Formation of double-walled microtubules and multilayered tubulin sheets by basic proteins.

Some basic proteins enable microtubule protein to form special assembly products in vitro, known as double-walled microtubules. Using histones (H1, core histones) as well as the human encephalitogenic protein to induce the formation of double-walled microtubules, we made the following electron microscopic observations: (1) Double-walled microtubules consist of an "inner" microtubule which is covered by electron-dense material, apparently formed from the basic protein, and by a second tubulin wall. (2) The tubulin of the second wall seems to be arranged as protofilaments, surrounding the inner microtubule in a helical or ring-like manner. (3) The surface of double-walled microtubules lacks the projections of microtubule-associated proteins, usually found on microtubules. (4) In the case of protofilament ribbons (incomplete microtubules), H1 binds exclusively to their convex sides that correspond to the surface of microtubules. Zn2+-induced tubulin sheets, consisting in contrast to microtubules of alternately arranged protofilaments, are covered by H1 on both surfaces. Furthermore, multilayered sheet aggregates appeared. The results indicate that the basic proteins used interact only with that protofilament side which represents the microtubule surface. In accordance with this general principle, models on the structure of double-walled microtubules and multilayered tubulin sheets were derived.

Effect of microtubule-associated proteins on the protofilament number of microtubules assembled in vitro.

In order to demonstrate the effect of microtubule-associated proteins on the protofilament number of microtubules, we used different systems of microtubule formation in vitro in which these proteins are either functionally eliminated (by DNA or glycerol) or absent (purified tubulin). The results obtained by electron microscopy of ultrathin-sectioned material indicate that under standard conditions in the presence of microtubule-associated proteins microtubules are formed consisting predominantly of 14 protofilaments. In cases of deficiency of microtubule-associated proteins, the mean value of the protofilament number is lower, and the protofilament number within the microtubule population varies remarkably. On the other hand, the action of microtubule-associated proteins is enhanced by histones resulting in increased protofilament numbers. A model is proposed illustrating that the quality and the quantity of microtubule-associated proteins bound to microtubules determine the curvature between the protofilaments and restrict the variety of their binding angles. In this way the microtubule-associated proteins may be regarded as an important factor in determining the structural fidelity of microtubules.

Physicochemical properties of salt-soluble, unsheared chromatin. Molecular weight studies.

A chromatin fraction, which can reproducibly be extracted from rat liver nuclei at moderate salt concentration (0.1 M (NH4)2SO4, 0.1 M Tris-HCl, 2 mM MnCl2, pH 7.9), was analyzed with regard to changes of its molecular weight in the range of (NH4)2SO4 concentrations between 0.1 M and 0.4 M. With the transition from 0.1 M to 0.2 M (NH4)2SO4 histone H1 is released and the molecular weight obtained from both sedimentation-viscosity and light scattering is reduced by approximately one-half. A spatial expansion of the resulting half-molecules is observed with further increasing salt concentration. On the basis of these results a double-fibrillar structure of this chromatin fraction is proposed.

Physicochemical properties of salt-soluble, unsheared chromatin. The mass per unit length by light scattering.

The mass per unit length of 7 X 10(3) (formula: see text) and the corresponding DNA packing ratio of about 14 for the chromatin soluble at moderate ionic strengths has been determined by light scattering. With the increase in ionic strength and corresponding release of histone H1 the DNA packing ratio has been found to decrease down to 4.4. The data obtained are consistent with the idea suggested previously that the salt-soluble chromatin is organized in double nucleosome chains arranged side-by-side and stabilized by H1. With salt-induced H1 release the double chain dissociates and the nucleosomal DNA partially unravels.

Physicochemical properties of salt-soluble, unsheared chromatin. Salt-dependent structural changes.

Salt-dependent structural changes of rat liver chromatin isolated by an extraction procedure not involving shear and exogenous nucleases were investigated by sedimentation and light scattering methods. The effects observed are complex involving changes in the molecular weight and expansion. Between 0.1 M and 0.2 M (NH4)2SO4 where histone H1 is released, a fragmentation into molecules of half molecular weight is found which is accompanied by an expansion into a more extended conformation gradually increasing to 0.4 M (NH4)2SO2. The H1-free chromatin does not exhibit the reduction in molecular weight but undergoes this expansion. The original conformation is not reversible on re-decreasing the salt concentration to 0.1 M (NH4)2SO4.

