Condensation of the chromatin gel by cations

Calf thymus chromatin gel, containing strongly bound nonhistone proteins, was used to study the effect of easily removable and tightly bound cations on the condensation of chromatin. The chromatin volume was found to be linearly dependent on the reciprocal square root of the concentration of easily removable cations (Tris X H+ + Na+ and Mg2+) except for the initial stages of condensation (up to 7-10 mM monovalent and 0.15-0.2 mM divalent cations). The effect of Mg2+ at the initial stage of condensation was not reproduced by Na+ and vice versa. At higher concentrations the effects of Na+ and Mg2+ were additive. The removal of tightly bound divalent cations by a treatment of the chromatin gel with 1,10-phenanthroline led to an approx. 50% increase in the volume of the chromatin gel, which was maintained at each concentration of easily removable cations. The 1,10-phenanthroline-caused decondensation of the chromatin gel was reversed by Ca2+ but not by Mg2+, Zn2+ and Cu2+. The chromatin gel pretreated with Ca2+ was not further decondensed by 1,10-phenanthroline.     

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Condensed chromatin in diploid and allopolyploid microseris species with different genome size: a quantitative electron microscopic study

Summary The species-specific proportion of chromatin in the condensed state was estimated by quantitative electron microscopic morphometry of nuclear sections in 9 diploid and 5 allopolyploid species of Microseris (Asteraceae). A positive correlation between the genome size (haploid DNA content, or C value) and the percentage of chromatin in the condensed state (as visible in ultrathin sections) was found in diploids (r=0.89). Nuclei of allopolyploid (tetraploid) species exhibit condensed chromatin in a percentage which corresponds to the average of the values found in the parents. This suggests that each parental genome controls chromatin condensation at interphase independently within the nucleus, and that the degree of condensation is not directly determined by the nuclear DNA content per se. Genome size differences among Microseris species may depend preferentially, but not entirely, on DNA fractions located in, and perhaps being the cause of, condensed chromatin.Dedicated to Professor F. Mechelke in honour of his 60^th birthday.     

Evidence that both kinetic and thermodynamic factors govern DNA toroid dimensions: effects of magnesium(II) on DNA condensation by hexammine cobalt(III)

Millimolar concentrations of divalent cations are shown to affect the size of toroids formed when DNA is condensed by multivalent cations. The origins of this effect were explored by varying the order in which MgCl(2) was added to a series of DNA condensation reactions with hexammine cobalt chloride. The interplay between Mg(II), temperature, and absolute cation concentration on DNA condensation was also investigated. These studies reveal that DNA condensation is extremely sensitive to whether Mg(II) is associated with DNA prior to condensation or Mg(II) is added concurrently with hexammine cobalt(III) at the time of condensation. It was also found that, in the presence of Mg(II), temperature and dilution can have opposite effects on the degree of DNA condensation. A systematic comparison of DNA condensates observed in this study clearly illustrates that, under our low-salt conditions, toroid size is determined by the kinetics of toroid nucleation and growth. However, when Mg(II) is present during condensation, toroid size can also be limited by a thermodynamic parameter (e.g., undercharging). The path dependence of DNA condensation presented here illustrates that regardless of which particular factors limit toroid growth, toroids formed under the various conditions of this study are largely nonequilibrium structures.     

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility

The effects of sodium and magnesium-ion interactions on chromatin structure and solubility were examined in isolated mouse liver nuclei. To facilitate this study, a simple assay of chromatin structure was developed, based on the absorbances at 260 nm (A260) and 320 nm (A320) of nuclei in test solutions. By subtracting the A320 from the A260, a single "spectral index" was obtained which served as a useful, but not absolute, indicator of chromatin structure. Electron microscopy verified the validity of this approach. The results indicate that either 200 mM NaCl or 0.5 mM MgCl2 were capable of preserving the native 20 to 30 nm chromatin fiber structure. Below 200 mM NaCl, the native fiber progressively uncoiled to the 10 nm unit fiber. The presence of 0.5 mM MgCl2 inhibited this uncoiling. Only divalent cations stabilized condensed chromatin (heterochromatin) within the nucleus. Monovalent and divalent cations interacted with one another at critical concentrations and modified their individual effects on chromatin structure; e.g., 10 to 25 mM NaCl interfered with the action of 0.5 to 1.5 mM MgCl2, causing a complete loss of condensed chromatin. Maximum solubility of micrococcal nuclease-digested chromatin occurred at 10 mM NaCl, which treatment allowed the chromatin to unfold to the 10 nm fiber. However, ionic conditions that disrupted condensed chromatin but maintained the native chromatin fiber morphology still resulted in relatively high yields of soluble chromatin. Minimum solubility occurred under conditions which preserved the structure of condensed chromatin.     

