THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

Structure and mode of formation of the nucleolus in meristematic cells of Vicia faba and Allium cepa

Interphase nucleoli from Vicia faba and Allium cepa meristematic cells are roughly classified into two categories: (a) those that commonly show a rather homogeneous texture (except for small light spaces of various sizes) and frequently contain dense particles 140 A in diameter; (b) those found more frequently in Vicia characterized by a very sharp boundary between a dense outer cortex and a much lighter central core. The dense particles are not found in such nucleoli. In Allium the boundary is more irregular and dense particles are sometimes observed in the outer layer. Many nucleoli show a structure intermediate between these two types. They are characterized by a gradient of increasing density from the center to the periphery and occasionally contain dense 140 A granules. During interphase, certain nucleoli are closely associated with segments of chromatin strands which undoubtedly represent nucleolar organizing regions. The dense 140 A granules are followed during the mitotic cycle. In Allium, they are first seen in loose clusters between arms of late anaphase chromosomes where they become more concentrated in early telophase. The substance within which they are scattered slowly increases in density during that time until finally, the particles are limited to small bodies of distinctive character. Evidence is presented suggesting that these small prenucleolar bodies fuse during telophase to give rise to the mature interphase nucleoli. Similar events are described in Vicia material except that a coating of dense substance appears around telophase chromosomes before the formation of prenucleolar bodies.     

A CORRELATED LIGHT AND ELECTRON MICROSCOPE STUDY OF THE NUCLEOLAR MATERIAL DURING MITOSIS IN VICIA FABA

Root meristematic cells of Vicia faba were examined, with both light and electron microscopes, in order to study the behaviour of the nucleolar material during the mitotic process. Under light microscopy, the preprophase nucleolus is seen to consist of a densely stained material in which are embedded several unstained vacuole-like structures of varying size. The electron microscope reveals that the dense nucleolar material is formed of two structurally distinct components, each segregated into irregularly shaped zones blending with one another. One of these components is represented by 150 A granules which, in places, are arranged into thread-like structures approximately 0.1 µ in diameter; the other component apparently consists of fibrils 60 to 100 A in diameter. The large and medium sized intranucleolar vacuoles contain loosely scattered granules and fibrils similar to those just described. The granular and fibrillar components of the denser portion of the nucleolus persist as such during prophase and disperse throughout the nuclear cavity at the time of nucleolar disintegration. After nuclear membrane breakdown, these granules and fibrils, as well as those of the nucleoplasm, mix freely with similar elements already present within the forming spindle. No evidence has been obtained that, during or after nucleolar disintegration, the structural components of the nucleolus become associated as such with the chromosomes to form an external or internal matrix. Our observations suggest the existence, of a matrix substance within late prophase, metaphase, and anaphase chromosomes, the fine structure of which bears strong resemblance to that of their constituent coiled chromonemata. Data are presented, moreover, that indicate that part of this matrix substance, presumably formed at some time during prophase, is released from the chromosomes during their anaphasic movement. A number of observations indicate that the main bulk of the next nucleolus is derived from a prenucleolar fibrillogranular material, arranged into thread-like structures some 0.1 µ in diameter, which collect in the interchromosomal spaces during early and midtelophase. Finally, our data would seem to favour the view that most of this prenucleolar material results from a resumption of the synthetic activity of the early and midtelophase chromosomes rather than from a mere shedding of a preexisting matrix substance.     

Nucleolus-like bodies in micronuclei of cultured Xenopus cells

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [³H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.     

A STUDY OF NUCLEOLAR VACUOLES IN CULTURED TOBACCO CELLS USING RADIOAUTOGRAPHY, ACTINOMYCIN D, AND ELECTRON MICROSCOPY

Previously it has been found that in tobacco callus cells nucleolar vacuoles repeatedly form and contract. In this study, nucleolar vacuoles were investigated by using radioautography, actinomycin D, and electron microscopy. It was found, from grain counts of nucleoli labeled with uridine-³H, that nucleoli containing vacuoles had more than three times as many grains/µ² of nucleolar substance as did nucleolei without vacuoles. Treatment of tobacco callus cells with various concentrations of actinomycin D caused the percentage of cells containing nucleolar vacuoles to decrease; with the highest concentration the percentage of these cells dropped from the normal level of about 70% to less than 10%. However, after removal of actinomycin D the cells regained nucleolar vacuoles up to the control level. When radioautography was used with actinomycin D, it was found that the actinomycin D inhibited the uptake of uridine-³H, i.e. inhibited RNA synthesis, in those nucleoli which lost their nucleolar vacuoles. In addition, after removal of the cells from actinomycin D, it was found that as the cells regained nucleolar vacuoles the nucleoli also began to incorporate uridine-³H. Electron micrographs showed the nucleoli to be composed of a compact, finely fibrous central portion surrounded by a layer of dense particles 100–150 A in diameter. Nucleolar vacuoles occurred in the fibrous central portion. Dense particles similar to those in the outer layer of the nucleoli were found scattered throughout the vacuoles and in a dense layer at their outer edge. These data suggest that in cultured tobacco callus cells the formation and contraction of nucleolar vacuoles is closely related to RNA synthesis in the nucleolus.     

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

ULTRASTRUCTURE AND CYTOCHEMISTRY OF METABOLIC DNA IN TIPULA

A DNA body is present in the females of the fly Tipula oleracea and is formed in contact with the sex chromosomes in the oogonial interphases. At each oogonial mitosis, the DNA body follows the chromosomes to one anaphase group and is included in one of the telophase nuclei. The body increases appreciably in size during the interphase of meiosis. All oocytes have the body, but only a few nurse cells possess it. The DNA body synthesizes its DNA at a different time than the chromosomes, as is shown by incorporation of tritiated thymidine, and contains 59% of the DNA of the nucleus, as is disclosed by spectrophotometric measurements. At late diplotene the DNA body disintegrates, releasing its DNA into either the nucleus or the cytoplasm. When studied in the electron microscope, the DNA body appears composed of a tight mass of intertwined fibrils. Demonstration that the main mass of the body is composed of DNA is obtained from cytochemical tests which reveal that the DNA body is Feulgen positive, stains green with azure B, incorporates H³-thymidine, and after digestion with DNase is Feulgen negative. The DNA of the body is complexed with histone, like the DNA of the chromosomes, as is revealed by an intense alkaline fast green staining. Electron microscope examination of oocytes reveals that one side of the DNA body is in close contact with the nuclear envelope and that the other side possesses an outer shell composed mainly of particles 150 to 250 A in diameter. Between the outer shell and the chromosomes there is a band of low electron opacity, 4000 to 7000 A thick. In the light microscope, this light band together with the outer shell is Feulgen negative and stains violet with azure B; this is confirmation of the presence of RNA. In the oocytes the nucleoli are found inside the DNA body. These nucleoli have a nucleolonema composed mainly of particles 150 to 250 A. The nucleoli are Feulgen negative, alkaline fast green negative, stain violet with azure B, and do not stain with azure B after RNase digestion, thus confirming their RNA content. The presence of the nucleoli inside the DNA body and of a band of RNA between the body and the chromosomes is indicative of a high RNA synthetic activity. Since the DNA of the body is complexed with histone, as in the chromosomes, and the nucleoli are located inside the body, the simplest interpretation of the DNA body is that it represents hundreds of copies of the operons of the nucleolar organizing region or neighboring regions. The situation found in Tipula has several basic features in common with the polytene chromosomes of other Diptera and with the hundreds of nucleoli present in Triturus oocytes. In all three cases, genes seem to be copied hundreds of times but are kept in different types of packages. A DNA body like the one in Tipula oleracea is found in other species of Diptera and in the Coleoptera. There is no indication, from the present investigation, that the DNA body is in any way associated with a virus.     

Nucleolus-like morphology produced during the in vitro reassociation of nucleolar components

Nucleoli, the sites of rRNA synthesis, rRNA processing, and the assembly of ribosomes, are dynamic organelles that, in most cells, disperse and reform during mitosis. The mechanisms that regulate nucleolar formation are unknown as is the relationship between nucleolar morphology and the pathway of ribosome biogenesis. In this report we describe the in vitro formation of nucleolus-like particles (NLPs) from soluble extracts of nucleoli. NLPs, which reached sizes comparable to nucleoli (1-3 microns), were found to contain 40% of the nucleolar DNA, RNA, and protein. The ultrastructure of NLPs resembled that of a number of in vivo structures including compact nucleoli, prenucleolar bodies, and pseudonucleoli. The particles were composed of two morphologically distinct regions. The core resembled the dense fibrillar component (DFC) of nucleoli while the cortex resembled the granular component (GC) of nucleoli. The cortex of NLPs contained numerous 15-20 nm osmophilic granules that resembled the preribosomes found in the GC of nucleoli. The distribution of nucleolar proteins in NLPs also resembled that in nucleoli. BN46/51, a component of the GC of nucleoli, was restricted to the GC-like cortex of NLPs. A mAb that bound to the DFC of nucleoli, bound only to the DFC-like core of NLPs while a second mAb that bound to both the DFC and GC of nucleoli, bound to both the core and cortex of NLPs. Thus solubilized components of nucleoli can reassociate in vitro to produce particles that resemble nucleoli in their size, ultrastructure, and protein distribution.     

CHANGES IN NUCLEOLI AT MATURATION OF NEWT OOCYTE

Changes in the nucleoli of maturing oocytes and the eggs of Cynops pyrrhogaster were studied with light and electron microscopy. Extrachromosomal nucleoli moved toward the center of the germinal vesicle in response to the maturation stimulus, released presumed ribosomal ribonucleoprotein, and further moved toward the center of the nucleus to form an aggregate with chromosomes which behaved in a similar manner. A few. ball-shaped nucleolar masses were formed from this aggregate, leaving the chromosomes and probably the extrachromosomal nucleolar organizer. The chromosomes then proceeded to the first meiotic metaphase. The nucleolar masses were surrounded by a layer of mitochondria and became smaller with formation of pinched-off fragments, which were also surrounded by mitochondria, during the time the egg was moving down the oviduct. Only fragments were observed in the subcortical area of the animal hemisphere of the egg after reaching the lowest part of the oviduct.     

The relationship of ribosomal RNA synthesis to the formation of segregated nucleoli and nucleolus-like bodies

The relationship of ribosomal RNA (rRNA) synthesis to nucleolar ultrastructure was studied in partial nucleolar mutants of Xenopus laevis. These mutations are the result of a partial deletion of rRNA genes and therefore alow studies on nucleolar structure and function without using drugs that inhibit rRNA synthesis. Ultrastructural studies demonstrated that normal embryos have reticulated nucleoli that are composed of a loose meshwork of granules and fibrils and a typical nucleolonema. In contrast, partial nucleolar mutants in which rRNA synthesis is reduced to less than 50% of the normal rate have compact nucleoli and nucleolus-like bodies. The compace nucleoli contain granules and fibrils, but they are segregated into distinct regions, and a nucleolonema is never seen. Since other species of RNA are synthesized normally by partial nucleolar mutants, these results demonstrate that nucleolar segragation is related specifically to a reduction in rRNA synthesis. The nucleolus-like bodies are composed mainly of fibrils,and the number of such bodies are composed mainly of fibrils, and the number of such bodies present in the different nucleolar mutants is inversely related to the relative rate of rRNA synthesis. Although the partial nucleolar organizers produce segregated nucleoli in these mutants, they organize morphologically normal, but smaller, nucleoli in heterozygous embryos. Alternative explanations to account for these results are discussed.     

小麦核仁在细胞周期中的变化

运用Bernhard染色方法研究了小麦根端分生组织细胞核仁在细胞周期中的变化。结果显示,间期核仁染色很深,能够区分出纤维中心（FC）、致密纤维组分（DFC）和颗粒组分（G）,而染色质被漂白,在染色质间可以观察到细小的RNP颗粒。进入前期,在染色质的边缘有小的RNP颗粒分布。中期,染色体周边分布着类似于间期核仁的深染的大RNP颗粒,形成一个不完全连续的“鞘”状结构;在染色体内部看不到类似核仁的深染颗粒。到了后期时,仍可见RNP“鞘”状结构的存在。进入末期,这些RNP植物逐渐由“鞘”脱离,最后参与新核仁的形成。这些结果表明,核仁解体后的物质直接转移到了中期染色的表面,并形成不连续的表层,没有进入染色体的内部。     

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.     

Localization of ribosomal genes and nucleolar activity in lymphocytes of swine (Sus scrofa domestica) stimulated by phytohemagglutinins

The Pig chromosomes that contain rDNA sites displayed a polymorphism in the distribution of the genes among the nucleolar organizers located on pairs Nos. 8 and 10. Two, or more often three, active sites were observed in the chromosomes of lymphocytes stimulated with phytohemagglutinin. Only 5% of the metaphases showed a 4th small active site. At the onset of stimulation most cells contained one-two nucleoli; four nucleoli were never observed. After prolonged stimulation, the number of nuclei containing three nucleoli increased. A 4th small nucleolus appeared in a few cells, presumably formed by activation of the smallest rDNA site.     

Characteristics of karyosphere formation in the lake frog. Light microscopy data

Morphological peculiarities of the oocyte nuclear organization were examined in R. ridibunda during winter and spring (February-March). Numerous nucleoli were seen to be assembled around regressive lampbrush chromosomes in the centre of the nucleus, and a central body was formed to which the chromosomes were attached. As result, a structural complex is constituted that involves a karyosphere and a capsule. Nucleoli are characterized by segregation and intensive fragmentation of their material. In result, a considerable part of nucleolar DNA is eliminated in the form of ring and polymorphous structures (micronucleoli). Besides the membranous component of nucleoli (nucleolar threads or tails) is lost. Towards the end of this period, nucleoli with complicated morphology become spherical again. The formation of the central body is started from the appearance of some small optically-light protein structures 5-20 nm in diameter (central body precursors-CBP). CBP are closely surrounded with ring micronucleoli to make intimate contact with the chromosomes and nucleolar threads. CBP commonly lie in one region of the nucleus not far from each other. The formation of a definitive central body obviously occurs due to a fusion of some small CBP. A conclusion is made of the nucleolar origin of the ring and polymorphous structures and of their essential role in the central body formation. The participation of chromosomal and eliminated nucleolar DNA in this process is discussed.     

Ultrastructural Changes of the Nucleolus During Cell Cycle in Triticum aestivum

The ultrastructural changes of the nticleolus during cell cycle in common wheat (Triticum aestivum L. ) were studied by an "en bloc" silver-staining method. It was observed that in interphase, the nucleolus was heavily stained, within which fibrillar centres, dense fibrillar component, granular component and nucleolar vacuoles could be identified. A large quantity of argentine fine granules were distributed in the condensed chromatin. Dur-ing prophase, along with the disintegration of the nucleolus and condensation of the chromatin, the larger heavily-stained granules gradually appeared at the periphery of the chromatin. At late prophase, the materials derived from the nucleolus were spread and deposited on the surface of the chromosomes. The silver-stained, larger granules, deriving from the disintegrated nucleolus, accumulated at the periphery of the metaphase chromosomes and formed an uneven and discontinuous "sheath"-like structure. This "sheath"-like structure was also observed at anaphase. In telophase, the silver-stained nucleolar materials were progressively separated from the "sheath' and fused with each other to form prenucleolar bodies, and at last, participating in the formation of new nucleoli. The results showed that the nucleolar materials were transferred directly to the surface of the chromosomes and formed a discontinuous coat, but not incorporated into the interior of the chromosomes. The silverstained granules inside the chromosomes were neither related to the nucleolus nor to the materials from the disintegrated nucleolus.