Formation of Chromoneme-like Fibrils Induced in Infusorian Somatic Nuclei by Magnesium Ions

A study was made of the effect of Mg²⁺ on higher-order chromatin structure in macronuclei (Ma) of infusoria Paramecium aurelia and Bursaria truncatella. In infusorian Ma, inactive chromatin is commonly packed in chromatin bodies sized 60–200 nm. When isolated chromatin or Ma were treated with Mg²⁺ (about 3 mM), chromatin bodies arranged into fibrils 100–300 nm in diameter, which resembled higher eukaryotic chromonemes. The dynamics of chromoneme-like fibril formation was described. The results testified to the similarity of chromatin bodies to chromomeres of higher eukaryotes. Structurally intact central chromomere cores proved to be essential for the formation of chromoneme-like fibrils. Chromatin organization in infusoria was shown to follow the discrete-level model (nucleosomes–nucleomeres–chromomeres–chromonemes) assumed for higher eukaryotes.     

Dynamics of structural changes of Paramecium caudatum and Bursaria truncatella macronuclear chromatin and nucleoli under hypotonic conditions

Dynamics of structural changes of nucleoli, complex nucleolar aggregates and chromatin bodies in macronuclei (Ma) of ciliates Paramecium candatum and Bursaria truncatella under hypotonic conditions was studied. It was shown that after a 3 min hypotonic treatment nuclei swelled and became highly vacuolated. 3D-reconstruction showed that such nucleoli were formed by nucleolonema-like threads about 100-200 nm in thickness. Intranucleolar chromatin bodies decompacted, but remained bound with fibrillar component of the nucleolus by thin fibres about 10 nm thick. After 6 min hypotonic treatment the nucleolar material loosened and had a "gauze", or network-like appearence. After 10 min hypotonic treatment nucleoli dissociated completely. It was shown that a transition of chromatin bodies from completely compact to partially and fully decompacted state occurred cooperatively in different regions of Ma. In particular, chromatin bodies in the central part of complex nucleolar aggregates decompacted much faster than those in the Ma karyoplasm. It evidences for a specific, well-ordered chromatin organization in Ma. Prolonged hypotonic treatment led to a complete dissociation of Ma components; fibres 6-10 nm thick were solely observed in such preparations. Such fibres may represent remnant structures of the nuclear matrix. Dynamics of Ma chromatin bodies decompaction correlates well with that of chromomeres in the nuclei of higher eukaryotes. Our data confirm that chromatin 100-200 nm bodies in the ciliate Ma are analogues of chromomeres--looped discrete chromatin domains, observed in the nuclei of higher eukaryotes.     

Nucleomeric organization of chromatin

Chromatin in the nuclei fixed in tissue and in the nuclei isolated by low ionic strength solutions in the presence of Mg2+ is represented by globular (nucleomeric) fibrils, 20-25 nm in diameter. The staphylococcal or endogenous nuclear nuclease splits the chromatin fibrils resulting in fragments corresponding to nucleomers and their multimers. Upon removal of firmly bound Mg2+ the nucleomers unfold to form chains consisting of 4-6-8 nucleosomes. Mild hydrolysis of nuclear chromatin by staphylococcal nuclease results in a split-off of mono-, di- and trimers of nucleomers sedimenting in a sucrose density gradient in the presence of EDTA as particles with the sedimentation coefficients of 37, 47 and 55S, respectively. The sedimentation coefficient for the mononucleomer in a sucrose density gradient with MgCl2 is 45S. Determination of the length of DNA fragments of chromatin split-off by staphylococcal nuclease showed that the nucleomer consists of 8 nucleosomes, while the dimer and trimer of the nucleomer consists of 14-16 and 21-24 nucleosomes, respectively. The nucleomeric monomer undergoes structural transition from the compact (45S) to the "loose" state (37S) after removal of Mg2+. This transition is completely reversible, when the nucleomer contains histone H1. The removal of the latter or dialysis of the nucleomer against EDTA in low ionic strength solutions results in a complete unfolding of the nucleomer into a nucleosomal chain fragment. A model for the nucleomer fibril structure in which the helical organization of the nucleosomal chain in the nucleomer (2 turns with 4 nucleosomes in each) is alternated with the impaired helical bonds between the nucleomers is discussed. The functional significance of the nucleomeric organization of chromatin may be an additional restriction of the site-specific recognition of DNA in chromatin with the possibility of local (at the level of one nucleomer) changes in chromatin conformation excluding this restriction.     

