Evidence of repetitive patterns of chromatin distribution in cell nuclei of rat liver

Samples of rat livers were fixed in glutaraldehyde, contrasted en bloc with phosphotungstic acid, embedded in an epoxy resin and serially sectioned. The study of three-dimensional models of 20 complete nuclei shows that all of them share some general features: they have more than one nucleolus (2-4), an irregular layer of compact chromatin adjacent to the nuclear membrane and well-delimited clumps of chromatin both in the nuclear sap and surrounding the nucleoli. A space of 8 sections containing the central nucleolus and a lateral one was studied in detail. In this space, 8 clumps of compact chromatin were found in 17 nuclei and 9 clumps in the other 3 nuclei. No other number of clumps was found in those zones. In all the nuclei studied the compact chromatin surrounding the central nucleolus contacts the nuclear envelope. This contact takes place in a region almost diametrically opposed to the lateral nucleolus in 13 nuclei. In 7 nuclei, these structures were at angles between 50 and 125 degrees. These results support the existence of nonrandom repetitive patterns of chromatin distribution in liver cells.     

Electron microscopic morphometric analysis of human T and B peripheral blood lymphocytes

Using the system of morphometric analysis described in this paper, human peripheral blood T and B lymphocytes, labeled with specific surface markers, can be compared on different analytical levels. They show differences in their surface and the eccentricity of cells, in the relative surfaces occupied by peripheral and central condensed chromatin, in the average surface of the central chromatin clumps and in the number of perichromatin granules per nuclear surface. The morphometric analysis reveals the importance of examining the nuclear and the surface parameters in the characterization of lymphocytes, confirming that a detailed analysis of the nuclear characteristics can contribute to the identification of T and B lymphocytes by transmission electron microscopy.     

牛肾细胞染色体中染色质纤维的包装及RNP的分布

应用培养的牛肾（ＢＫ）细胞及分离的ＢＫ细胞染色体做常规透射电镜样品,经表面舒展技术和临界点干燥制备ＢＫ细胞染色体的扫描电镜样品,并结合电镜细胞化学研究了ＢＫ染色质纤维的包装及核糖核蛋白（ＲＮＰ）的分布。观察到ＢＫ染色体具有多级双股螺旋结构。在染色体横切面中,可见染色体中央有一低电子密度的无染色质区,该区内有大量ＲＮＰ物质。在染色质区ＲＮＰ较少,分布在染色质纤维间,与中央轴区的ＲＮＰ相连续,表明ＲＮＰ在染色体中呈不均匀分布。     

Chromatin structure in somatic nucleus of ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis>

The structural organization of macronuclear chromatin of the ciliate Didinium nasutum was studied. The macronuclear genome of D. nasutum is represented by DNA molecules of subchromosomal size. At interphase, macronuclear chromatin is organized into chromatin of 100–200-nm clumps. Some of these clumps form short, thick fibers that consist of several chromatin clumps. Using the differential staining of nucleic acids on ultrathin sections, we revealed perichromatin fibers and granules on the surface of many chromatin clumps. A 3D model of the spatial distribution of chromatin clumps in the macronucleus was built based on serial ultrathin sections and peculiar features of chromatin spatial organization were studied.     

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.     

Cytology of lepidoptera. VI. Immunolocalization of microtubules in detergent-extracted apyrene spermatocytes of Ephestia kuehniella Z

Apyrene meiosis was studied in two wild-type strains, L and Sbr, of the Mediterranean mealmoth, Ephestia kuehniella, using anti-tubulin immunofluorescence. The observations were supplemented by phase-contrast light microscopy of living spermatocytes from strain L. The study revealed that nuclear envelope breakdown, centrosome separation, migration of chromatin elements towards the poles, and spindle elongation also occur in apyrene spermatocytes. However, a conventional metaphase plate is never formed, and chromatin segregation is irregular and delayed. Chromosome laggards are frequent. As a rule, apyrene spindles have a low microtubule content. The two strains, L and Sbr, differ regarding the chromatin behavior during meiosis. In strain L, the developing spindles contain numerous small chromatin clumps which segregate asynchronously. The resulting daughter cells possess about the same amount of chromatin. In contrast, large chromatin clumps exist in strain Sbr at the onset of spindle formation. The chromatin blocks transiently occupy an equatorial position and elongate subsequently parallel to the spindle axis. These elongated chromatin bodies often divide highly unequally. As a consequence, secondary spermatocytes in strain Sbr differ greatly in chromatin content. Subjective assessment shows that the size of the microtubular cytoskeleton is positively correlated with the chromatin content of the cell. Hence, it is hypothesized that the chromatin content determines spindle size. This possibly comes about the number of available kinetochores which are exposed and able to stabilize microtubules of centrosomal origin attached with the kinetochores. However, a direct bearing of chromatin on spindle size is similarly conceivable. Other Lepidoptera species examined so far are compatible with a 'type L' or a 'type Sbr' pattern of apyrene meiosis.     