The superstructure of chromatin and its condensation mechanism

Synchroton radiation X-ray scattering experiments have been performed on chicken erythrocyte chromatin fibres over a wide range of ionic conditions and on various states of the fibres (i.e. "native" in solution, in gels and in whole nuclei; chromatin depleted of the H1 (H5) histones and chromatin with bound ethidium bromide). A correlation between the results obtained with the various chromatin preparations provides evidence for a model according to which at low ionic strength the chromatin fibre already possesses a helical superstructure, with a diameter comparable to that of condensed chromatin, held together by the H1(H5) histone. The most significant structural modification undergone upon an increase of the ionic strength is a reduction of the helix pitch, this leads to condensation in a manner similar to the folding of an accordion. The details of this process depend on whether monovalent or divalent cations are used to raise the ionic strength, the latter producing a much higher degree of condensation. Measurements of the relative increase of the mass per unit length indicate that the most condensed state is a helical structure with a pitch around 3.0-4.0 nm. In this paper we give a detailed presentation of the experimental evidence obtained from static and time-resolved scattering experiments, which led to this model.     

Counterion-induced condesation of deoxyribonucleic acid. a light-scattering study.

The addition of the trivalent or tetravalent cations spermidine or spermine to a solution of T7 DNA in aqueous solution causes an alteration of the DNA from its extended coil form to a condensed form. If performed at low DNA concentration and at low ionic strengths, this transformation results in a monomolecular collapse to form a particle with a hydrodynamic radius of about 500 A. We have monitored this change using quasielastic and total intensity light scattering. In a solution of 50% methanol in water, the divalent cations Mg2+ and putrescine also can cause the condensation of DNA. Using Manning's (1978) counterion condensation theory, we calculate a striking unity among these disparate ions: the collapse occurs in each case when from 89 to 90% of the DNA phosphate charges are neutralized by condensed counterions.     

多价阳离子诱导的DNA缩合研究进展

DNA分子在多价阳离子作用下,可以缩合成紧密有序的纳米级缩合体。常用的多价阳离子包括阳离子聚合物,蛋白质,高价无机离子等。缩合主要是由于67～90%的DNA磷酸基负电荷被中和而引起的,缩合的过程伴随着微粒的聚集过程;常见的缩合体是圆环体状,其尺寸一般在50～300nm之间;缩合主要受缩合剂正电荷的影响,缩合剂的结构也对缩合过程有一定的影响;DNA分子的大小和碱基成分也是影响缩合的因素,同时也决定缩合体的体积大小。     

Real-time observation of nucleoplasmin-mediated DNA decondensation and condensation reveals its specific functions as a chaperone

Fertilization requires decondensation of promatine-condensed sperm chromatin, a dynamic process serving as an attractive system for the study of chromatin reprogramming. Nucleoplasmin is a key factor in regulating nucleosome assembly as a chaperone during fertilization process. However, knowledge on nucleoplasmin in chromatin formation remains elusive. Herein, magnetic tweezers (MT) and a chromatin assembly system were used to study the nucleoplasmin-mediated DNA decondensation/condensation at the single-molecular level in vitro. We found that protamine induces DNA condensation in a stepwise manner. Once DNA was condensed, nucleoplasmin, polyglutamic acid, and RNA could remove protamine from the DNA at different rates. The affinity binding of the different polyanions with protamine suggests chaperone-mediated chromatin decondensation activity occurs through protein–protein interactions. After decondensation, both RNA and polyglutamic acid prevented the transfer of histones onto the naked DNA. In contrast, nucleoplasmin is able to assist the histone transfer process, even though it carries the same negative charge as RNA and polyglutamic acid. These observations imply that the chaperone effects of nucleoplasmin during the decondensation/condensation process may be driven by specific spatial configuration of its acidic pentamer structure, rather than by electrostatic interaction. Our findings offer a novel molecular understanding of nucleoplasmin in sperm chromatin decondensation and subsequent developmental chromatin reprogramming at individual molecular level.     

Comparative study of the condensation of chicken erythrocyte and calf thymus chromatins by di- and multivalent cations

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers

To assess the influence of chromatin structure on the frequency of radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to nuclear and nucleoid monolayers. These chromatin substrates were prepared by treating human fibroblasts grown as monolayers with the nonionic detergent Triton X-100 and varying concentrations of cations. The chromatin structure was modified either by a stepwise removal of DNA-bound proteins by extraction in increasing concentrations of monovalent salt, or by the addition or deletion of mono- and divalent cations to condense or decondense the chromatin, respectively. It was found that the stepwise removal of DNA-bound proteins from the chromatin dramatically increased the frequency of radiation-induced DNA strand breaks. The DNA-bound proteins showed a qualitative difference in their ability to protect the DNA where proteins removed by salt concentrations above 1.0 M exerted the greatest protection. Furthermore, the frequency of radiation-induced DNA strand breaks was found to be 6 times lower in condensed chromatin than in decondensed chromatin and about 80 times lower than in protein-depleted chromatin. It is concluded that the presence of DNA-bound proteins and the folding of the chromatin into higher-order structures protect the DNA against radiation-induced strand breaks.     

Effects of various ionic media on extraction of soluble nuclear proteins and on nuclear ultrastructure

Isolated liver nuclei were extracted 3 times at pH 7.2 with solutions containing either (1) monovalent cations, (2) both mono- and divalent cations, or (3) sucrose solutions containing only divalent cations. The extracted proteins were analysed by two-dimensional acrylamide gel electrophoresis and the ultrastructural alterations of the treated nuclei were examined by electron microscopy. The solutions containing Na⁺ or K⁺ monovalent and Ca²⁺ and Mg²⁺ divalent ions extracted the same amount (18–22 %) of the nuclear proteins. The two-dimensional gel electrophoretic patterns of these extracts were nearly identical and the structures of the nuclear components were well preserved even after 3 times repeated extractions. The solution containing only Na⁺ extracted less protein (14–15 %) than the solutions containing both mono- and divalent cations. Extraction with isotonic NaCl solution altered the nuclear and nucleolar morphology; unlike the other solutions employed, this solution extracted some DNA and histones. The isotonic sucrose solution containing only divalent cations extracted less protein than the other solutions (9–11 %) and produced marked condensation of the chromatin. These analytical and electron microscopic studies showed that mono- and divalent cations play a role in structural organization of chromatin.     

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved.

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.     

B-X transition in synthetic and natural (dA-dT)n.(dA-dT)n sequences

AB-X transition of polyh(dA-dT).poly(dA-dT) was observed to occur in methanol-water mixtures with methanol concentrations higher than 50% in the presence of a specific combination of monovalent and divalent cations. In the presence of Na+, divalent cations induce denaturation of poly(dA-dT).poly(dA-dT) accompanied by condensation and/or aggregation, and effect similar to that observed previously with random sequence DNA (Votavová, Kucerová, Felsberg and Sponar, J. Biomol. Struct. Dyn. 4,477-489, 1986). In the presence of Cs+ cations a B-X transition was induced by addition of Ca2+ or Mn2+ but not Mg2+ or Ni2+ ions. Circular dichroism and ultraviolet spectroscopy demonstrate that the X conformation is a double stranded form of poly(dA-dT).poly(dA-dT) belonging presumably to the B family which, however has an altered base stacking. The X conformation of poly(dA-dT).poly(dA-dT) found in methanol-water mixtures is a condensed and/or aggregated form. In contrast, the X conformation characterized by similar CD spectra observed in high salt concentrations is not aggregated up to a concentration of 6 M CsF. In methanol-water mixtures (A+T)-rich bacterial DNA behaves essentially as a random sequence DNA revealing no detectable amount of the X form. On the other hand crab (Cancer pagurus) satellite and crab non-satellite DNAs containing varying amounts of (dA-dT)n.(dA-dT)n sequences were shown to undergo a B-X transition, at least partly, in both methanol-water mixtures and 6 M CsF solutions.     

Comparative Study of the Condensation of Chicken Erythrocyte and Calf Thymus Chromatins by Di- and Multivalent Cations

Abstract

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidityJulprecipitation and electric dichroism measurements. For all the cations investigated (Mg²⁺, Tb³⁺, Co(NH₃)₆ ³⁺, spermidine Spd²⁺ and spermine Sp⁴⁺) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg²⁺ and Tb³⁺. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a “precondensed” state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the “cross-linker” models. Tb³⁺ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that “30 nm fibers” were formed. Very little aggregation was produced by Tb³⁺. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

Organization in the cell nucleus: divalent cations modulate the distribution of condensed and diffuse chromatin

The organization of rat liver nuclei in vitro depends on the ionic milieu. Turbidity measurements of nuclear suspensions in the presence of varying concentrations of divalent cations have been correlated with nuclear ultrastructure. The concentration of MgCl2 (2 mM) at which turbidity of nuclear suspensions is maximal and chromatin condensation appears most extensive is the same concentration that reportedly (Gottesfeld et al., 1974, Proc. Natl. Acad. Sci. U. S. A. 71:2193-2197) precipitates "inactive" chromatin. Thus, a mechanism is suggested by which chromatin activity and ultrastructural organization within the nucleus may be mediated. The nuclear organizational changes attendant upon the decrease in divalent cation concentration were not entirely reversible.     

The superstructure of chromatin and its condensation mechanism

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and the polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb and Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chloride and alkylammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg^-- concentration at which precipitation occures on chromatin concentration and confirm the differences between cations observed by X-ray scattering.The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.