DNA-dependent RNA polymerase activity of isolated zooflagellates (Crithidia oncopelti) nucleoli

A fraction of nucleoli is isolated from zooflagellates (Crithidia oncopelti) nuclei, its DNA-dependent RNA polymerase activity is studied at different temperature, ionic strength and Mg2+, Mn2+ and antibiotic concentrations. The effect of some factors and alpha-amantine on RNA polymerase activity of exonucleolar chromatin was studied as a control. A comparison of heat denaturation of nucleoli and chromatin RNA polymerase activities within the temperature range 30--55 degrees C has revealed a higher thermosensitivity of nucleoli RNA polymerase. Substitution of Mg2+ with equivalent amount of Mn2+ results in a considerable decrease of rRNA synthesis in nucleoli. Nucleoli RNA polymerase activity in the presence of Mg2+ is sensitive to the elevation of ionic strength from 0.12 to 1.30 u; chromatin RNA polymerase activity in the presence of Mn2+ is maximal at high ionic strength (1.30 mu). alpha-Amantine and cycloheximide at high concentrations (10 and 200 mkg/ml) practically do not affect RNA polymerase activity of nucleoli. Nucleoli RNA polymerase of zooflagellates (Crithidia oncopelti) is similar to the A-form of the enzyme in higher eukaryotes.     

Condensation of the chromatin gel by cations

Calf thymus chromatin gel, containing strongly bound nonhistone proteins, was used to study the effect of easily removable and tightly bound cations on the condensation of chromatin. The chromatin volume was found to be linearly dependent on the reciprocal square root of the concentration of easily removable cations (Tris X H+ + Na+ and Mg2+) except for the initial stages of condensation (up to 7-10 mM monovalent and 0.15-0.2 mM divalent cations). The effect of Mg2+ at the initial stage of condensation was not reproduced by Na+ and vice versa. At higher concentrations the effects of Na+ and Mg2+ were additive. The removal of tightly bound divalent cations by a treatment of the chromatin gel with 1,10-phenanthroline led to an approx. 50% increase in the volume of the chromatin gel, which was maintained at each concentration of easily removable cations. The 1,10-phenanthroline-caused decondensation of the chromatin gel was reversed by Ca2+ but not by Mg2+, Zn2+ and Cu2+. The chromatin gel pretreated with Ca2+ was not further decondensed by 1,10-phenanthroline.     

Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy.

The higher-order assembly of the approximately 30 nm chromatin fibers into the characteristic morphology of HeLa mitotic chromosomes was investigated by electron microscopy. Transmission electron microscopy (TEM) of serial sections was applied to view the distribution of the DNA-histone-nonhistone fibers through the chromatid arms. Scanning electron microscopy (SEM) provided a complementary technique allowing the surface arrangement of the fibers to be observed. The approach with both procedures was to swell the chromosomes slightly, without extracting proteins, so that the densely-packed chromatin fibers were separated. The degree of expansion of the chromosomes was controlled by adjusting the concentration of divalent cations (Mg2+). With TEM, individual fibers could be resolved by decreasing the Mg2+ concentration to 1.0-1.5 mM. The predominant mode of fiber organization was seen to be radial for both longitudinal and transverse sections. Using SEM, surface protuberances with an average diameter of 69 nm became visible after the Mg2+ concentration was reduced to 1.5 mM. The knobby surface appearance was a variable feature, because the average diameter decreased when the divalent cation concentration was further reduced. The surface projections appear to represent the peripheral tips of radial chromatin loops. These TEM and SEM observations support a "radial loop" model for the organization of the chromatin fibers in metaphase chromosomes.     

The condensing action of Mg2+, hexamminecobalt3+ and spermidine3+ on chromatin with gene regions activated by 17-beta estradiol.

1. Hepatic vitellogenin synthesis was induced by injecting 17-beta estradiol into fish. Liver nuclei were incubated with the endonuclease EcoRI at an increasing concentration of Mg2+ (0.15-1.50 mM), hexamminecobalt3+ or spermidine3+ (0.10-1.00 mM). Chromatin was separated into a 5000 g supernatant, S-fraction, and pellet fraction. 2. The release of chromatin into the S-fraction was higher for the induced than the control chromatin. Hybridization of the vitellogenin gene retained in the pellet fraction of the controls was unaffected by the individual cations. After activation of the vitellogenin gene, Mg2+ at its lowest concentration retained a high amount of the vitellogenin gene in the pellet fraction. The level of hybridization decreased by increasing the Mg2+ concentration. Retention of the gene rose by adding hexamminecobalt3+ and more so by adding spermidine3+. 3. The condensing action of spermidine3+ was extended to the activated vitellogenin gene regions of chromatin.     

