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1.
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.  相似文献   

2.
Condensation of chromatin: role of multivalent cations   总被引:4,自引:0,他引:4  
D Sen  D M Crothers 《Biochemistry》1986,25(7):1495-1503
We have used electric dichroism to investigate the influence of multivalent cations upon the compaction of chicken erythrocyte chromatin from the unfolded, 10-nm fiber to the 30-nm solenoid and subsequent aggregation. The pattern of condensation, which consists of compaction plus aggregation, is found to be strikingly similar for a variety of cations of differing charge, including the physiologically important polyamines spermine and spermidine. With a few exceptions such as Cu2+ and Gd3+, an optimally compacted fiber with reproducible hydrodynamic properties is produced prior to the onset of aggregation. We report the concentrations of di-, tri-, and tetravalent cations required for optimal condensation; in addition, for tri- and tetravalent cations, we were able to estimate the extent of charge neutralization produced by their binding to the optimally compacted fiber. The results show that the multivalent ion concentration required for optimal compaction decreases as cationic charge increases. In addition, the effect of a mixture of dilute mono- and multivalent cations on chromatin condensation is synergistic, rather than competitive as has been found for the multivalent cation induced condensation of DNA or the B----Z conformational transition. A simple calculation indicates that the entropy of ion uptake in chromatin condensation is surprisingly constant for a range of ionic conditions; this factor may be a dominant one in determining the folding equilibrium.  相似文献   

3.
Condensation and precipitation of chromatin by multivalent cations   总被引:2,自引:0,他引:2  
The condensation and the precipitation of rat liver chromatin upon addition of spermine4+, spermidine3+, hexamminecobalt(III)3+ and Mg2+ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg2+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg2+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin induced by spermine, triamines NH3+(CH2)iNH+(CH2)jNH3+, designated as much less than i, j much greater than: much less than 3, 4 much greater than (spermidine), much less than 3, 3 much greater than, much less than 2, 3 much greater than, much less than 2, 2 much greater than; the diamines putrescine and cadaverine and MgCl2. It is found that the different polyamines affected DNA and chromatin in a similar way. The degree of compaction of the chromatin fibers induced by spermine, triamines except much less than 2, 2 much greater than and Mg2+ has been found to be identical. The triamine much less than 2, 2 much greater than and the diamines studied do not condense either chromatin of DNA. Such a big difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations are important for their interactions with DNA and chromatin. The stoichiometry of polyamine binding to chromatin at which condensation occurred is found to be 2 polyamine molecules per DNA helical turn. Polyamines are supposed to bind to the exposed sites of core DNA every 10 b.p. The extent of DNA phosphate neutralization by the histones is estimated to be about 55%. It has been shown that a mixture of mono- and multivalent cations affected DNA and chromatin condensation competitively and not synergistically, as claimed in a recent report by Sen and Crothers.  相似文献   

7.
Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.  相似文献   

8.
Polyamine-DNA interactions. Condensation of chromatin and naked DNA   总被引:2,自引:0,他引:2  
We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.  相似文献   

9.
The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Friend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations by the use of flow linear dichroism and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA--2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA--for chromatins with linker DNA 10-30 b.p. it is partially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric fields do not have an effect on chromatin structure, while higher electric fields (more than 7 kV/cm) distort the structure of chromatin.  相似文献   

10.
Identical samples containing polynucleosomal chains of chicken erythrocyte (CE) and Ehrlich ascites tumour (EA) chromatin were studied under various ionic conditions with regard to electric linear dichroism (ELD) and flow linear dichroism (FLD). Both orientation techniques consistently confirmed that, in the limit of very low ionic strength and in the absence of multivalent cations, the reduced linear dichroism of chromatin is negative in the DNA-base absorption band, as expected for an extended zig-zag polynucleosomal conformation. With increasing electrolyte content, both ELD and FLD decreased drastically in amplitude, but in contrast to the ELD which remains negative in an intermediate range of low ionic strength (0.1-0.5 mM Mg2+) the FLD changes sign and becomes positive. The ELD and FLD amplitudes decrease with higher Mg2+ concentrations and FLD even vanishes in the region of 0.2-0.4 mM; both signals are positive above 0.4-0.5 mM Mg2+. The origin of the dissimilarities between ELD and FLD observations is still not fully understood. Several possibilities are considered: ELD signals are more influenced than FLD by the presence of short chromatin chains, nucleosomes and small pieces of naked DNA, while FLD is more susceptible to the presence of large, easily orientable, scattering aggregates. Different preferred orientation directions of the chromatin fibre with respect to electric and hydrodynamic fields may also be involved. Finally, FLD and ELD probably "see" different features of the chromatin structure.  相似文献   

11.
The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.  相似文献   

12.
Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.  相似文献   

13.
We have measured forces generated by multivalent cation-induced DNA condensation using single-molecule magnetic tweezers. In the presence of cobalt hexammine, spermidine, or spermine, stretched DNA exhibits an abrupt configurational change from extended to condensed. This occurs at a well-defined condensation force that is nearly equal to the condensation free energy per unit length. The multivalent cation concentration dependence for this condensation force gives the apparent number of multivalent cations that bind DNA upon condensation. The measurements show that the lower critical concentration for cobalt hexammine as compared to spermidine is due to a difference in ion binding, not a difference in the electrostatic energy of the condensed state as previously thought. We also show that the resolubilization of condensed DNA can be described using a traditional Manning–Oosawa cation adsorption model, provided that cation–anion pairing at high electrolyte concentrations is taken into account. Neither overcharging nor significant alterations in the condensed state are required to describe the resolubilization of condensed DNA. The same model also describes the spermidine3+/Na+ phase diagram measured previously.  相似文献   

