Interpretation of the X-ray scattering profiles of chromatin at various NaCl concentrations by a simple chain model.

In order to interpret the change in the X-ray scattering profiles from rat thymus chromatin, extensive model calculation was carried out. Chromatin is modelled as a string of subunits (nucleosomes) in which disorder is introduced into the positions of adjacent subunits. Disposition parameters characterizing the arrangement of subunits were estimated for various states of chromatin, so that the main feature of the scattering profiles is described. The result indicated that the structure of chromatin changes, as the NaCl concentration increases, from the extended "beads-on-a string" structure to the condensed helical structure. The latter has an outer diameter of about 26 nm with 3-4 nucleosomes per turn. In the intermediate state, it has a loose helical structure. The estimation of disorder suggested that the arrangement of subunits is appreciably disordered even in the condensed helical filament at 50 mM NaCl. Our model for chromatin condensation seems to support models of the "crossed linker" type.     

Organisation of subunits in chromatin.

There is considerable current interest in the organisation of nucleosomes in chromatin. A strong X-ray and neutron semi-meridional diffraction peak at approximately 10 nm had previously been attributed to the interparticle specing of a linear array of nucleosomes. This diffraction peak could also result from a close packed helical array of nucleosomes. A direct test of these proposals is whether the 10 nm peak is truly meridional as would be expected for a linear array of nucleosomes or is slightly off the meridian as expected for a helical array. Neutron diffraction studies of H1-depleted chromatin support the latter alternative. The 10 nm peak has maxima which form a cross-pattern with semi-meridional angle of 8 to 9 degrees. This is consistent with a coil of nucleosomes of pitch 10 nm and outer diameter of approximately 30 nm. These dimensions correspond to about six nucleosomes per turn of the coli.     

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.     

The superstructure of chromatin and its condensation mechanism

Electric dichroism and X-ray scattering measurements on solutions of uncondensed and condensed chicken erythrocyte chromatin were interpreted on the basis of model calculations. Information about the state of uncondensed fibers in the conditions of electric dichroism measurements was obtained from scattering patterns recorded as a function of pH, in the presence of spermine and at very low monovalent cation concentrations. Electric dichroism measurements on a complex of uncondensed chromatin with methylene blue were made to determine the contribution of the linker and of the nucleosomes to the total dichroism.A new approach to calculate the dichroism from realistic structural models, which also yields other structural parameters (radius of gyration, radius of gyration of the cross-section, mass per unit length) was used. Only a restricted range of structures is simultaneously compatible with all experimental results. Further, it is shown that previous interpretations of dichroism measurements on chromatin were in contradiction with X-ray scattering data and failed to take into account the distribution of orientation of the nucleosomes in the fibers. When this is done, it is found that the linker DNA in chicken erythrocyte and sea urchin chromatin must run nearly perpendicularly to the fibre axis. Taken together with the dependence of the fibre diameter on the linker length, these results provede the strongest evidence hitherto available for a model in which the linker crosses the central part of the fibre.     

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.     

Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin

We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher- order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101- 107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.     

Possible nucleosome arrangements in the higher-order structure of chromatin

Consideration has been given to possible sequences of nucleosomes which can produce a ‘thick fibre’-like structure. Only a few basic requirements were imposed: (i) the thick fibre is a regular single helix with about 7 nucleosomes per turn; (ii) the nucleosomes are equidistant along the polynuclesome chain; (iii) the helix is flexible having variable pitch. It was found that in addition to the straightforward sequential arrangement there is only one other nonsequential arrangement which satisfies these requirements. This is a helix with around 8 nucleosomes per turn in which all nucleosomes are identically placed. It is possible in the region of 200 to 218 ± 10 base pairs (b.p.) DNA repeats lengths. The linker DNA is straight or almost straight and crosses the internal ‘hollow’ cylinder which is not occupied by nucleosomes. This structure satisfies the experimental data for the distance distribution function, and the observed mass per unit length and changes noted in the mass per unit length. Further, if it is assumed that the core particle axis of symmetry is in the plane of the two linkers and bisects them then this makes the core particles oblique to the thick fibre radii with alternate angles of ± 20 to 30°. This orientation of the nucleosomes can explain the DNA digestion patterns obtained with DNase II and with DNase I.     

Cryo-electron microscopy of vitrified SV40 minichromosomes: the liquid drop model.

The structure of SV40 minichromosomes has been studied by cryo-electron microscopy of vitrified thin layers of solution. In high-salt buffer (130 mM NaCl), freshly prepared minichromosomes are condensed into globules 30 nm or more in diameter. On the micrograph, they appear to be formed by the close packing of 10 nm granules which give rise to a 10 nm reflection in the optical diffractogram. The globules can adopt many different conformations. At high concentration, they fuse into a homogeneous 'sea' of closely packed 10 nm granules. In low-salt buffer (less than 10 mM NaCl), the globules open, first into 10 nm filaments, and then into nucleosome-strings. The 'liquid drop' model is proposed to explain the condensed structure of the minichromosome in high-salt buffer: nucleosomes stack specifically on top of one another, thus forming the 10 nm filaments. 10 nm filaments in turn, tend to aggregate laterally. Optimizing both these interactions results in the condensation of 10 nm filaments or portions thereof into a structure similar to that of a liquid. Some implications of this model for the structure of cellular chromatin are discussed.     

A defined structure of the 30 nm chromatin fibre which accommodates different nucleosomal repeat lengths

Earlier work on the condensation of chromatins of different repeat lengths into the 30 nm fibre has been surveyed and it is shown that the external geometry of the fibre must be the same for all the chromatins. This can only be fitted by a helical coiling of nucleosomes into a solenoid with the linker DNA disposed internally. On this basis, various models were calculated and compared with published electric dichroism data. The only good fit is found with a 'reverse-loop' model, where the linker DNA forms a complete turn into the hole of the solenoid, of opposite hand to the nucleosomal DNA superhelix. This gives a topological linking number of one per nucleosome and would resolve the 'linking number paradox' if the DNA screw is the same in chromatin as in solution. The feasibility of a reverse-loop for short linkers (down to 15 base pairs) was investigated by model building and kinks of approximately 120 degrees into both DNA grooves are described, which will allow such packing. There will, however, be a 'forbidden' range for the linker DNA length, between approximately 1 and 14 bp, corresponding to nucleosomal repeats of 163 and 176 bp.     

RNA sequences specifically associated with mouse intracisternal A particles.

Electron microscopic examination of the histone H1-depleted, folded genomes of Drosophila melanogaster reveals that they are composed of long cylindrical cables of about 100 Å diameter. Limited single-strand nicking with DNAase I relaxes the 100 Å fibers to a “beads-on-a-string” structure, showing the nucleosomes and internucleosome DNA.Based on these results and other available data, we have constructed a detailed space-filling model for the higher order DNA coiling in chromatin, starting with the symmetrical nucleosome core previously described (Weintraub, Worcel and Alberts, 1976). The model defines the path of the DNA helix and the nucleosome arrangement along the DNA coil for both the 100 Å and the 200–300 Å fibers.Following Sobell et al. (1976), we believe that the DNA is coiled in the 100 Å nucleofilament in a uniform left-handed supercoil of about 90 base pairs (bp) per turn and 47 Å pitch; the 140 bp symmetrical nucleosome cores align themselves along this uniform DNA superhelix so that the isologous outer surfaces of adjacent nucleosomes touch and the internucleosome spacer DNA coils between them. A few single-strand discontinuities [about one nick per 85 kilobases (kb); Benyajati and Worcel, 1976] in the H1-depleted 100 Å fiber can thus relax the negatively supercoiled internucleosome DNA generating the “beads-on -a-string” appearance.We propose that histone H1 binds to the 100 Å diameter superhelix and coils it into tightly packed, 110 Å pitch super-superhelices (“solenoids;” Finch and Klug, 1976) of variable diameter (between 200–300 Å). In our model, the “thick” 200–300 Å fiber is stabilized at metaphase by histone H1-H1 heterologous interactions between adjacent helical turns of the nucleofilament, and the internucleosome spacer DNA is located on the outside. Symmetry considerations demand that changes in the length of the repeat should lead to variations in the number of nucleosomes per helical turn and in the handedness of these turns in the 200–300 Å metaphase fiber.     

The fine structure of chromatin in Paranosema grylli (Microsporida)

Nucleosomes were found for the first time in the nuclear chromatin of Microsporida--organisms known among the smallest eukaryotes on Earth. Chromatin of Paranosema grylli sporoplasm was studied by Miller's technique. On low ionic-strength cell spreads, this chromatin was represented by 10 nm nucleosome filaments, 20 nm filaments, and "smooth" (nucleosome-free) filaments of 3-4 nm in diameter. Nucleosome filaments display structural heterogeneity seen as irregular arrangement of nucleosome particles along the filament length. Different nucleosome filaments show 13-30 nucleosomes per 1 microm with the length of linker DNA ranging from 10 to 45 nm. The present results suggest that microsporidian chromatin is weakly condensed. Only lower-order chromatin packaging levels displayed some structural peculiarities.     

