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1.
The metabolism of histone fractions. VI. Differences in the phosphorylation of histone fractions during the cell cycle 总被引:3,自引:0,他引:3
Phosphorylation of histone fractions in the presence and absence of DNA synthesis was measured using the new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G1-phase. Using preparative electrophoresis, histone f1 phosphorylation was found to be dependent upon cell-cycle position, being absent in G1-arrested and G1-traversing cells and active in the S-phase. The absence of f1 phosphorylation in G1-arrested cells, which are known to exhibit f1 turnover, indicates that f1 phosphorylation is not an obligatory part of the f1 turnover process. In contrast to histone f1, it was found that histone f2a2 phosphorylation is independent of cell-cycle position, occurring with equal magnitude in the G1-traversing state when DNA synthesis is essentially absent and in the S-phase when DNA synthesis is active. When cells were arrested in the G1-state by isoleucine deprivation, f2a2 phosphorylation continued to be active, occurring at 56% of the rate observed in the G1-traversing state. These results indicate that phosphorylation of histone f2a2 is independent of f2a2 synthesis, independent of DNA synthesis, and independent of histone f1 phosphorylation. Because f2a2 is actively phosphorylated in G1-arrested cells known to be active in the synthesis of various types of RNA (including messenger) as well as in G1-traversing and S-phase cells, we feel that phosphorylation of histone f2a2 should continue to be considered in models concerning activation of DNA template activity. 相似文献
2.
Hsueh Jei Li Ali W. Hu Rose A. Maciewicz Patricia Cohen Regina M. Santella Catherine Chang 《Nucleic acids research》1977,4(11):3839-3854
Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones. 相似文献
3.
R. J. Inglis T. A. Langan H. R. Matthews D. G. Hardie E. M. Bradbury 《Experimental cell research》1976,97(2):418-425
The previous observation that growth-associated histone kinase (HKG) from Ehrlich ascites cells brings forward mitosis in Physarum polycephalum has been confirmed with more step 1 histone kinase and a more purified (step 2) histone kinase and the statistical significance of the results assessed. The mitosis appears normal in the phase contrast microscope and DNA synthesis is initiated after mitosis as usual. In vitro the growth-associated histone kinase phosphorylates chromatin, the phosphate appearing in F 1 histone. The results are interpreted as providing support for the hypothesis that growth-associated histone kinase controls the initiation of mitosis through F 1 histone phosphorylation and chromosome condensation. 相似文献
4.
Robert C. Krueger 《Molecular biology reports》1986,11(3):189-192
Nucleosomal subunits isolated from rabbit thymus nuclei in 0.04 M K2SO4-0.02 M Tris, pH 7.4 were devoid of histone H1, while whole chromatin prepared in the same buffer contained the full complement of histone H1. The question is asked why histone H1 dissociates from the subunits but not from the high molecular weight material. We propose that, at physiological salt concentrations, histone H1 is not bound to linker DNA as depicted in the current models; rather, alternate attachment sites, present only in the polymer, are involved. 相似文献
5.
1. Experiments were carried out to determine the extent of dissociation of histone from deoxyribonucleohistone as a result of irradiation with γ-rays from 60Co. 2. The bulk of the nucleohistone was removed from the irradiated solutions either by sedimentation or by precipitation with dilute sodium chloride solution. The supernatants were then analysed for DNA and histone. 3. The ratios of histone to DNA in these supernatants were less than for the original nucleohistone. This indicated that histone was dissociated by the irradiation, and then aggregated either with itself or with other nucleohistone molecules, and so was removed with the bulk of the nucleohistone during sedimentation or precipitation. 相似文献
6.
