The structure of inactive interphase macronuclear chromatin of the ciliate Bursaria truncatella. Radial loops in the structure of chromatin clumps

The organization of chromatin in macronuclei of Bursaria truncatella cells that completed their growth and differentiation was electron microscopically studied. The data obtained showed that (1) inactive macronuclear chromatin was organized in compact chromatin clumps 120 to 180 nm in diameter linked by one or several chromatin fibres, and (2) in low salt buffer the chromatin clumps gradually unraveled, radial loops of supranucleosomal or, more often, nucleosomal structure appearing around chromatin clumps. Upon prolonged incubation in low salt buffer chromatin clumps were completely transformed into nucleosomal fibres. The data obtained evidenced in favour of a loop-packed structure of chromatin clumps.     

The globular domain of histone H5 is internally located in the 30 nm chromatin fiber: an immunochemical study.

The location of the globular domain of histone H5 relative to the axis of the 30 nm chromatin fiber was investigated by following the accessibility of this region of the molecule in chicken erythrocyte chromatin to specific antibodies as a function of chromatin structure. Antibodies to the globular domain of H5 as well as their Fab fragments were found to react with chromatin at ionic strengths ranging from 1-80 mM NaCl, the reaction gradually decreasing upon increase of salt concentration. If, however, Fab fragments were conjugated to ferritin, no reaction of the complex with chromatin was observed at salt concentrations higher than 20 mM. The accessibility of the globular part of H5 in unfolded chromatin to the Fab-ferritin complex was also demonstrated with trypsin-digested chromatin. The experiments were carried out by both solid-phase immunoassay and inhibition experiments. The data obtained are consistent with a structure in which the globular domain of H5 is internally located in the 30 nm chromatin fiber.     

The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers

To assess the influence of chromatin structure on the frequency of radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to nuclear and nucleoid monolayers. These chromatin substrates were prepared by treating human fibroblasts grown as monolayers with the nonionic detergent Triton X-100 and varying concentrations of cations. The chromatin structure was modified either by a stepwise removal of DNA-bound proteins by extraction in increasing concentrations of monovalent salt, or by the addition or deletion of mono- and divalent cations to condense or decondense the chromatin, respectively. It was found that the stepwise removal of DNA-bound proteins from the chromatin dramatically increased the frequency of radiation-induced DNA strand breaks. The DNA-bound proteins showed a qualitative difference in their ability to protect the DNA where proteins removed by salt concentrations above 1.0 M exerted the greatest protection. Furthermore, the frequency of radiation-induced DNA strand breaks was found to be 6 times lower in condensed chromatin than in decondensed chromatin and about 80 times lower than in protein-depleted chromatin. It is concluded that the presence of DNA-bound proteins and the folding of the chromatin into higher-order structures protect the DNA against radiation-induced strand breaks.     

Sperm chromatin integrity of bucks transgenic for the WAP bGH gene

The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13-18 months old. Semen was collected at 1-week intervals and 3-7 ejaculates from each rabbit were examined in total. Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes. The mean value of COMPalpha(t) was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.alpha(t) for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (-0.68, P<0.05) was found between S.D.alpha(t) of NTG males and percentages of pregnant does. The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene.The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.     

Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.     

Effects of aluminum and other cations on the structure of brain and liver chromatin

The reactivity of aluminum and several other divalent and trivalent metallic cations toward chromatin from rat brain and liver has been investigated. Two criteria are used to determine the relative reactivity of these cations toward chromatin. The first involves the ability of the ions to compact the chromatin fibers to the point where chromatin precipitates. The second criterion measures the ability of cations to interfere with the accessibility of exogenous structural probes (nucleases) to chromatin. Of the divalent cations tested, nickel, cobalt, zinc, cadmium, and mercury were the most reactive toward chromatin, on the basis of their ability to induce precipitation of chromatin in the micromolar concentration range. The divalent cations magnesium, calcium, copper, strontium, and barium were much less effective, although all cations precipitate chromatin if their concentration is increased. Of the trivalent cations tested, aluminum, indium, and gallium were very effective precipitants, whereas iron and scandium were without effect at the concentrations tested. Of all the cations tested, aluminum was the most reactive. Aluminum's ability to alter the structure of chromatin was investigated further by testing its ability to interfere with nuclease accessibility. This test confirmed that aluminum does induce considerable changes in chromatin structure at micromolar concentrations. Furthermore, chromatin from cortical areas of the brain was much more sensitive to aluminum than chromatin from liver. These results are discussed in light of the known toxicity of these cations, with particular emphasis on the possible role of aluminum in Alzheimer's disease.     

The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.     

DNase I hypersensitivity and expression of the Shrunken-1 gene of maize

The local chromatin structure of the Shrunken-1 (Sh) gene of maize was probed by analyzing DNase I hypersensitivity. Sh encodes the gene for sucrose synthetase, a major starch biosynthetic enzyme, which is maximally expressed in the endosperm during seed maturation. In addition to general DNase I sensitivity, specific DNase I hypersensitive sites were identified in endosperm chromatin that mapped near the 5 end of the Sh gene. The pattern of hypersensitive sites and their relative sensitivity were altered in other non-dormant tissues that produce little or no enzyme. However, some changes in chromatin structure appear to be independent of Sh gene expression and may reflect general alterations associated with plant development. The chromatin structure of several sh mutations, induced by Ds controlling element insertions, was also analyzed. Although the insertions perturbed expression of the gene, there were no notable effects on local chromatin structure.     

Changes in the compactness of nucleosome chromatin from the bovine liver during aging

Electrophoresis methods used to study the fragments of chromatin revealed under the effect of Ca,Mg-dependent endonuclease on it have permitted establishing that stability of chromatin to the nucleosome level increases with aging. Changes in the compactness of the chromatin structure with aging make the accessibility of the linker DNA to nuclease lower the size of DNA fragments corresponding to mononucleosomes increasing from 192 +/- 5 pairs of bases to 209 +/- 5 pairs. Stabilization of the chromatin structure begins from certain nucleosomes which become more compact with the age. When analyzing basic proteins of chromatin by electrophoresis in different systems no qualitative changes were found in the subfraction composition of histones with aging, that permits supposing nonhistone proteins of chromatin and histone H1 to participate in the change of the chromatin structure compactness with the age.     

Gene-specific differences in the supranucleosomal organization of rat liver chromatin

Rat liver chromatin was separated into a solubilized portion and insoluble nuclear material, and the solubilized portion was fractionated by sucrose gradient sedimentation. The chromatin encompassing three transcribed genes (albumin, tryptophan oxygenase, and alpha-fetoprotein), which are expressed at very different levels, partitions preferentially with insoluble nuclear material and possesses a disrupted nucleosome structure. On the contrary, the chromatin encompassing three inactive genes fractionates into the solubilized chromatin portion and exhibits a canonical nucleosome repeat structure. By sucrose gradient sedimentation, all size classes of inactive chromatin particles are found to contain internal cleavages in the linker region between nucleosomes; they are probably held together by histone H1 and mono- and divalent cations. When the chromatin encompassing two flanking sequences of the tyrosine aminotransferase gene is studied, the 0.35-kilobase upstream-located chromatin exhibits features of active genes, while the 2.55-kilobase upstream-located chromatin partitions preferentially with solubilized chromatin and sediments in internally cleaved particles.     

Selective release of HMG nonhistone proteins during DNase digestion of Tetrahymena chromatin at different stages of the cell cycle.

The possible role of LG-1, a Tetrahymena specific HMG protein found in the macronuclear chromatin (Hamana, K. and Iwai, K. (1979) J. Biochem. 86, 789-794), was examined in relation to the chromatin structure. The chromatin isolated from cells synchronized at different stages of the cell cycle contained about one molecule of LG-1 per nucleosome. Limited digestion of the chromatin with DNase I or micrococcal nuclease selectively released LG-1 with the nucleosomal core histones and H1 remained insoluble, bound to the resistant DNA. Depending on the cell stages several types of chromatin structure were distinguished by their nuclease sensitivity. However, the chromatin at different stages exhibited the similar behavior of the LG-1 release with the nucleases as a function of the degree of chromatin solubilization. The results suggest that LG-1 proteins play a role in the chromatin organization which is rather independent of the cell stages.     

Organization of the newly replicated chromatin in the vicinity of the replication fork

Ehrlich ascites tumour cells were pulse-labelled with [³H]thymidine for 1 min or were treated with cycloheximide and labelled with [³H]thymidine for 45 min. The kinetics of digestion with micrococcal nuclease of both pulse-labelled and cycloheximide chromatins showed that they exhibited increased susceptibility towards the enzyme. At the same time their release from the nucleus was retarted and this was interpreted to mean that, unlike the bulk of chromatin, they were tightly bound to a fixed nuclear structure. When subjected to an equilibrium metrizamide-triethanolamine density gradient centrifugation both pulse-labelled and cycloheximide chromatins banded at higher density than control chromatin, which was an indication of their higher protein to DNA ratio. After a mild trypsinization, eliminating H1 and the nonhistone proteins, the pulse-labelled chromatin sedimented to the same density as control chromatin, and the cycloheximide chromatin sedimented to a density which was intermediate between those of control chromatin and free DNA. This result showed that the newly replicated chromatin had the same, and the cycloheximide chromatin half the amount of core histones present in control chromatin.     

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.     

Changes of nuclear structure induced by increasing temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.     

The arrangement of proteins on the deoxyribonucleic acid in chromatin

The arrangement of the protein component on the DNA of the chromatin complex was studied by comparing the rate of release of oligonucleotides and of protein after addition of deoxyribonuclease I and deoxyribonuclease II to rat thymus chromatin. Also the action of deoxyribonuclease I on normal chromatin and on chromatin depleted of non-histone protein was compared, to elucidate the role of the latter protein in chromatin structure. As a preliminary to the above, the rate of action of deoxyribonuclease I on DNA and on chromatin at the same DNA concentration, and the dependence of the action of this enzyme on the Mg(2+) concentration, were studied. It was found that: (1) little if any DNA in chromatin is present in extensive, truly ;free' zones, i.e. completely uncovered by protein; (2) at relatively low concentrations of added Mg(2+), deoxyribonuclease I degrades chromatin more rapidly than DNA; (3) the non-histone protein is not attached directly to the DNA in chromatin.