Chromatin organization during mouse oocyte growth

We investigated the changes in the organization of oocyte nuclear chromatin and nucleolar-associated chromatin throughout folliculogenesis. Zona-free oocytes were isolated from ovaries, grouped into seven classes according to size and chromatin organization, and analyzed after staining with Hoechst 33342. We show that oocyte differentiation from the dictyate stage to the conclusion of maturation is associated with either of two chromatin configurations. Initially, all oocytes are in the NSN configuration (nonsurrounded nucleolus oocytes; characterized by a Hoechst positive-chromatin pattern of small clumps forming a network on the nuclear surface, with a nucleolus nonsurrounded by chromatin). While growing, some of these NSN oocytes continue their development in the NSN configuration, whereas others shift (from class IV on) into the SN configuration (surrounded nucleolus oocytes; characterized by a threadlike chromatin organization that may partially surround the nucleolus or project towards the nuclear periphery). The percentage of SN oocytes increases both with increasing size of the oocyte (class I–III, 10–40 μm in diameter: 100% NSN vs. 0% SN; class VII 70–80 μm in diameter: 47.3% NSN vs. 52.3 SN, in 4–6-week-old females), and with aging (class VII: 94.1% NSN vs. 5.9% SN in 2-week-old females; 11.8% NSN vs. 8.2% SN in 56-week-old females). Further, we suggest as a working hypothesis that those oocytes that switch to the SN chromatin organization early in maturation may not be ovulated, even though this particular chromatin structure normally occurs just prior to ovulation. © 1995 Wiley-Liss, Inc.     

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.     

Discrimination of osteoarthritic and rheumatoid human synovial cells in culture by nuclear image analysis.

Rheumatoid arthritic (RA) and osteoarthritic (OA) synovial cells in culture differ in their metabolic and proliferative behaviour. To assess links between these properties and nuclear changes, we used image analysis to study chromatin texture, together with nuclear morphometry and densitometry of OA and RA cells in primary culture. Chromatin pattern at the third day (D3) was heterogeneous and granular with chromatin clumps whereas at the final stage (D11) of culture a homogeneous and finely granular chromatin texture was observed. This evolution indicates global chromatin decondensation. These characteristics were more marked for RA than for OA nuclei. At each culture time, RA nuclei could be discriminated with high confidence from OA ones from parameters evaluating the organization of the chromatine texture. Nuclear image analysis is thus a useful tool for investigating synovial cell biology.     

Distinct nuclear organization, DNA methylation pattern and cytokinin distribution mark juvenile, juvenile-like and adult vegetative apical meristems in peach (Prunus persica (L.) Batsch)

Chromatin organization, nuclear DNA methylation and endogenous zeatin localization were investigated in shoot apical meristems (SAM) during juvenile and adult phases of peach (Prunus persica (L.) Batsch). The aim was to examine the extent to which these parameters could discriminate the juvenile and adult SAMs. Seedlings (juvenile, cannot flower), basal shoots (called juvenile-like, because they exhibit juvenile macroscopic traits) and apical shoots (competent to form flowers) of adult plants were chosen. Nuclear chromatin exhibited chromocentres that were peripherally distributed in SAMs of juvenile and juvenile-like shoots, but were diffusely spread in those of adult shoots. These patterns coincided with a peripheral labelling of DNA methylation in juvenile and juvenile-like meristem nuclei versus a diffuse labelling pattern in adult meristem nuclei. During vegetative growth (from March to June), the level of nuclear DNA methylation was higher in adult meristems than in juvenile and juvenile-like ones. The immunolocalization of zeatin in juvenile SAM was in the subapical region, but adult meristems exhibited a widespread localization or a signal confined within the boundaries of the central zone. The extent to which the acquisition of a strongly zonated pattern of these parameters as markers of floral competence in adult SAMs is discussed.     