Synergistic interactions between the genetically modified bacterial polysaccharide P2 and carob or konjac mannan

Rheological studies have confirmed that the bacterial polysaccharide P2, a genetically modified variant of the Acetobacter xylinum polysaccharide acetan, undergoes synergistic gelation with either of the plant polysaccharides carob or konjac mannan. X-ray fibre diffraction data shows that P2 can form a 5-fold helical structure of pitch 4.7nm and an axial rise per disaccharide repeat of 0.92nm. Optical rotation data demonstrate that P2 undergoes a coil-helix transition in solution and that deacylation enhances the stability of the helical structure in solution. Studies made on mixtures prepared at different temperatures and ionic strengths suggest that denaturation of the P2 helix favours interaction and gelation. Deacetylation of P2 enhances gelation. X-ray diffraction data for oriented fibres prepared from deacetylated P2-konjac mannan mixed films reveal a 6-fold helical structure of pitch 5.54nm with an axial rise per disaccharide repeat also of 0.92nm. This mixed helix provides direct evidence for binding between the two polysaccharides. P2 contains two sites of acetylation: one on the backbone and one on the sidechain. The former site of acetylation inhibits helix formation for P2. It is suggested that this site of acetylation also inhibits formation of the mixed helix, explaining the enhanced gelation of mixtures on deacetylation.     

Interaction of chromatin with NaCl and MgCl2: Solubility and binding studies,transition to and characterization of the higher-order structure

Chicken erythrocyte chromatin containing histones H1 and H5 was carefully separated into a number of well-characterized fractions. A distinction could be made between chromatin insoluble in NaCl above about 80 mm, and chromatin soluble at all NaCl concentrations. Both chromatin forms were indistinguishable electrophoretically and both underwent the transition from the low salt “10 nm” coil to the “30 nm” higher-order structure solenoid by either raising the MgCl₂ concentration to about 0.3 mm or the NaCl concentration to about 75 mm. The transitions were examined in detail by elastic light-scattering procedures. It could be shown that the 10 nm form is a flexible coil. For the 30 nm solenoid, the assumption of a rigid cylindrical structure was in good agreement with 5.7 nucleosomes per helical turn. However, disagreement of calculated frictional parameters with values derived from quasielastic light-scattering and sedimentation introduced the possibility that the higher-order structure, under these conditions, is more extended, flexible, or perhaps a mixture of structures. Values for density and refractive index increments of chromatin are also given.To understand the interaction of chromatin with NaCl and with MgCl₂, a number of experiments were undertaken to study solubility, precipitation, conformational transitions and binding of ions over a wide range of experimental conditions, including chromatin concentration.     

Chromatin structure. Evidence that the 30-nm fiber is a helical coil with 12 nucleosomes/turn

Sedimentation analysis has been used to compare the structure of 30-nm chromatin fibers, isolated and digested under conditions that maintain the native structure, with relaxed-refolded chromatin. The native chromatin fibers show sharp, ionic strength-dependent changes in sedimentation coefficient that are not apparent in relaxed-refolded fibers. The first transition at approximately 20 mM ionic strength reflects the organization of the 10-nm polynucleosome chain into a loose helically coiled 30-nm fiber. Between 20 and 60 mM ionic strength there is considerable interaction between nucleosomes within the coils to generate a stable helical array with 12 nucleosomes/turn. Above 60 mM ionic strength the helical coil continues to condense until it precipitates at ionic strengths slightly greater than those considered physiological, indicating that there is no end point in fiber formation. The data is incompatible with a solenoid model with 6 nucleosomes/turn and also rules out the existence of a beaded subunit structure.     

3-D reconstruction of bluetongue virus tubules using cryoelectron microscopy.

Bluetongue virus (BTV) forms tubules in infected mammalian cells. These tubules are virally encoded entities which can be formed with only one protein, NS1. The NS1 protein does not form a part of virus particles, and its function in viral infection is uncertain. Expression of the NS1 gene in insect cells by recombinant baculovirus yields high amounts of NS1 tubules (ca. 50% of cellular proteins) which are morphologically and immunologically similar to authentic BTV NS1 and can be isolated to about 90% purity. The structure of these synthetic NS1 tubules was investigated by cryoelectron microscopy. NS1 tubules are on average 52.3 nm in diameter and up to 100 nm long. The structure of their helical surface lattice has been determined using computer image processing to a resolution of 40 A. The NS1 protein is about 5.3 nm in diameter and forms a dimer-like structure, so that the tubules are composed of helically coiled ribbons of NS1 "dimers," with 21 or 22 dimers per turn. The surface lattice displays P2 symmetry and forms a one-start helix with a pitch of 9.1 nm. The NS1 tubules exist in two slightly different pH-dependent conformational states.     

Higher-order structure of nucleosome oligomers from short-repeat chromatin

Sedimentation measurements and electron microscopy at a series of ionic strengths suggest that chromatin from neurons of the cerebral cortex is able to form condensed structures in vitro that are probably several turns of a solenoid with about six nucleosomes per turn. Since neuronal chromatin has a short nucleosomal repeat (approximately 165 bp) allowing virtually no linker DNA between nucleosomes, and yet forms apparently 'normal' elements of solenoid, the packing of nucleosomes in the solenoid must be highly constrained. This permits only a limited number of possible models, and enables tentative suggestions to be made about the location of the linker DNA in the typical solenoid.     

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators.