Rat liver chromatin was moderately digested by micrococcal nuclease and analysed by centrifugation in isokinetic sucrose gradients and electron microscopy. Two classes of particles sedimenting with about 33S and 60S were characterized. Kinetics of their appearance and disappearance during progressive digestion suggests that they represent monomers and dimers cleaved from a higher order (supranucleosomal) structure of chromatin. Biochemical and electron microscopical results suggest that the monomers and dimers contain eight and sixteen nucleosomes, respectively, which are densely packed into 23 nm (monomer) and 29 nm (dimer) globules.

Animals↗

PubMed1977

Elevated cyclic GMP concentrations during estrogen induced differentiation of the chick oviduct.

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Aging↗

PubMed1976

Relaxation of chromatin structure by ethidium bromide binding: determined by viscometry and histone dissociation studies.

The effects of ethidium bromide intercalation on chromatin structure were monitored by viscometry and analysis of histone dissociation. Investigation of the NaCl concentration dependence of chromatin viscosity showed that the reduced viscosity (etared) was very low up to 0.4 M NaCl and increased gradually when the salt concentration was raised further. In chromatin intercalated by ethidium bromide, etared was not significantly different at low salt concentrations (up to 0.2 M NaCl). However, when the salt concentration was raised further, the viscosity response curve increased sharply to reach viscosities about 4-5 times higher than those for nonintercalated chromatin. The increase in viscosity was proportional to the increase in fluorescence intensity, when the ratio of ethidium bromide to DNA mucleotide was raised. The transition of intercalated chromatin into the relaxed form was reversible, dependent on the nature of the electrolyte and cooperative, as indicated by the small increase in salt concentration required to obtain chromatin relaxation. Investigation of the NaCl concentration dependence of histone dissociation revealed that total histones and each individual histone fraction were released from intercalated chromatin at much reduced NaCl concentrations. The midpoints of the dissociation curves of the individual histones ranged from 0.30 to 0.45 M NaCl and fell within the same range where the drastic viscosity change occurred. These results indicate that intercalation of ethidium bromide labilizes chromatin structure to relaxation by moderately elevated salt concentrations. It is suggested that the labilization is caused by changes in the DNA helix conformation due to dye intercalation decreasing the stability of histone-DNA interactions.

Animals↗

PubMed1976

Studies on the structure and function of chick-oviduct chromatin. 1. Fractionation by ECTHAM-cellulose chromatography and physico-chemical characterization.

Chick oviduct chromatin was separated into a ribonucleoprotein fraction and two chromatin fractions (early and late eluting). We utilized a gentle procedure in which moderately hydrated chromatin was subjected to chromatography on a weak ionic-exchange resin (ECTHAM-cellulose) eluted with a combined pH-salt gradient. Chemical analysis of the early (fraction I) and late (fraction II) eluting fractions revealed that their histones were identical and their nonhistone proteins were markedly different. Control experiments showed that these differences were not due to protein rearrangements during chromatin preparation and/or fractionation. The physical properties of fraction I and II differed in certain aspects. The aggregation response of fraction I to increasing concentrations of monovalent cations was five times lower than that of fraction II but the aggregation response to divalent cations was identical. Thermal denaturation assays of DNAs isolated from fractions I and II revealed identical derivative profiles of hyperchromicity vs temperature, thereby indicating similar base composition in the two fractions. Circular dichroism, spectra of the purified DNAs isolated from both fractions showed identical B-type conformations. However, DNA renaturation kinetics analyzed by computer technique indicated that fraction I DNA contained less than half the amount of highly repetitive sequences as compared to either unfractionated chromatin or fraction II. Circular dichroism spectra of fraction I and II chromatins (at room temperature) showed significant differences in a wavelength region were only DNA is optically active (i.e. 255-320 nm). These results indicated that the DNA complexed to proteins in fraction II assumed a more C-type conformation than the DNA in fraction I. The differences in the circular dichroism spectra could not be accounted for by differences in the RNAs or protein chromophores contained in fraction I and fraction II. When the circular dichroism spectra of fraction I and II were recorded at 55 degrees C, the differences between the two fractions were abolished. These results were interpreted to mean that the differences in the DNA conformations found in fractions I and II were due to the differences in their nonhistone proteins. These proteins were effective in maintaining DNA conformation differences only when they were in their native form but not when heated to 55 degree C. Comparison of the sedimentation coefficients of fractions I and II with their calculated molecular weights suggested a more extended structure in fraction I as compared to a more compact structure in fraction II. Only small differences were observed between fraction I and fraction II with respect to either buoyant density analysis in a metrizamide gradient or in the number of phosphate charges accessible to polylysine.

Animals↗

PubMed1976

Studies on the structure and function of chick-oviduct chromatin. 2. Biochemical characterization of two chromatin fractions isolated by ECTHAM-cellulose chromatography.

Chromatin prepared at various stages of hormone-mediated development of the chick oviduct was investigated for the relative proportions of transcriptionally active (fraction I) and repressed (fraction II) fractions by ECTHAM-cellulose chromatography. During primary stimulation with estrogen, the amount of chromatin DNA in fraction I plotted as a function of time of stimulation showed a bell-shaped profile, similar to the profile obtained earlier for the number of chromatin sites available to RNA polymerase for initiation of RNA synthesis. Chromatin form a transcriptionally inactive system, hen erythrocytes, eluted mainly (98%) as fraction II. The transcriptionally active fraction I of estrogen-stimulated oviduct contained a 4-fold greater RNA polymerase II activity than was found in fraction II. This could be explained by a differential inhibition of RNA polymerase activity in fraction II since enzyme preparations extracted and purified from both chromatin fractions showed equal activities. In support of this finding, fraction I eluted from ECTHAM-cellulose showed a 4-fold greater concentration of rifampicin-resistant RNA chain initiation sites as compared to fraction II. When chromatin from oviduct mince incubated with labeled progesterone and 17 beta-estradiol and was chromatographed on ECTHAM-cellulose, the transcriptionally active fraction also contained a 4-fold greater concentration of bound hormone (per weight DNA) as compared to the repressed fraction.

Animals↗

PubMed1976

Stimulation of transcription on chromatin by polar organic compounds.

Polar organic compounds, including DMSO, increase RNA synthesis on isolated chromatin by E. coli RNA polymerase and RNA polymerase II from calf thymus. Transcription is stimulated on chromatin from Friend-virus-infected erythroleukemia cells and from various other sources. Using procedures which inhibit specifically the formation of a stable initiation complex, it is shown that the stimulation does not result from an increase in initiation of both E. coli and the eukaryotic RNA polymerase. After separation of chromatin into template active and inactive fractions, DMSO increases RNA synthesis by a factor of about 1.5 using the template inactive fraction, while stimulation of transcription on the template active portion is lower (factor of 1.2). It is suggested that the effect on RNA synthesis is mediated by a weakening of the apolar interactions between histones in chromatin subunits, releasing transcription partially from the constraints imposed by histones.

Animals↗

PubMed1967

[Insulin from the hagfish (Myxine glutinosa: Cyclostomata)].

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Adipose Tissue↗