Selective removal of histone H1 from nucleosomes at low ionic strength

The method for removal of histone H 1 from chromatin by treatment with ion-exchange resin AG 50 WX 2 in the presence of 100 mM NaCl and 50 mM phosphate buffer (Thoma and Koller, 1977, Cell, 12, 101–107) results in production not only of H1-depleted chromatin but also free DNA. We have now modified this procedure so that the nucleosome is treated with the cation exchange resin in two steps, first in 50 mM sodium phosphate buffer and then in 50 mM sodium phosphate and 50 mM NaCl whereby histone H 1 is selectively removed without a release of free DNA at low resin concentrations.Abbreviations NaP Sodium phosphate buffer of molarities and pH as stated in the text - SDS Sodium dodecyl sulfate     

Precollapse of T7 DNA by spermidine at low ionic strength: A linear dichroism and intrinsic viscosity study

At low salt ([Na⁺] = 10^?3M), spermidine is capable of transforming DNA from a highly extended random coil to a compact particle. The transition takes place at a spermidine concentration of around 25 μM and the compact particle has been previously studied in considerable detail for several different DNAs. The objective of the present study is to see what effect, if any, spermidine has on T7 DNA conformation prior to collapse using flow dichroism and intrinsic viscosity. We conclude that increasing the spermidine concentration from 0 to the collapse transition point (above 20 μM) makes DNA increasingly nondraining. Furthermore, the persistence length dropped from 785 (±42) to 560 (±32) to 445 (±26) Å on increasing the ambient spermidine concentration from 0 to 1 to 10 μM. These results are in good agreement with counterion condensation theory and Odijk's theory of the electrostatic contribution to the persistence length of DNA. Nonetheless, it is concluded that counterion condensation is not entirely responsible for DNA collapse and that crosslinking promotes the transition to the compact state.     

Specific histone-histone contacts are ruptured when nucleosomes unfold at low ionic strength.

The ordered unfolding of the nucleosome core within chromatin at low ionic strengths has been studied. The results show that, when nuclei are lysed gently in solutions of very low ionic strength, their constituent nucleosomes rupture at a major H2B-H4 binding site but remain unperturbed at the site of the H2A-H2B interaction. These conclusions are based on data which show that at least four separate but closely spaced H2B-H4 contacts, identifiable by contact-site cross-linking in intact nuclei, are broken when nuclei are suspended in very dilute buffers. Appropriate controls on purified nucleosomes monomers demonstrate that the H2B-H4 contacts being broken are indeed intranucleosomal. Sedimentation of nucleosomes in the ultracentrifuge at various salt concentrations reveals that a significant conformational transition occurs in the range of ionic strength over which the H2B-H4 binding site ruptures.     

Optico-hydrodynamic properties of high molecular weight DNA from steady-state flow birefringence and viscosity at extremely low velocity gradients

The flow birefringence, extinction angles, and shear-dependent viscosity over a velocity gradient range of approximately 0.1–3 sec^?1 have been obtained for T2 bacteriophage DNA at low concentration in neutral aqueous buffer. The data are found to be interpretable and self-consistent in terms of subchain dynamical theory, including hydrodynamic, interactions and excluded volume, and the parameters characterizing these phenomena are in good agreement with the results of other hydrodynamic experiments and theoretical calculations. No necessity for modification of the subchain model in terms of limited extensibility or internal viscosity is found for high molecular weight DNA at the velocity gradients studied, although the latter cannot be ruled out on the basis of the present data. The Kuhn statistical segment length is determined from the intrinsic optical anisotropy and is estimated as 930 Å. Implications of these findings and their relation to appropriate dynamical models for DNA are discussed.     

Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1.

Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.     

Measurement of flow birefringence at very low velocity gradients

Instrumentation and associated techniques are described for the measurement of flow birefringence and extinction angles of high molecular weight polymer systems at extremely low velocity gradients. Precision of less than one degree in the extinction angles is obtained for birefringence smaller than 10^?11, based upon studies of very dilute high molecular weight DNA solutions over a range of velocity gradients from 0.1 to 3 sec^?1. Extinction angles approaching 44° are also observed with at least this degree of reliability for these systems. A relatively simple method of data reduction is outlined which permits explicit correction for solvent effects and for residual instrument birefringence and background; only the latter are found to be necessary at the shear rates reported in this communication. Applicability of the techniques to other macromolecules of biological interest is discussed.     