Isolation and characterization of salt-soluble, unsheared chromatin.

From rat liver nuclei depending on the extraction time, 10 to 20% of total chromatin has been extracted with a solution containing 0.1 M ammonium sulfate, 2 mM MnCl2, 0.1 M Tris-HCl, pH 7.9. We term this chromatin chromatin S. It has a protein: DNA ratio of 1.3, the full amount of the 5 histones in an undegraded state, and a RNA: DNA ratio of approximately 0.2. Its nonhistone protein pattern, obtained by gel electrophoresis exhibits a rich spectrum of proteins in a broad range of molecular weights. Electrophoretic analysis of the DNA fragments obtained by micrococcus nuclease digestion of chromatin S yields the same digestion pattern as that of nuclei. Thus, chromatin S fulfils an essential criterion of unsheared chromatin. In contrast to other chromatin preparations described so far, this chromatin is soluble at a salt concentration of 0.1 M ammonium sulfate. We have shown previously that it exhibits a compact conformation, low intrinsic viscosity and low radius of gyration obtained by light scattering measurements. Its mean molecular weight was determined to be nearly 10(8).

Salt-and histone H1-induced structural changes of reconstituted minichromosomes.

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.

Altered histone-DNA interactions in rat liver chromatin containing 5-bromodeoxyuridine-substituted DNA.

The extractability of the different histone types from rat liver chromatin was studied following the incorporation of bromodeoxyuridine (BrUdR) in liver DNA. This was accomplished by a continuous application of 20 mumol BrUdR/ml/h 17--41 after partial hepatectomy. As a result, thymidine (TdR) replacement by BrUdR of about 80% in the newly-synthesized DNA strand of approx. 30% of total liver DNA was obtained; this causes remarkable changes in the histone--DNA interactions as determined from the release of histones from liver nuclei by ammonium sulfate and ethidium bromide (EB), respectively. In particular, the relative amounts of the two slightly lysine-rich histones H2A and H2B remaining on the BrUdR chromatin proved to be about 3-fold higher than those remaining on the control chromatin of TdR-treated animals. Similarly, histones H1 and H3 tend to bind closer to BrUdR-containing DNA. These results may be of interst with regard to the well-known selective effects of BrUdR on differentiation processes.

[X-ray small angle scattering and x-ray wide angle scattering of single nucleosomes. Preliminary results]. / Röntgen-Kleinwinkelstreuung und Röntgen-Weitwinkelstreuung von eizelnen Nukleosomen. Vorläufige Ergebnisse.

The X-ray scattering diagram from single chromatin subunit particles is registered within a scattering vector intervall from s = 0 to s = 1 1/A. Preliminary results concerning the dimensions and the structure of the nucleosome core particle are communicated.

A sedimentation study of the interaction of superhelical SV40 DNA with H1 histone.

By moving boundary sedimentation it is shown that the interaction of H1 histone with superhelical circular SV40 DNA results in the formation of giant heterogeneous aggregates. The size of these aggregates grows with increasing H1 concentration. s20,w values of some 10 000 S were measured. As compared with open relaxed circular DNA a preferential interaction of superhelical DNA with H1 histone is observed, irrespective of the sign of the superhelical turns which was reversed by the addition to DNA of ethidium bromide. The addition to the H1 complexed aggregates of ethidium bromide effects a progressive breakdown of the aggregates. Furthermore, the superhelicity of DNA is not changed by the addition of small amounts of H1 histone.

Evidence of altered histone interactions, as investigated by removal of histones, in chromatin isolated from rat liver nuclei by a conventional method.

It is shown that the release of the slightly lysine-rich histones f2a2 and f2b by 0.4 M ammonium sulfate from conventionally isolated chromatin is diminished in comparison to the lysed nuclei. The change in extractability is further demonstrated by the application of ethidium bromide. At a molar input ratio of 0.09 (moles ethidium bromide/moles nucleotide) and 0.4 M ammonium sulfate the slightly lysine-rich histones are released from the chromatin to 70 - 80% if the lysed nuclei are used. At 0.1 M ammonium sulfate ethidium bromide effected also a release of 50 % of histone f1. Comparable effects could not be observed with chromatin prepared in a conventional way but instead a tendency towards loss of histone f3 in the presence of ethidium bromide was observed.