Magnesium binding and conformational change of DNA in chromatin

The structure of chromatin in the presence of Mg2+ ions was examined by circular dichroism and equilibrium dialysis. Circular dichroism (CD) shows that above 260 nm the intensity of the spectrum of DNA in nucleoproteins decreases as the Mg2+ concentration increases. This change is an intrinsic characteristic of DNA since it is also observed in protein-free DNA and has been attributed to a change in the winding angle of base pairs around the DNA axis. Some structural elements of the DNA in the nucleosome core, therefore, are as movable as those of protein-free DNA. The basic organization of H1-depleted chromatin, 146 base pairs (bp) of DNA wound around core histones and a residual 49 bp in the linker region in the repeating unit, is maintained both in the presence and in the absence of Mg2+ ions, as shown by the fact that the CD spectrum of H1-depleted chromatin has the same type of linear combination between the spectrum of protein-free DNA and that of the nucleosome core in 0.2 mM MgCl2-10 mM triethanolamine (pH 7.8) as it has in 1 mM ethylenediaminetetraacetic acid-10 mM tris(hydroxymethyl) aminomethane (pH 7.8). The ellipticity of chromatin shows a smaller decrease relative to the other nucleoproteins and protein-free DNA upon the addition of Mg2+ ions. Therefore, some structural elements of chromatin are apparently somewhat protected against the conformational change induced by these ions. The spectrum of chromatin becomes almost indistinguishable from that of H1-depleted chromatin in 0.2 mM MgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exchange of H1 histone depends on aggregation of chromatin, not simply on ionic strength

Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.     

The ultrastructure and possible role of the vacuolar component of hepatocyte nucleoli

A study was made of the nucleolar vacuoles in guinea pig hepatocytes that are poorly investigated for animal cells. A comparison of ultrathin sections, contrasted by heavy metals, with those treated according to Bernhard allowed to reveal the following intravacuolar structures: 1) fibrils 10-15 nm and 20-30 nm thick similar to perinucleolar chromatin fibrils; 2) RNP-granules 15-20 nm in diameter resembling the granular component of the nucleolus; 3) RNP-fibrils 15-20 nm thick with high electron density. The latter were visualized for the first time, their function still remains obscure. Upon stimulation of hepatocytes with partial hepatectomy, the vacuolar component changed. In 2.5 and 5 hours after the operation the vacuoles became smaller, the number of RNP structures of two types increased. Further, in 9 hours, the enlarging of vacuoles was accompanied by a sharp decrease in the number of these RNP-structures. The results obtained allow to suppose that the vacuoles of nucleolonemic nucleoli may be functioning elements, linking intra- and perinucleolar chromatin fibrils. They are depots for the RNP synthesized in the nucleolus; the rRNP is transported through the vacuolar system.     

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Linear dichroism of chromatin fibers

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Friend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations by the use of flow linear dichroism and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA--2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA--for chromatins with linker DNA 10-30 b.p. it is partially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric fields do not have an effect on chromatin structure, while higher electric fields (more than 7 kV/cm) distort the structure of chromatin.     

毛竹茎秆纤维细胞发育过程中的细胞程序性死亡

利用TUNEL检测、细胞学及细胞化学方法,对毛竹茎秆纤维细胞发育过程中的细胞程序性死亡进行了研究。在次生壁形成的早期,纤维细胞出现染色质凝聚、细胞器膨胀、液泡膜解体和细胞质泡状化等典型的细胞程序性死亡形态学特征;TUNEL检测反应呈阳性,显示此时的纤维细胞核DNA发生了片段化。此时,在纤维细胞裂解的液泡膜、降解的细胞质和凝聚的染色质上具有ATPase活性。纤维细胞质的Ca^2＋水平会随着次生壁的形成而逐渐升高,随后Ca^2＋聚集成块状。在初生壁形成后期,纤维细胞染色质上的酸性磷酸酶（APase）活性增强。随着纤维次生壁的持续增厚,ATPase、酸性磷酸酶和Ca^2＋将在裂解的细胞质和凝聚的染色质上持续存在多年。结果表明,毛竹茎秆纤维细胞的次生壁形成过程是一个主动自溶的细胞程序性死亡过程。初生壁形成后期染色质上酸性磷酸酶活性增强及次生壁形成期胞质Ca^2＋的聚集,与纤维细胞的程序性死亡密切相关。ATPase,Ca^2＋和APase参与了纤维细胞程序性死亡过程中原生质体的降解。     

Mn2+-dependent endonuclease activity of chromatin

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.     

In vitro hexagonal assembly of R-form lipopolysaccharides: effect of pH on the Mg+2-mediated hexagonal assembly

The R-form lipopolysaccharide (LPS) from Escherichia coli K-12, from which cationic material had been removed by electrodialysis and the pH of which had fallen to 3.6, formed a rough hexagonal lattice structure with the lattice constant of about 19 nm. The rough hexagonal structure was maintained in buffers at pH 5 or lower but disintegrated into the ribbon-like structures in buffers at pH 6 or higher. However, in the presence of 10 mM Mg2+, the hexagonal lattice structure was not disintegrated even at alkaline pH levels but conversely it became more dense. At pH 8.3 to 8.9, the hexagonal lattice structure with the shortest lattice constant (15 nm) was formed. The same optimal pH levels were obtained for formation of the dense hexagonal lattice structure (lattice constant, 14 to 15 nm) by the electrodialyzed LPS from Klebsiella pneumoniae strain LEN-111 (O3-:K1-). The ability of Mg2+ to induce formation of the dense hexagonal lattice structure of the K-12 LPS depends upon the presence of buffers showing the optimal pH levels, since a very high concentration of Mg2+ such as 500 mM was required for the lattice formation in distilled water. The amount of the magnesium bound to the K-12 LPS did not significantly differ throughout the pH range of 3 to 9. Therefore, the optimal pH range is another essential factor for formation of the dense hexagonal lattice structure of the LPS in addition to binding of the magnesium to the LPS.     

Detection of endonuclease activity and several features of chromatin autolysis in brain cell nuclei]

The presence of Ca2+, Mg2+-dependent endonuclease activity in isolated brain cell nuclei was demonstrated and a comparison of some peculiarities of chromatin autolysis in rat brain and liver cell nuclei was carried out. Endogenous brain nuclease hydrolyzes chromatin into its structural subunits; its specific activity is 10,5 times as low as compared to the endogenous nuclease activity in rat liver nuclei. The dependency of the chromatin autolysis rate on pH and ionic composition of the incubation medium in isolated rate brain and liver nuclei appeared to be the same. The presence of Mn2+ changed the autolysis nature both in brain and in liver cell nuclei, the relative (as compared to Mg2+-dependent) Mn2+-dependent activity being higher in the brain cell nuclei. Possible differences of brain and liver chromatin structure (e. g. the presence of regions free of nucleosomic organization in brain chromatin) are assumed.     

Sequence composition of the template-active fraction of rat liver chromatin.

Rat liver chromatin has been separated into nuclease-sensitive and -resistant fractions after mild digestion with DNAase II. The nuclease-sensitive material is further fractionated into Mg2+ -soluble and -insoluble chromatin fractions. The kinetics of production of these chromatin fractions have been investigated. After a brief enzyme treatment (5 min at 10 enzyme units/A260 unit of chromatin at pH 6.6), 11% of the input chromatin DNA is found in the Mg2+ -soluble fraction. This DNA has a weight-average single-strand length of about 400 nucleotides and, as determined by renaturation kinetics, comprises a subset of nonrepetitive DNA sequences and a subset of families of middle repetitive sequences. This demonstrates the nonrandom distribution of repetitive and single copy sequences in the Mg2+ -soluble fraction of chromatin. Previous studies have shown that the Mg2+ -soluble fraction is enriched in nonrepeated sequences which are transcribed in vivo (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F., and Bonner, J. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). We now report that the Mg2+ -soluble fraction of liver chromatin contains a low proportion of sequences in common with the Mg2+ -soluble fraction of brain chromatin. Thus, fractionation does not depend on some general property of chromatin but is specific with regard to the template activity of the tissue from which the chromatin was obtained.     

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.