Physical studies of chromatin. The recombination of histones with DNA.

Experiments have been carried out to define clearly which histone combinations can induce a higher order structure when combined with DNA. The criterion for a higher order structure being the series of low-angle X-ray diffraction maxima nominally at 5.5 nm, 3.7 nm, 2.7 nm and 2.2 nm. Such a pattern, with resolution similar to that of H1-depleted chromatin, is readily attainable by recombining histones H2A + H2B + H3 + H4 with DNA using a salt-gradient dialysis method. However, the use of urea in the recombination procedure is shown to be detrimental to the production of a higher order structure. Low-angle ring patterns are not obtained by recomgining DNA with single pure histones or any combination of histone pairs exept H3 + H4. The diffraction maxima from the latter are, however, weaker than those from chromatin and there are pronounced semi-equatorial arcs. The presence of a third histone, either H2A or H2B in the H3 + H4 recombination mixture tends to distort the recognised low-angle pattern. It is concluded that the histone pair H3 + H4 is essential for the formation of a regular higher order structure in chromatin, although for a complete structural development the presence of H2A + H2B is also required.     

PHYSICAL STUDIES OF ISOLATED EUCARYOTIC NUCLEI

The degree of chromatin condensation in isolated rat liver nuclei and chicken erythrocyte nuclei was studied by phase-contrast microscopy as a function of solvent pH, K⁺ and Mg⁺⁺ concentrations Data were represented as "phase" maps, and standard solvent conditions selected that reproducibly yield granular, slightly granular, and homogeneous nuclei Nuclei in these various states were examined by ultraviolet absorption and circular dichroism (CD) spectroscopy, low-angle X-ray diffraction, electron microscopy, and binding capacity for ethidium bromide Homogeneous nuclei exhibited absorption and CD spectra resembling those of isolated nucleohistone. Suspensions of granular nuclei showed marked turbidity and absorption flattening, and a characteristic blue-shift of a crossover wavelength in the CD spectra. In all solvent conditions studied, except pH < 2 3, low-angle X-ray reflections characteristic of the native, presumably superhelical, nucleohistone were observed from pellets of intact nuclei. Threads (100–200 A diameter) were present in the condensed and dispersed phases of nuclei fixed under the standard solvent conditions, and examined in the electron microscope after thin sectioning and staining Nuclei at neutral pH, with different degrees of chromatin condensation, exhibited similar binding capacities for ethidium bromide. These data suggest a model that views chromatin condensation as a close packing of superhelical nucleohistone threads but still permits condensed chromatin to respond rapidly to alterations in solvent environment.     

Visualization of chromatin substructure: upsilon bodies

Spread chromatin fibers, from isolated eucaryotic nuclei, reveal linear arrays of spherical particles (upsilon bodies), about 70 A in diameter, connected by thin filaments about 15 A wide. These particles have been observed in freshly isolated nuclei from rat thymus, rat liver, and chicken erythrocytes. In addition, upsilon bodies can be visualized in preparations of isolated sheared chromatin, and in chromatin reconstructed from dissociating solvent conditions (i.e., high urea-NaCl concentration). As a criterion for perturbation of native chromatin structure low-angle X-ray diffraction patterns were obtained from nuclear pellets at different stages in the preparation of nuclei fro electron microscopy. These results suggest that the particulate (upsilon body) structures observed by electron microscopy may be closely related to the native configuration of chromatin.     

Higher-order structure of long repeat chromatin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.     

Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.     

X-ray small angle scattering study of chromatin as a function of fiber length

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.     

A Structural Analysis of Nerve Myelin

A structure analysis of the low-angle X-ray diffraction data from nerve myelin is described. The low-angle X-ray data are interpreted in terms of an electron density strip model which has five parameters, these refer to the dimensions of the membrane pair and their component electron densities. Three sets of low-angle X-ray data from peripheral nerve swollen in media of different electron densities are analyzed and membrane pair dimensions and component electron densities on an absolute scale are assigned. Membrane pair dimensions are given for a variety of peripheral nerve myelins and central nervous system myelins.     

Physicochemical studies of the folding of the 100 A nucleosome filament into the 300 A filament. Cation dependence

The cation-induced refolding of the 100 A nucleosome filament into the 300 A filament has been studied over a wide range of concentrations of Na+, Mg2+, Co(NH3)3+6 and other cations. X-ray diffraction, electron microscopy and analytical ultracentrifugation have been used to determine the conditions under which the 300 A filament is formed. It is shown that cations induce chromatin refolding by acting as general DNA counterions. The concentration of any cation required to induce refolding is greatly dependent on the valence of that cation. Na+ (and, presumably, other monovalent cations) has dual effects: at high concentrations (greater than 45 to 65 mM) it stabilizes the 300 A filament state of chromatin; however, at low concentrations (less than approximately equal to 45 mM), when cations of higher valence are present and stabilizing the 300 A filament state, Na+ has the opposite effect, competing with the higher-valence cation for binding to the chromatin and destabilizing the 300 A filament state. It is shown that further addition of cations to chromatin in the 300 A filament state causes a further folding of the chromatin in which the sedimentation coefficient increases and the X-ray diffraction bands resulting from nucleosomal packing sharpen. This may reflect subtle structural changes within the 300 A filament, or it may reflect a shift in equilibrium constant for chromatin fluctuating between the 100 A and 300 A filament states. It is also shown that, with continued addition of cation, the 300 A filaments precipitate before any "endpoint" is reached in this further folding. The tendency of 300 A filaments to aggregate in vitro appears to be a built-in property, and may reflect the packing of 300 A filaments within metaphase chromosomes in vivo.     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys33, Leu67)100-Orn20, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 A, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimensional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

Effect of urea, dimethylurea, and tetramethylurea on the phase behavior of dioleoylphosphatidylethanolamine

The phase behavior of dioleoylphosphatidylethanolamine in aqueous solutions of urea, N,N'-dimethylurea (DMU), and N,N,N',N'-tetramethylurea (TMU) has been characterized by synchrotron X-ray diffraction and differential scanning calorimetry. All three solutes stabilize the lamellar liquid-crystalline phase at the expense of lamellar-gel phase and inverted hexagonal phase of the phospholipid when present in concentrations up to 3 M. X-ray diffraction data demonstrated that the repeat spacing of DOPE increased with increasing urea concentration, but decreased as the DMU and TMU concentrations increased. The repeat spacing of DOPE in the liquid-crystal phase dispersed in the three solutes is d(urea)>d(DMU)>d(TMU). The molecular mechanisms underlying these observations are discussed in terms of either membrane Hofmeister effect, where urea acts as a water structure breaker, or a direct insertion effect of the amphiphilic DMU and TMU molecules into the lipid head groups in the interfacial region of the phospholipid bilayer.     

Urea-induced structural changes in chromatin obtained by sedimentation.

Sedimentation coefficients have been determined for fractionated preparations of whole and stripped (depleted of very lysine-rich histones and non-histone proteins) chicken erythrocyte chromatin fragments in 0-10 M urea. Significant differences in urea effects are observed between these preparations; differences which can be interpreted structurally by use of Kirkwood's dynamical theory of the translational frictional coefficient. This type of analysis implies that urea-induced chain-swelling in stripped chromatin is due largely to the urea effect upon the constituent nu-bodies, whereas the much larger swelling observed in whole chromatin appears to involve also the effect of urea upon the region between adjacent nu-bodies.     

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.     

Specific phosphorylation of yeast hexokinase induced by xylose and ATPMg. Properties of the phosphorylated form of the enzyme.

Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit γ globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no cross-reactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 m urea, but was reduced by exposure to 6.0 m urea plus 0.4 m guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.     

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.     

Degradation of chromosomal proteins during dissociation and reconstitution of chromatin

Histones and nonhistone chromosomal proteins are degraded when chromatin is exposed to 2 M NaCl-5 M urea (pH 6–8) which has been most often used for disscciation and reconstitution of chromatin. Histones and nonhistone proteins are also degraded in 5 M urea (pH 6–8).     

The integrity of the histone-DNA complex in chromatin fibres is not necessary for the maintenance of the shape of mitotic chromosomes

In this study we addressed the question of whether scaffold structures produced from purified mitotic chromosomes are an artefact of dehistonization, and whether the integrity of the chromatin fibres is necessary for the maintenance of the well-known shape of mitotic chromosomes. Purified mitotic chromosomes from Friend erythroleukemia cells were treated either with increasing NaCl concentrations up to 500 mM, or with 6 M urea in the presence or absence of 10 mM 2-mercaptoethanol. The main criterion for the intactness of the overall chromosome shape as seen by electron microscopy was the characteristic X-or U-like appearance with clearly discernable chromatid axes. Histone H1 is known to be essential for the integrity of chromatin fibres. Its removal in sucrose gradients containing 500 mM NaCl did not lead to loss of the overall chromosome shape. However, treatment of chromosomes in sucrose gradients containing 10 mM 2-mercaptoethanol and 6 M urea led to loss of the structure probably due to dissociation (or denaturation) of shape-determining (scaffolding) components. Under these conditions most of the histones remained bound to the chromosomes, and the fibres in this chromatin material, after removal of excess urea and 2-mercaptoethanol, still showed condensation of the nucleosome filaments into the characteristic fibre structures upon increasing ionic strength. Our observations are compatible with the model that specific non-histone components, independently of histone-DNA interactions, organize or stabilize the structure of metaphase chromosomes.     

Chromatin nu bodies: isolation, subfractionation and physical characterization.

Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study.     

Reconstitution of endogenous DNA synthesis in isolated nuclei and template activity of chromatin with respect to mode of inhibition by aphidicolin

We succeeded in reconstituting the endogenous nuclear DNA synthesis of the sea urchin. Endogenous DNA synthesis of isolated nuclei was reconstituted by mixing the salt-treated nuclei (chromatin exhibiting essentially no endogenous DNA synthesis) and the salt extract containing DNA polymerase-alpha. DNA synthesis in this reconstitution system showed a level of activity and a mode of inhibition by aphidicolin similar to those of the original isolated nuclei (noncompetitive with respect to dCTP). On the other hand, the inhibitory mode was competitive with respect to dCTP in DNA synthesis in the reconstituted system obtained from the chromatin and purified DNA polymerase-alpha, indicating that some other factor(s) in addition to DNA polymerase-alpha is necessary for the reconstitution with reference to the inhibitory mode of aphidicolin. We also studied the template activity of the chromatin. When chromatin was used as a template, inhibition by aphidicolin of DNA polymerase-alpha was noncompetitive and uncompetitive with respect to the template at high and low concentrations, respectively. Treatment of chromatin with 5 M urea gave urea-treated chromatin (nonhistone protein-deprived chromatin) and the extract (mainly nonhistone protein fraction). Inhibition by aphidicolin of DNA polymerase-alpha was uncompetitive with respect to the urea-treated chromatin. However, when chromatin reconstituted from the urea-treated chromatin and the extract was used as a template, the inhibitory mode by aphidicolin was similar to that with original chromatin, indicating that the nonhistone protein fraction contained factor(s) which modified the inhibitory mode of aphidicolin. Thus, the inhibitory mode of aphidicolin is a useful parameter for monitoring the resolution and reconstitution of endogenous DNA synthesis of isolated nuclei.