The presence of highly basic proteins (histones or protamines), causes an increase in the rate of the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs2) with the tripeptide model glutathione. This effect is explained by considering that polycationic molecules, such as histones or protamines, can attract the negatively charged reacting molecules, thus producing a catalytic effect. This effect disappears at high ionic strength due to a shielding of the charges; Urea causes a shift to the K2(app)vs. pH curve for the histone H3-Nbs2 reaction. This shift (2.1 units of pH for 8 m urea) indicates that urea denatures, at least to some extent, the tertiary structure of the microenvironments containing cysteine of histone H3, but it is unable to eliminate an unspecific electrostatic effect (similar to that caused by polycations in the GSH-Nbs2 reaction), which also contributes to the increase of the reaction rate. Combined effects of urea and ionic strength on the reaction of GSH and of histone H3 with Nbs2 gives rise to shifts of both curves of K2(app)us. pH, approaching one to the other very closely. This is interpreted as due to the appearance of shielding effects on the electrostatic charges of the histone, and also of the small molecules. The greater efficiency of guanidine hydrochloride, compared to that of urea, in causing a shift of the rate constant curve of histone H3 is interpreted as due to a combined effect of denaturation and electrostatic shielding in the case of guanidine hydrochloride. 相似文献
7.
Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1 总被引:20,自引:0,他引:20
Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed. 相似文献
8.
Chinese hamster cells (line CHO) were labeled in suspension culture with 3H-lysine and 32PH3PO4. Preparative polyacrylamide gel electrophoresis of histone fractions from these cells was performed in the presence of 8 urea, 6 mM Triton X-100, and 0.9 N acetic acid. This method separates histones f2a2 and f2b by a Large distance, thus making it possible to resolve the controversy concerning which histone -- f2b or f2a2 -- is phosphorylated. It is shown that the two most highly phosphorylated histones in interphase CHO cells are f1 and f2a2. Histones f2b and f3 are shown to contain no significant incorporation of 32PO4 in interphase cells, while histone f2a1 contains a small but detectable amount of incorporated 32PO4. Binding of the nonionic detergent Triton X-100 to hydrophobic centers appears to be greatest for histones f2a2 and f3, thus significantly retarding the mobility of these two histones during electrophoresis. 相似文献
9.
Transport of histone intoEscherichia coli 总被引:1,自引:0,他引:1
Histone appears to be transported intoEscherichia coli cells in two ways: by an adsorption-like process and by a system which is temperature-dependent and is inhibited by 2,4-dinitrophenol.
The adsorbed component cannot be washed out with a salt solution but can be partly exchanged for labelled histone and can
be displaced with Tween 80. The actively transported histone can be replaced with unlabelled histone and with polylysine,
and released by a high concentration of Mg2+ or by Tween 80. The active transport is observed only at concentrations greater than 100 μg histone/ml; the adsorption-like
process occurring at lower concentrations. 相似文献
10.
A method distinguishing between the concentration effect due to the cell wall and that due to the protoplasm is described: the importance of this lies in the fact that if the protoplasm shows a concentration effect one or both ions of the salt must tend to enter its outer surface. Studies on the concentration effect of KCl with living protoplasm of Nitella show that when P.D. is plotted as ordinates and the logarithm of concentration as abscissæ the graph is not the straight line demanded in the ideal case by theory but has less slope and is somewhat concave to the axis of the abscissæ. With a variety of salts the dilute solution is positive, which indicates that the cation has a greater mobility in the protoplasm than the anion or that the partition coefficient of the cation (Ac) increases faster than that of the anion (Aa) as the concentration increases. If the result depended on the partition coefficients we should say that when Ac ÷ Aa increases with concentration the dilute solution is positive. When Ac ÷ Aa decreases as the concentration increases the dilute solution is negative. In either case the increase in concentration may be accompanied by an increase or by a decrease in the relative amount of salt taken up. Theoretically therefore there need be no relation between the sign of the dilute solution and the relative amount of salt taken up with increasing concentration. Hypothetical diagrams of the electrical conditions in the cell are given. If we define the chemical effect as the P.D. observed in leading off at two points with equivalent concentrations of different salts we may say that the chemical effect of the protoplasm is very much greater than that of the cell wall. 相似文献
11.
Using giant axons of squid, Doryteuthis, available in Hokkaido, Japan, it was shown that axons internally perfused with a dilute sodium salt solution undergo an abrupt transition from a resting to a depolarized state on addition of KCl to an external medium containing CaCl2. Under internal perfusion with a dilute solution of sodium or cesium salt, it was possible to induce abrupt transitions between the two (i.e., resting and depolarized) states of the membrane by changing the temperature. “Giant fluctuations” in the state of the axon membrane were demonstrated at and near the critical points of the axon membrane. These findings are interpreted as supporting the view that an abrupt change in the membrane potential and conductance is an electrochemical manifestation of a phase transition of the membrane macromolecules. 相似文献
12.
Interactions of calf-thymus histone fractions in aqueous solution with 8-anilinonaphthalene-1-sulphonic acid 总被引:4,自引:3,他引:1
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D J Laurence 《The Biochemical journal》1966,99(2):419-426
1. The interactions of histone fractions with 8-anilinonaphthalene-1-sulphonic acid were investigated by fluorimetry and spectrofluorimetry and the results were interpreted with the aid of equilibrium-dialysis techniques. 2. Characteristic differences were found between the various histone fractions, and with fractions F3 and F2a the binding was found to be salt-dependent. 3. Evidence was obtained indicating a slow change of the physical state of fractions F3 and F2a in the presence of salt, and the binding by these two fractions in the presence of salt was greater by an order of magnitude than by fractions F1 and F2b. 4. Conditions favouring binding were also those favouring histone aggregation; SO(4) (2-) ions activated binding at a lower concentration than Cl(-) ions; urea, guanidinium ions and high concentrations of I(-) ions were inhibitory to binding. 5. After histones had been kept in the presence of salt for a long time the reversal of interaction on decreasing the salt concentration was incomplete. 6. The inhibition of binding by fraction F2a in the presence of urea or fraction F2b depended on the time sequence of addition of the reagents. 7. Artificial nucleoproteins made by precipitating DNA with the histone fractions in neutral 0.14m-sodium chloride showed the same order of interaction as was found for the fractions in solution. 8. Comparison of the binding by fraction F2a with that by bovine plasma albumin showed that in both cases there were a large number of weakly binding sites but that fraction F2a lacked the small number of strongly binding sites found in albumin. No slow change of binding in the presence of salt was found for albumin. 9. Binding by fraction F2b increased the affinity of the protein for further molecules of the adsorbate. 10. The results are discussed in relation to the close relationship between binding and aggregation and the possible role of non-polar interactions as determined by the balance between polar and non-polar amino acids in the histone fractions. 相似文献
13.
The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped. 相似文献
14.
M Bttger S Scherneck C U von Mickwitz H Fenske R Lindigkeit 《Nucleic acids research》1979,6(11):3581-3597
Structural changes of reconstituted SV 40 minichromosomes have been studied in relation to the salt concentration and addition of histone H1 by sedimentation and electron microscopy. Sedimentation data are represented as functions of the NaCl concentration and the Debye-Hückel electrostatic screening radius 1/alpha. The latter representation which proved to provide more information revealed three structural states of the SV 40 reconstitutes which can be additionally characterized by electron microscopy as follows: Expanded or relaxed conformation including free DNA spacers between the nucleosomes at low salt concentration (approx. 0.001 M-0.05 M NaCl), increasing condensation at moderate salt concentration (approx. 0.05 M-0.3 M NaCl) and expansion of this condensed state above approx. 0.3 M NaCl. The condensation of the reconstitutes at moderate salt concentration does not require the presence of histone H1. H1 seems to stabilize the condensed state against electrostatic expansion. The condensation might be promoted by salt-dependent conformational changes of naked superhelical DNA as revealed by sedimentation measurements. 相似文献
15.
It is found that T4 phage DNA complexed with histone H1 assembled into a string-of-bead structure, when the complex is prepared by a gentle diluting procedure from a high salt solution (2 M NaCl) to a low salt solution (50 mM NaCl). We used fluorescence microscopy to perform the real-time observation on formation and motion of a string-of-bead structure. Spatial histone H1 distribution on the DNA-H1 complex is observed by immuno-fluorescence microscopy. 相似文献
16.
Qiu Y Tereshko V Kim Y Zhang R Collart F Yousef M Kossiakoff A Joachimiak A 《Proteins》2006,62(1):8-16
The structure of Aq_328, an uncharacterized protein from hyperthermophilic bacteria Aquifex aeolicus, has been determined to 1.9 A by using multi-wavelength anomalous diffraction (MAD) phasing. Although the amino acid sequence analysis shows that Aq_328 has no significant similarity to proteins with a known structure and function, the structure comparison by using the Dali server reveals that it: (1) assumes a histone-like fold, and (2) is similar to an ancestral nuclear histone protein (PDB code 1F1E) with z-score 8.1 and RMSD 3.6 A over 124 residues. A sedimentation equilibrium experiment indicates that Aq_328 is a monomer in solution, with an average sedimentation coefficient of 2.4 and an apparent molecular weight of about 20 kDa. The overall architecture of Aq_328 consists of two noncanonical histone domains in tandem repeat within a single chain, and is similar to eukaryotic heterodimer (H2A/H2B and H3/H4) and an archaeal histone heterodimer (HMfA/HMfB). The sequence comparisons between the two histone domains of Aq_328 and six eukaryotic/archaeal histones demonstrate that most of the conserved residues that underlie the Aq_328 architecture are used to build and stabilize the two cross-shaped antiparallel histone domains. The high percentage of salt bridges in the structure could be a factor in the protein's thermostability. The structural similarities to other histone-like proteins, molecular properties, and potential function of Aq_328 are discussed in this paper. 相似文献
17.
Fractionation of a hyaluronic acid preparation in a density gradient. Some properties of the hyaluronic acid 总被引:2,自引:2,他引:0
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1. Hyaluronic acid was isolated from ox synovial fluid by sedimentation equilibrium in a caesium chloride density gradient (Silpananta, Dunstone & Ogston, 1967). The product was almost free from chondroitin sulphate and from protein. 2. Its composition did not differ significantly from that of the carbohydrate part of the protein-containing material isolated by filtration. Its physicochemical properties and molecular configuration were similar, except for its viscosity, which showed markedly reduced concentration-dependence and shear-dependence. This suggests that the associated protein tends to form links between molecules of hyaluronic acid. 3. The accurate measurement of viscosity at very low velocity gradient, by use of the damping of oscillations in a Couette viscometer, is described. 4. A method is described for measuring, approximately, the thermodynamic non-ideality of a solute from the shape of its schlieren curve at sedimentation equilibrium in a density gradient. 5. A value for the partial specific volume of hyaluronic acid in dilute salt solution was calculated from its isopycnic density in a caesium chloride gradient. 相似文献
18.
A protein kinase (ATP: histone phosphotransferase) with high specificity for the phosphorylation of the very lysine-rich histone H1 has been partially purified and characterized from soybean hypocotyl. The enzyme has a molecular weight of about 48,500. Its activity and sedimentation behavior are refractory to cyclic nucleoside monophosphates. No significant amount of cyclic AMP or cyclic GMP binding activity could be detected in the crude or partially purified enzyme preparations. Km for ATP and histone H1 are 0.4 μM and 0.7 μM, respectively. The enzyme requires Mg2+ or Mn2+ for activity, while addition of 0.5 mM Ca2+, Zn2+ or Hg2+ results in 50% inhibition. Arginine-rich histones H3 and H4 are inhibitory to histone H1 phosphorylation; these histones affect the Vmax of the enzyme, but not the Km for histone H1. 相似文献
19.
20.
Dirk Stigter 《Cell biochemistry and biophysics》1987,11(1):139-158
This is a review of applications of the McMillan-Mayer-Hill virial theory and the ionic double-layer theory to dilute colloidal
solutions, in particular, solutions of DNA. Interactions of highly charged colloidal rods are developed in terms of the second
virial coefficients between two rods, and between one rod and one small co-ion. The relevant cluster integrals are evaluated
with interaction potentials based on the Poisson-Boltzmann equation. The treatment is extended to the intrachain repulsion
responsible for the statistical swelling of coiled DNA (excluded volume effect). The theory is compared with three sets of
experimental data: The salt distribution in Donnan membrane equilibria of DNA-salt solutions, sedimentation equilibria of
short DNA fragments at different ionic strengths, and the intrinsic viscosity of T7 DNA in NaCl solutions. In all cases the
theory agrees well with the experiments. The agreement is not convincing for the sedimentation equilibrium at low ionic strength,
because here the experimental DNA concentration is too high for the truncated dilute solution expansion of the DNA-salt repulsion. 相似文献