Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry.

Human erythroblasts which are prereticulocyte maturation stages of red blood cells were studied by light microscopic cytochemistry and electron microscopy to provide more information on the ultrastructure of the micronucleoli which are terminal stages of nucleolar changes found during maturation of these cells. As indicated by light microscopy of smeared cells, micronucleoli were virtually the only types of nucleoli present in the last stages of maturing erythroblasts, i.e., polychromatic and orthochromatic (late polychromatic) erythroblasts. Accordingly, they were not portions of the periphery of other nucleoli. Inasmuch as most of the micronucleoli exhibited characteristic segregation of nucleolar fibrillar and granular components they presumably are producing little if any preribosomal RNA, since such segregation generally reflects inhibition of nucleolar RNA synthesis.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

Further studies of the nucleolar material in salivary gland nuclei of Sciara coprophila

The nucleolar-like bodies or micronucleoli of Sciara coprophila salivary gland nuclei have been studied with phase contrast, the Nomarski optics, Azure B staining, and electron microscopy. In the late fourth instar the main nucleolus formed at the X-chromosome may become extensively fragmented and may appear as a large aggregate of micronucleoli. At about the same time large numbers of micronucleoli in a more peripheral location are also found. Studies in partially squashed and stained nuclei, as well as in unfixed glands have shown that, at a time when the nucleolar material is abundant, the X-NOR is highly ramified with its branches permeating much of the nuclear space. These observations make it appear probable that most or all of the nucleolar material, even the more peripherally located, is actually in contact with the main nucleolar organizer or its branches. On the other hand, many chromosomal bands are also in close association with micronucleoli. At the level of electron microscopy some of the associations between chromo somal bands and micronucleoli are very intimate with the nucleolar material often found deep within the band. In other instances there seems to be physical continuity between extensions of band chromatin and certain areas of the fibrillar component. The bands in question could be the sites of secondary nucleolar organizers. In the electron microscope a large aggregate of micronucleoli, interspersed with portions of chromatin can often be seen in an approximately central location. This is interpreted as the main nucleolus with portions of its NOR. Both the main nucleolus and the more peripheral micronucleoli are indistinguishable in their fine structure and show the components typically found in nucleoli, i.e., fibrils and granules. On the other hand the fine structure of both RNA and DNA puffs is strikingly different.Supported by funds from Public Service Grants GM 12191 from the National Institute of General Medical Sciences and 5 RO 1 AM 10016-06 from the National Institute of Arthritis and Metabolic Diseases (to Dr. A. M. Garcia).     

Studies on nucleoli of maturing frog erythroblasts

Summary Frog erythroblasts were studied in summer animals with a very active as well as reduced erythropoiesis due to experimental hibernation, the latter being administered in order to get more information on the frequency of various nucleolar types in maturing cells. The results suggest that nucleoli with nucleolonemata are a transitional nucleolar type between compact and ringshaped nucleoli. Since micronucleoli represent final nucleolar maturation changes and compact nucleoli are present in most immature cells, the sequence of nucleolar changes based on the frequency of investigated nucleolar types is as follows: compact nucleolinucleoli with nucleolonemataringshaped nucleolimicronucleoli. The experimental hibernation produces a shift of nucleoli to less active and maturer nucleolar types in all stages of the erythroblastic maturation. In addition, the experimental hibernation produces the formation of ringshaped nucleoli in the first stages of the erythroblastic maturation which in summer animals usually contain compact nucleoli and/or nucleoli with distinct nucleolonemata.     

Nucleoli in the cells of the erythrocyte and granulocyte series in the albino rat

In albino rats, as in other mammals, nucleoli with nucleolonemata and compact nucleoli in the stem cells of the erythrocyte and granulocyte series are progressively replaced during maturation and differentiation first by ring shaped nucleoli and then by micronucleoli with low and finally with inhibited RNA synthesis. There are, however, differences between the nucleolar coefficient values and the proportion of ring shaped nucleoli and micronucleoli in maturing neutrophils in the albino rat, the mouse and man. In the case of the erythroblasts, the differences between the proportion of various nucleolar types in the three given species are smaller. The results thus indicate that the developmental trend of nucleolar changes related to cell maturation and differentiation is the same, but that there are interspecies differences because of which the results obtained in one species cannot be applied mechanically to another, i.e. results obtained in a laboratory animal cannot be applied automatically to man.     

Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria

We have examined the distribution of RNA processing factors in the germinal vesicle (GV) of the common frog Rana temporaria during early vitellogenesis by immunostaining on light- and electronmicroscopic levels and by in situ nucleic acid hybridization. Small nuclear RNPs (snRNP) and factor SC35 involved in pre-mRNA splicing occur in lampbrush chromosome loops and numerous granules 1-3 microns in size. These granules are identical to B snurposomes of Xenopus laevis and Notophtalmus viridescens described earlier (Wu et al., 1991). Some of B snurposomes are attached to homologous loops of lampbrush chromosomes. Immunofluorescent study of Cajal bodies/coiled bodies (CB) showed that sometimes CB have B snurposomes attached to their surface. In this case splicing factor SC35 is found in B snurposomes and B-like inclusions in CB matrix. In CB without attached B snurposomes splicing factor SC35 localizes throughout the whole organelle. Staining of GV spreads with antibodies against nucleolar protein NO38 revealed this protein in CB, nucleoli and micronucleoli. Using in situ nucleic acid hybridization and immunofluorescent staining we have found that on GV spreads from hibernating frogs B snurposomes contact nucleoli. Nucleoli contain snRNP. These data suggest that nucleoli may be storage sites of snRNPs during natural inactivation of RNA synthesis. During winter season in Rana temporaria GV nucleoli become compacted and a number of micronucleoli (less than 2 microns) dramatically increases. Analysis of micronucleoli showed that they contain rRNA, protein NO38, trace amount of U3 small nucleolar RNA and do not contain fibrillarin, involved as U3 in pre-rRNA processing. We suggest that decrease of rRNA synthesis during frog hibernation results in transformation of part of nucleoli in micronucleoli.     

Localization of fibrillarin, 53 kDa protein and Ag-NOR proteins in the nuclei of giant antipodal cells of the wheat Triticum aestivum

Distribution of nucleolar argentophylic proteins, fibrillarin and 53 kDa protein, in highly polyploid nuclei of antipodal cells of Triticum aestivum L. was studied at different stages of the embryo sac development. The main results are as follows. 1. Ag-NOR proteins and fibrillarin form clusters are distributed in the giant nucleoli, whereas 53 kDa protein is mainly localized on the nucleolar periphery. Ag-NOR proteins and fibrillarin are accumulated as globular nucleolar-like particles--micronucleoli. 2. Dynamics of Ag-NOR proteins, fibrillarin and 53 kDa protein depends on the proliferative activity of endosperm cells. In embryo sacs with non-dividing endosperm cells at interphase stages, Ag-NOR proteins and fibrillarin were observed only within nucleoli and micronucleoli. In embryo sacs with dividing endosperm cells, fibrillarin and 53 kDa protein formed heterogeneous globular bodies varying in size. Simultaneously, some argentophylic material was observed in giant chromosomes. This may be due, presumably, to a partial or complete disappearance of the nucleoli of antipods and transition of some nucleolar components into the peripheral material of giant polytene chromosomes. We suggest that giant nuclei of antipodal cells may undergo cyclic transformation similar to those in the nuclei of dividing cells.     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.     

The effect of the Bacillus thuringiensis exotoxin on the fine nucleolar morphology and ultrastructure

Depending on the dose administred to the experimental mice, the Bacillus thuringiensis exotoxin produces striking changes in the nucleolar morphology of hepatocytes such as the formation of ring-shaped nucleoli, micronucleoli and the segregation of nucleolar components. Such changes are apparently related to the decrease and inhibition of nucleolar biosynthetic activities in the production of the nucleolar RNA. In addition, the Bacillus thuringiensis exotoxin causes the formation of nucleolar peripheral dense plaques and, at higher concentrations, the segregation of two distinctly separated granular areas in the nucleolus. Both these light and dense granular areas showed positive staining with Bernhard's EDTA procedure for the preferential demonstration of RNA-containing structures. In some segregated nucleoli the granular components of light granular areas seemed to leave the nucleolus. The presence of discontinuous filamentous shell around micronucleoli produced by the high dose of exotoxin suggests the nucleolar origin of nuclear granular bodies which are surrounded by similar but continuous filamentous shell characteristic of these structures.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry.
Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.     

TPA, tumor promoter-induced suppression of immunoglobulin secretion in human blood lymphocytes

12-O-Tetradecanoylphorbol-13-acetate (TPA) modulates DNA synthesis and differentiation of normal and malignant human lymphoid cells. Using the reverse plaque forming assay and radioimmunoassay, we showed that nontoxic concentrations of TPA (5 to 10 ng/ml) inhibited Ig secretion of peripheral blood lymphocytes. This inhibition was dependent on T lymphocytes and not monocytes; TPA treatment of the B cell-enriched fraction slightly enhanced Ig secretion. