Interaction of histone H1 with superhelical DNA. Conformational studies and influence of ionic strength.

The interaction of histone H1 with superhelical SV40 DNA at low ionic strength (approximately 0.02 M NaCl) results in the formation of DNP double-fibers and bundle- and cablelike twisted side-by-side associates of several of these double-fibers. On the basis of simple cylindrical or ellipsoidal models the sedimentation properties of these structures can be calculated in accordance with the experiment allowing a direct assignment of electron microscopical and hydrodynamic results. Sedimentation measurements in dependence on the ionic strength indicate a redistribution of H1 resulting in the formation of associates at 0.04 M NaCl and of aggregates at higher salt concentration. Double-fibers are present up to physiological salt concentrations.

Studies on histone H1 condensing properties in complexes with DNA and polydeoxyribonucleotides using netropsin as a probe.

The binding of histone H1 with DNA and synthetic DNA duplex polymers with respect to its property to induce higher ordered structures has been studied using the DNA binding antibiotic netropsin as a probe. It was shown that the formation of distinct steps of different condensed structures (double-fibers, cable- and stem-like forms) is influenced by the ionic strength. CD titration data of DNA-H1 complexes with netropsin at 20 mM NaCl indicated no change in the binding to strong affinity sites (dA X dT clusters) as compared to free DNA's, while weak netropsin binding regions are strongly affected by competition interaction with H1. At low histone concentration the presence of netropsin favours the formation of double fibers. CD and electron microscopic findings indicated that at 20 mM NaCl the occurrence of condensed structures of DNA histone H1 complexes is not dependent on the base content. The major groove interaction of H1 most probably plays the major role in the formation of higher ordered structures. However, the minor groove binding might be involved as a secondary event. A hierarchy of relevant morphological structures observed for DNA-H1 complexes is presented.

In vitro transformation and mutation of Chinese hamster cells by different SV40 nucleoprotein complexes.

SV40 minichromosomes (MCH) either isolated from SV40 infected CV-I monkey cells (native MCH) or reconstituted in vitro from viral DNA and the H1 depleted calf thymus histone fraction could transform and mutate Chinese hamster (CH) cells in vitro. Whereas reconstituted MCH transformed and mutated CH cells with about the same efficiency as purified SV40 DNA, approximately 10-200-fold increase in the transforming activity had been demonstrated for native MCH. All transformed cell colonies and a major part of the isolated mutant cell clones recovered after inoculation of CH cells with SV40 MHC expressed the SV40 T antigen. Addition of H1 to both purified SV40 DNA and reconstituted MHC drastically diminished the transforming capacities of both agents. Possible reason(s) for the inhibition effect of H1 histone is discussed.

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes.

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native 'minichromosomes'. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.

Discrimination of several classes of nonhistone proteins in chromatin fractions from liver and thymus nuclei of rats.

Nonhistone proteins (NHPs) of salt-soluble chromatin (Chromatin S) and of the residual nuclei (Chromatin P) from rat liver and thymus were studied by SDS-polyacrylamide gel electrophoresis. The two chromatin fractions of the liver showed significant differences in their NHP patterns with most of the hnRNP and matrix proteins occurring in Chromatin P. In accordance with the low protein content of thymus nuclei, the corresponding thymus fractions exhibited electrophoretic patterns with a markedly lower amount of NHPs than in liver. Chromatin P from thymus, in contrast to the liver fraction, revealed only a very low content of hnRNP-specific proteins of molecular weight 30,000-40,000 (30 K to 40 K) (informosomal proteins) consistent with the significantly lower RNA content of thymus nuclei. In the region of the matrix proteins (60-75 K) Chromatin P showed only two bands of about 64 K and 73 K in thymus, whereas in liver five strong bands at 64 K, 66 K, 69 K, 73 K, and 75 K were found. RNase digestion was employed to discriminate hnRNP-specific protein from "real" chromosomal NHPs. At least about 65% and 25% of the NHPs from Chromatin P and S of liver, respectively, were found to be RNP-specific. The two chromatin fractions were further fractionated by sucrose gradient centrifugation and isopycnic banding in metrizamide. After centrifugation the main peaks, both of Chromatin S and P, contained only minor amounts of NHPs with a predominating protein of 38 K. By the centrifugation procedures described in this paper, a small subfraction of chromatin could be separated which was enriched in newly synthesized RNA, informosomal proteins, matrix- and other high molecular weight proteins. This subfraction might be related to transcriptionally active chromatin.

Interaction of histone H1 with superhelical DNA. Sedimentation and electron microscopical studies at low salt concentration.

Complexes of histones H1 with superhelical SV40 DNA obtained by direct mixing were studied in 0.1 SSC buffer corresponding to 0.02 M Na+. Depending on the molar input ratio H1/DNA three classes of sedimenting species were observed: (1) a component sedimenting similar to superhelical DNA with a sedimentation coefficient s2o,w of 25 S observable up to 335 Mol H1/Mol DNA (w/w = 2); (2) a component with s2o,w = 120 S appearing at 135 Mol H1/Mol DNA and (3) growing amounts of heterogeneous aggregates greater than 1000 S. Electron micrographs revealed the 25 S component to consist of double-fibers formed from one DNA molecule and the 120 S component to consist of bundles of several such double-fibers. The aggregates represent cable-like structures. The addition of ethidium bromide to 25 S complexes induces the formation of bundles, if H1 is present in a quantity which alone is not sufficient to bring about this effect. This result indicates that ethidium bromide effects a redistribution of H1 molecules and that H1 is responsible for the bundle formation.

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.

Complexes of DNA with arginine-rich and slightly lysine-rich histones. Transcription and electron microscopy.

Calf thymus DNA was reconstituted with arginine-rich histones H3 and H4 and slightly lysine-rich histones H2A and H2B respectively. Complexes containing histones H3 and H4 exhibit nucleosome-like structures when examined in the electron microscope and show a restriction of in vitro transcription similar to that obtained for reconstitutes made up from the four histones H2A, H2B, H3 and H4. One the contrary, complexes of DNA and histones H2A and H2B create different morphological structures of short stretches of bound histones and do not cause a template restriction in vitro.

Electron microscopical investigations on the glycocalyx of cultivated cells after incubation with extracts from rapidly growing normal and tumour tissues.

Electron microscopical investigations by means of the ruthenium red method on Huggin-tumour cells cultivated in vitro resulted in a different thickness of the glycocalyx depending on the fixation of the cells in monolayer or, after mechanical detachment, in suspension. Cells fixed in suspension display a thicker glycocalyx, which is supposed to be due to a contraction of the cells before fixation. Tissue extracts from rapidly growing tissues (mammary gland of pregnant cows, tumour tissue) cause a significant reduction of the glycocalyx in transformed hamster fibroblasts and Huggin-tumour cells after an 18-hour period of action. These findings are in accordance with previous findings on the glycolytic effect of tissue extracts on sections of cartilaginous tissue. It is discussed whether the action of the tissue extracts on the glycocalyx is due to its detachment or to an inhibition of its renewal.