Kinetics of nucleosome unfolding at low ionic strength

The kinetics of a conformational change which occurs in nucleosome core particles at about 1 mM ionic strength have been studied by observing changes in the fluorescence of labeled histone H3. The unfolding reaction is intramolecular since no concentration dependence is observed. However, the kinetics are unexpectedly complicated and reveal evidence of at least three relaxation times. It is possible to fit the kinetics observed under several conditions to a consistent four-state cyclic mechanism in which folded and unfolded forms can inter-convert by two parallel pathways, each involving a distinct intermediate. While the data are not sufficient to establish this mechanism as a unique choice, they exclude many simpler possibilities. The cyclic mechanism is quite reasonable in view of what is currently known about the structures of the folded and unfolded forms.     

The roles of H1, the histone core and DNA length in the unfolding of nucleosomes at low ionic strength.

Calf thymus nucleosomes exhibit two different and independent hydrodynamic responses to diminishing salt concentration. One change is gradual over the range 40 to 0.2 mM Na+ and is accompanied by decreases in contact-site cross-linking efficiency. The other change is abrupt, being centered between 1 and 2 mM Na+. We found only one abrupt change in sedimentation rate for particles ranging in DNA content fom 144 to 230 base pairs. This response to decreasing ionic strength is similar for particles of both 169 and 230 base pairs. Core particles (144 base pairs) exhibit a somewhat diminished response. The abrupt change is blocked by formaldehyde or dimethylsuberimidate cross-linking. The blockage by dimethylsuberimidate demonstrates that the abrupt conformational change requires the participation of the core histones. H1 completely blocks the abrupt but not the gradual conformational change. Thus H1 uncouples the different responses to low ionic strength and exerts an important constraint on the conformational states available to the nucleosome core.     

Thermal stability of nucleosomes studied in situ by flow cytometry: effect of ionic strength and n-butyrate

Heating of cells permeabilized with ethanol and resuspended in aqueous media increases accessibility of DNA to intercalating dyes such as acridine orange (AO). The curves, representing increase in binding of AO as a function of rise in temperature, indicate that the transitions are cooperative. The transitions are sensitive to ionic strength and occur at lower temperatures when cells are suspended in media of increasing ionic strength. Extraction of histones raises accessibility of DNA to intercalators at room temperature, and heating has little effect on additional binding. The results are interpreted as indicating thermal destruction of nucleosomal structure in nuclear chromatin; dissociation of DNA from core histones results in its increasing ability to intercalate AO, most likely due to increased topological freedom to undergo unwinding and elongation following binding of the intercalator. Preincubation of cells with n-butyrate, known to induce histone hyperacetylation, lowers the heat stability of nucleosomes by about 5 degrees C. On the other hand, no differences are observed between chromatin of mitotic vs interphase cells tested over a wide range of ionic strengths (0.1-0.7 N NaCl). The method appears to be useful as a probe of chromatin structure at the nucleosomal level.     

Crystallization of tuna ferricytochrome c at low ionic strength

Previous crystallographic studies of tuna ferricytochrome c have employed crystals grown from solutions of ammonium sulfate, corresponding to an ionic strength of 9.5 M (Takano, T., and Dickerson, R. E. (1981) J. Mol. Biol. 153, 95-115). To obtain a structure at a lower ionic strength, the ferric tuna protein was crystallized at neutral pH with polyethylene glycol at an ionic strength of 45 mM, These crystals (space group P2(1), a = 37.11 A, b = 107.66 A, c = 55.75 A, beta = 105.3 degrees) contain four molecules/asymmetric unit and grow to dimensions of 0.2 X 0.4 X 1.0 mm in 2-4 weeks. They diffract to beyond 1.8 A and are stable in the x-ray beam. We have recorded 28,198 unique Bragg reflections (83% of those possible) to a resolution of 1.89 A from a native crystal. We are undertaking a solution of this structure by the molecular replacement method.     

Multiple conformations of deoxyribonuclease A. Their separation at alkaline pH and low ionic strength in the presence of Ca2+.

Gel filtration on Sephadex G-100 at pH 9.0 in 1 mM Tris buffer produces denaturation and inactivation of pancreatic DNAase A. Limiting concentrations of Ca2+ in the suspension and elution buffer, reactivates some of the enzyme molecules in an amount proportional to the calcium added. Stable active and inactive forms were separated on Sephadex columns. A model for the conformational role of Ca2+ on DNAase A demonstrates that at least one Ca2+ is involved (Kapp = 8.3 . 10(-5) M) in the correct folding of the polypeptide chain. Na+ was unable to reactivate the enzyme.