14.
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.  相似文献   

15.
Electric birefringence of DNA and chromatin. Influence of divalent cations.   总被引:5,自引:0,他引:5  
The effects of divalent cations on the DNA and chromatin conformation have been investigated by electric birefringence and birefringence relaxation measurements at low and constant ionic strength (0.001). An important decrease of the intrinsic optical anisotropy of DNA has been found in the presence of Mn2+ and Cu2+, but not with Mg2+. A complex variation of the mean relaxation time with the ratio I/P of ion to DNA-phosphate molar concentration has been evidenced in the presence of Mn2+ and Cu2+, while the mean relaxation time monotonously decreased in the presence of Mg2+. These observations are interpreted in terms of a specific organization of DNA in a compact, rigid structure, in the presence of Mn2+ and Cu2+, and a non-specific coiling in the presence of Mg2+. Drastic conformational changes encountered by chromatin in the presence of Mg2+ and Mn2+ cations have also been evidenced through electric birefringence measurements. They are interpreted by the formation of a superhelical compact arrangement of nucleosome strings which yielded a reversal of the birefringence sign with respect to the negative anisotropy observed in the presence of Na+ ions. The removal of the histone H1 prevented the appearance of this quaternary structure. More extended fragments of the chromatin chain obtained by ECTHAM chromatography of sonicated chromatin could not afford such compact arrangements.  相似文献   

16.
Abstract

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentraitons by the use of flow linear dichroism (LD) and electric dichroism.

We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA—2 mM NaCl and close positive values in the range of 2–100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5–10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers.

The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA—for chromatins with the linker DNA of 10–30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible.

Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin.

Presented resutls explain the contradictory data obtained by electrooptical and hydrooptical methods.  相似文献   

17.
The condensation of chromatin and histone H1-depleted chromatin by spermine   总被引:2,自引:0,他引:2  
At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.  相似文献   

18.
Measurements of binding of certain divalent cations to yeast apoenolase were made using a pH-meter, chromatography, a divalent cation electrode, and ultrafiltration. The binding of the activating metal ions Mg2+ and Co2+ and the nonactivator Ca2+ were studied as functions of the presence or absence of substrate/product, phosphate, and fluoride or level of Tb3+. The data suggest phosphate and fluoride increase Mg2+ binding but not Ca2+ binding. Substrate/product appears to increase Ca2+ binding as well as that of Mg2+ and Co2+. In the presence of substrate, Co2+ binding was 5-6 mol/mol dimer. In the absence of substrate/product, Tb3+ reduced Co2+ binding from 4 mol/mol to 2. These data are interpreted in terms of binding to "conformational," "catalytic" (substrate/product dependent), and "inhibitory" sites. Measurements of Tb3+ fluorescence quenching by Co2+ suggested that the distance between "conformational" sites on the two subunits was large, while the distance between "conformational" and "inhibitory" sites was ca. 17 +/- 4 A. Potentiometric titrations of apoenzyme with Ca2+ and Mg2+ showed that the metal ions produced the same proton release in the presence or absence of substrate/product. If phosphate and fluoride were present, then more protons were released if Ca2+ was the titrant rather than Mg2+, suggesting a difference in ionization state in the complex with the activating metal. Electron paramagnetic resonance studies of Co2+ binding to the various sites in the enzyme are presented. The Co2+ bound to all three sites appears to be high spin, consistent with a preponderance of oxyligands in an octahedral environment. Substrate, citrate, and a strongly binding substrate analogue strongly enhance the hyperfine structure of conformational Co2+. This is interpreted as the result of a change in interaction of an axial ligand to conformational Co2+ produced by carbon-3 of substrate or analogue.  相似文献   

19.
P Colson  C Houssier 《FEBS letters》1989,257(1):141-144
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.  相似文献   

20.
Multivalent cations were tested for their ability to replace the Ca2+ requirements of aggregation factor (AF) complex in activity, stability, and integrity assays. The ability of each cation to replace the Ca2+ required for the cell aggregation-enhancing activity of AF was examined by replacing the usual 10 mM Ca2+ with the test cation at various concentrations in the serial dilution assay of the AF. The other alkaline earth cations, Mg2+, Sr2+, and Ba2+, could not replace Ca2+; two transition elements, Mn2+ and Cd2+, partially replaced calcium. All 15 of the available lanthanides (including La3+ and Y3+) produced normal activity but only at 10-400-fold lower cation concentrations than Ca2+. An AF preparation is stable and remains active for months in 1 mM Ca2+ but decays rapidly when Ca2+ is lowered. Sr2+ and Ba2+ at 20 mM but not at 1 mM could replace 1 mM Ca2+ and give long term stability. AF was not stable in the presence of Mg2+, even at 100 mM. High Mn2+ concentrations did not stabilize AF even though AF was partially active in Mn2+. Cd2+ gave full stability at 75 mM and La3+ at about 0.1 mM. When Ca2+ is chelated, the macromolecular subunits of the AF slowly dissociate. Permeation chromatography and analytical ultracentrifugation showed that the cations that stabilized activity maintained the integrity of AF complex while those that failed to stabilize activity allowed the complex to dissociate into subunits, indicating that these two Ca2+ requirements are related. The cation specificities for activity and for stability-integrity are different indicating that these are separate Ca2+-dependent functions.  相似文献   

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