core histones in the presence of DNA topoisomerase I. In the absence of core histones, the direct binding of HMG proteins could induce negative superhelical turns in DNA at low ionic strength, but not at physiological ionic strength. The nucleosome formation in the higher histone-to-DNA ratios at physiological ionic strength was not facilitated by HMG proteins, in contrast to poly(L-glutamic acid). HMG proteins suppressed the nucleosome assembly in the moderate histone-to-DNA ratios, resulting in the reduction of fully supercoiled DNA topoisomers. The suppression by HMG proteins was not cancelled by poly(L-glutamic acid). These suggest that the highly acidic carboxy terminal of HMG proteins does not act as an assembly factor, and that the HMG proteins, on the contrary, suppress the nucleosome formation, probably by binding to DNA in a way to inhibit the assembly into core particles.     

Structure and optical activity of the DNA-aminoacridine complex

The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (～3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.     

Optimal conditions and specificity of interaction of a distinct class of nonhistone chromosomal proteins with DNA.

A subclass of nonhistone chromatin proteins with high DNA affinity has been isolated from rat liver. The interaction of the isolated proteins with DNA in vitro was characterized utilizing a nitrocellulose filter binding technique. The temperature, time, concentration, ionic strength, and pH dependence were characterized. Optimal interaction was observed at 0.19 M naCl, pH 7.5 with a protein to DNA ratio of 13 (w/w). Equilibrium and kinetic competition experiments indicated that these proteins interact optimally with A-T rich and single-stranded DNA. The data also suggest that these proteins might affect the helixcoil transiton of DNA.     

Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.     

Effect of UV-light on the structure of soluble deoxyribonucleoprotein-200 A]

The effects of UV-light (253,7 nm) on the structure of DNP and its protein and nucleic components were studied. The formation of protein-DNA covalent bonds in DNP-200 A at low ionic strength was confirmed. Under certain irradiation conditions more than 80% of protein may be linked to the DNA; all histone fractions were linked to the same extent and at the same rates. The local denaturation of DNA in the region of photo-induced thymine-thymine dimers and other photoadducts dramatically changed the rate and specificity of the effects of staphylococcal nuclease, which directly affected the composition and size of the fragments formed. A possible application of UV-irradiated DNP for various structural investigations is discussed.     

Neutron scattering studies of nucleosome structure at low ionic strength

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Electrophoresis of adsorbed DNA

The electrophoresis mobilities of native calf thymus DNA adsorbed on the charged solid particles were measured by a micro-electrophoretic method as functions of pII, ionic strength, and DNA concentration. The mobility data confirm the adsorption of DNA both on the positively charged alumina and negatively charged resin particles at wide range of pH and ionic strength. The mobility data also indicate significant DNA adsorption by negatively charged glass in the acidic range of pH. The electrophoretic mobilities of DNA adsorbed on different substrate particles under identical conditions do not differ widely, indicating the major role of the adsorbed DNA rather than the covered substrate in controlling the charge behavior of the particle. The mobilities of the adsorbed DNA at salt pH are of a comparable order of magnitude to those for the dissolved DNA in solution. The mobility of the adsorbed heat-denatured and alkali-denatured DNA is lower than that of the native adsorbed DNA under identical conditions of pH and ionic strength.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

The conformation of the chromatin core particle is ionic strength-dependent.

We have examined the susceptibilities of the histones within the HeLa chromatin core particle to covalent modification by a diol-epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon benzo(a)-pyrene. Core-particle histones exhibit substantial variation in their relative susceptibilities to modification, depending upon the ionic strength of the environment. In contrast, the relative susceptibilities of either purified histones or histones in urea-denatured core particles are insensitive to changes in ionic strength. The variations in the pattern of modification of core particle histones occur primarily at ionic strengths at which the histones remain associated with core-particle DNA (0 to 0.6 M NaCl). Non-histone proteins influence the ionic strength-dependent variations in histone modification. The results imply that the ionic strength of the environment affects the conformation of the core particle and that the nucleoprotein has a flexible structure.     

Physico-chemical and functional characteristics of a subfraction of low-density lipoproteins isolated by ion-exchange column chromatography

Fractionation of human blood plasma low density lipoproteins (LDL) was performed by ion-exchange chromatography, using a linear NaCl gradient. It was shown that the binding of LDL subfractions eluted with a low ionic strength buffer (i.e., containing the particles with a lower negative charge) to B, E-receptors of fibroblasts was more effective than that of subfractions eluted with a high ionic strength buffer (i.e., containing the particles with a higher negative charge). The LDL particles with a lower negative charge had lower values of flotation coefficients (according to analytical ultracentrifugation data), smaller dimensions (according to gradient gel electrophoresis data) and a lower phospholipid/protein ratio (w/w). The experimental results suggest that LDL subfractions having different electrical parameters of the particle surface also differ in other physicochemical properties and seem to play a different role in atherogenesis.     

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.     

Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities.

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of polyelectrolyte theory to the study of the B-Z transition in DNA (1)

We have used the polyelectrolyte theory to study the ionic strength dependence of the B-Z equilibrium in DNA. A DNA molecule is molded as an infinitely long continuously charged cylinder of radius a with reduced linear charge density q. The parameters a and q for the B and Z forms were taken from X-ray data: aB = 1nm, qB = 4.2, aZ = 0.9 nm and qZ = 3.9. A simple theory shows that at low ionic strengths (when Debye screening length rD much greater than a) the electrostatic free energy difference FelBZ = FelZ - FelB increases with increasing ionic strength since qB greater than qZ. At high ionic strengths (when rD much less than a) the FelBZ would go on growing with increasing ionic strength if the inequality qB/aB greater than qZ/aZ were valid. In the converse case when qZ/qB greater than aZ/aB the FelBZ value decreases with increasing salt concentration at high ionic strength. Since X-ray data correspond to the latter case, theory predicts that the FelBZ value reaches a maximum at an intermediate ionic strength of about 0.1 M (where rD approximately a). We also performed rigorous calculations based on the Poisson-Boltzmann equation. These calculations have confirmed the above criterion of nonmonotonous behaviour of the FelBZ value as a function of ionic strength. Different theoretical predictions for the B-Z transition in linear and superhelical molecules are discussed. Theory predicts specifically that at a very low ionic strength the Z form may prove to be more stable than the B form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.