Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.     

The Size of DNA Molecules and Chromatin Organization in the Macronucleus of the Ciliate Didinium nasutum (Ciliophora)

Pulsed‐field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short “gene‐sized” DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80–265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.     

Three-dimensional structure of the ciliate Didinium nasutum nucleoli

The nucleolar organization in ciliate Didinium nasutum somatic interphase nuclei was studied using serial ultrathin sections and compared for various physiological states of the cell, namely, fed ciliates, starved ciliates, and dormant cysts. It has been shown that the interphase nucleoli are large structures with a complex architecture: the fibrillar component forms an intricate network in the macronucleus space, while the granular component is located inside this network. The structures looking as individual nucleoli in single sections are actually parts of branched nucleolar networks. The intricate nucleolar networks do not disintegrate after a 30-h starvation; however, the granular component becomes denser and develops numerous cavities filled with fine fibrils of a nonribonucleoprotein nature. In fed D. nasutum, the fibrillar structures on the periphery of nucleoli contain numerous pores (virtually absent in starved cell nucleoli), which can potentially serve for transporting newly synthesized rRNP. Branched nucleolar networks are undetectable in cysts. Their nucleoli are individual structures consisting mainly of the fibrogranular component.     

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.     

Structure of the interphase chromatin in the ciliata Bursaria truncatella macronucleus. II. Loop organization of inactive chromatin clumps

Electron microscopic study of chromatin organization in isolated macronuclei of a ciliate Bursaria truncatella showed macronuclear chromatin to be organized in compact clumps 120--180 nm in diameter linked with each other by one or several chromatin fibres. Macronucleus being dispersed in a solution of low ionic strength, radial loops basically of nucleosomal structure start appearing around chromatin clumps. Long-time dispersing of macronuclear chromatin brings complete decompactization of chromatin clumps into a set of nucleosome fibres. The way the fibres of interphase chromatin are packed in a chromatin clump is discussed.     

Structural organization of rat hepatocyte chromatin as visualized in thin frozen sections selectively stained for DNA

We studied the structure of rat hepatocyte chromatin in situ using thin frozen sections selectively stained for DNA after aldehyde fixation. Our results indicate that intranucleolar chromatin is arranged into three different organization levels, confirming the observations on Epon-embedded chromatin. These are: completely extended DNA filaments, with a thickness of approximately 3 nm, clustered in loose, roundish agglomerates, very long fibers with a thickness ranging from 15 to 35 nm and compact chromatin clumps. Both the fibers and the chromatin clumps frequently appeared to be composed of nucleosome-like particles. In the extranucleolar chromatin, agglomerates of extended DNA filaments and long fibers were never visualized. In contrast to data from Epon-embedded chromatin, we noticed that in frozen sections neither the nucleolar nor the extranucleolar compact chromatin appear to be organized into discrete, 20 to 30 nm superordered fibers.     

Trout sperm chromatin. II. Ultrastructural aspects after salt dissociation of proteins

The ultrastructural organization of the trout sperm nucleus was studied in ultrathin sections and spread preparations after partial decondensation of the nucleus with increasing NaCl concentrations. The obtained results suggest that the organization of the trout sperm chromatin is much more complex than a pure nucleoprotamine. Three types of complexes were observed. The first one results from the association of DNA with protamines. This complex appears as a fibrous network when partially decondensed nuclei are digested with DNase I indicating that at least a part of DNA remains protected by protamines and favours models accepting a colinear alignment of the latter on the DNA molecules. The second type of structures represent the DNA-protamine fibers compacted into dense clumps which appear as separate compaction units seen upon partial decondensation of the sperm nucleus. A third type are complexes of the ring-shaped granular bodies tightly associated with DNA and resisting high salt-urea and detergent treatment.     

Identification of 10 nm non-chromatin filaments in the macronucleus ofEuplotes eurystomus

Distinct in situ 10 nm non-chromatin fibers exist within the macronucleus of the ciliated protozoanEuplotes eurystomus. Their presence is detected after permeabilizing cells in a cytoskeleton-stabilizing buffer and then fixation with glutaraldehyde-tannic acid, followed by OsO₄. The 10 nm fibers are primarily localized within condensed chromatin and within the forward zone of the replication band. Although their functional role is unclear, it is suggested that they may constitute a structural framework for organization of the very large number (ca. 10⁸) of macronuclear minichromosomes.     

The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus

The formation of polytene chromosomes during macronuclear development of the ciliate Stylonychia mytilus was examined in spread electron microscopical preparations. The chromatin organization of early macronuclear anlagen closely resembles the organization of micronuclear chromatin. In the course of polytenization 300 A chromatin fibers become organized in loop-like structures laterally attached to a thinner axial fiber. It is suggested that this reorganization of chromatin during polytenization is a necessary event for the subsequent chromatin elimination.     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

Capturing native interactions: intrinsic methods to study chromatin conformation

The 3D organization of chromatin controls gene expression through spatial interactions between genomic loci. FISH and 3C‐based methods that are commonly used to study chromatin organization utilize chemical crosslinking, a step that may introduce biases in detectable chromatin interactions. In their recent study, Papantonis and colleagues (Brant et al, 2016 ) developed alternative new methods of detecting chromatin contacts without the use of chemical crosslinking agents. These tools increase the resolution and confidence at which interactions can be identified, and may be informative for chromatin interaction dynamics.     

Hierarchical Chromatin Structure of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> Revealed by Atomic Force Microscopy

Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear. Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S. pombe chromatin have not been clarified owing to its small nucleus. We used atomic force microscopy for nano-scale imaging of chromatin isolated from S. pombe. Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains. The average diameters of the domain and the nodule were 363 ± 85.2 nm and 46.2 ± 9.30 nm. Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level. The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S. pombe.     

Chromatin pattern of isolated human small thymocytes. A morphometric and stereologic study

Human small thymocytes, isolated by the unit gravity velocity sedimentation technic, were analyzed in ultrathin sections to define their chromatin pattern using morphometric and stereologic methods. Condensed chromatin in these cells represents about 70% of the nuclear volume and, is distributed in peripheral (75%) and central clumps (25%). The analysis of the distribution pattern of these clumps shows that peripheral clumps are distributed more irregularly than central clumps with respect to both size and relative position. Comparison of these results with those previously described for human peripheral blood T lymphocytes shows significant differences, probably related to the different maturation states of the two cell types.     

Visualization in situ of extended DNA filaments in nucleolar chromatin of rat hepatocytes

Using the osmium-ammine DNA tracer (Cogliati, R & Gautier, A, Compt rend acad sci ser D 276 (1973) 3041 [1]), we were able to study the structure of intranucleolar chromatin in thin sections of rat hepatocytes fixed in situ after partial hepatectomy and cortisol treatment. Three levels of chromatin organization were observed: (1) highly compact clumps; (2) fibres with a thickness varying from 11 to 25 nm; (3) loose agglomerates of extended DNA filaments with a thickness of 2–3 nm. Both the clumps and the fibres of chromatin showed nucleosomal configuration which was absent in the loose agglomerates. These latter structures appeared to be peculiar to nucleolar chromatin.     

A thermal denaturation study of macronuclear chromatin in Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida)

The macronuclear chromatin of the ciliate Blepharisma japonicum, in two starvation states, was studied by thermal denaturation analysis. The behaviour of B. japonicum chromatin, native and reconstituted with Tetrahymena pyriformis H1 histone, was analysed. The data obtained are consistent with the hypothesis that B. japonicum macronuclear chromatin contains a H1-like peptide associated with the linker DNA, although this peptide is reduced in amount and/or chromatin stabilising ability when compared to Tetrahymena macronuclear H1.