The effect of caspase-inhibitors on radiation induced apoptosis in human peripheral blood lymphocytes: an electron microscopic approach

Resting lymphocytes are sensitive to radiation damage and die by apoptosis. We investigated the effect of caspase-inhibitors on radiation induced apoptosis in human peripheral blood lymphocytes. Lymphocytes were irradiated in vitro with 5 Gy ⁶⁰ Co--rays and cultured for 24 hours in the presence or absence of the caspase-inhibitors zVAD-fmk and zDEVD-fmk. Cell death was evaluated by electron microscopy. Irradiation in the absence of the inhibitors resulted in about 30% dead cells, almost all showing typical apoptotic morphologies. Addition of either one of the inhibitors could not rescue cells from death. Part of the dead lymphocytes (about 65%) still showed typical nuclear characteristics of apoptotic cells: sharply marginated, condensed chromatin, clumped into one sphere or into a crescent shaped mass. The remaining part of the dead cells had ultrastructural characteristics, aberrant from apoptic cells: clumping of the chromatin was less pronounced and less sharply marginated. Irregular clumps were formed. Data indicate that part of the lymphocytes go in apoptosis in a caspase-independent way. The other part shows caspase-dependent apoptosis with respect to the nuclear events.     

SPATIAL DISTRIBUTION OF GENETIC INDIVIDUALS IN THICKETS OF ALNUS INCANA SSP. RUGOSA,A CLONAL SHRUB

Thickets of speckled alder (Alnus incana ssp. rugosa (Du Roi) Clausen) consist of numerous discrete clumps of stems. Presumably all stems in a single clump are part of a single genetic individual, but a genet could comprise more than one clump. Starch-gel electrophoresis was used to identify genetic individuals in four alder populations in central New York. A single genetic marker, a tetramer with three alleles, could discriminate five genotypes. Nearest neighbor analysis revealed that genotypes were distributed randomly. That is, the pattern of genotypes was statistically indistinguishable from a model where each clump is considered a unique individual and where clump genotypes are randomly distributed. Calculation of Morisita's index of dispersion confirmed that clumps of a single genotype were not aggregated. Although alder is capable of forming root suckers and offsets, lateral expansion of genets is apparently ineffective. Apparently, spatial distribution of genetic individuals within alder thickets is not influenced by clonal growth or by other factors acting to cause patterns in the genetic structure of plant populations.     

A comparative study of chromatin from lymphocyte nuclei upon activation of transcription by irradiation from an He-Ne-laser or phytohemagglutinin]

The influence of He-Ne laser radiation (632.8 nm, 56 J/m2, t = 10 s) and phytohaemagglutinin (PHA, 2 micrograms/ml) on chromatin structure in human lymphocytes was studied by electron microscopy using ultrathin cell sections. Morphometric analysis of extranuclear condensed chromatin masses was performed 1 h after the irradiation or after the beginning of PHA treatment. In the irradiated cells the following insignificant changes were revealed: decrease in the relative area of the nucleoplasmic chromatin, increase in the relative area of decondensation zones as well as increase in the number of clumps of nucleoplasmic chromatin and relative length at their boundary with nucleoplasma. The tendency of these morphological changes may be interpreted as functional activation of extranucleolar RNA synthesis in response to irradiation by red laser light. Action of PHA results in significant changes of the surfaces of chromatin clumps, namely increase in relative length of nucleoplasmic chromatin boundary and decrease in relative length of perimembranous chromatin boundary with nucleoplasma as well as some less expressed delamination of the chromatin masses from the nuclear membrane. These essential changes may reflect chromatin activation by proliferative stimulus. Peculiarities of the ultrastructural reorganisation in the condensed chromatin after irradiation and PHA-treatment probably reflect the differences in the processes of gene activation caused by the two agents.     

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.     

The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal.III Structural heterogeneity of chromatin and argyrophilic zone of the nucleolus in stretched membrane-free nuclei and chromatin bodies from human peripheral lymphocytes

Viewed by light microscopy, the majority of lymphocytes in smears of human peripheral blood display a deep staining (with any chromatin- or DNA-specific dye) of the nucleus consisting of densely aggregated chromatin in addition to one or several small nucleoli with a dot- or spot-like argyrophilic zone. Amembraneous nuclei and "free chromatin" structures were isolated from intact lymphocytes gently treated with Triton X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol--glacial acetic acid (3:1), resulted in spatial separation of thin and thick chromatin or argyrophilic fibres, nucleoli, intranuclear bodies, polymorphous aggregations of chromatin or argyrophilic fibres and incidentally observed splitted or beaded thick chromatin fibres and the chromocenter. The light microscopic pattern of chromatin fibres of stretched amembraneous nuclei, isolated from peripheral lymphocytes, well compares with that of deconvolved images of intact lymphocyte nucleus obtained with optical tomography.     

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.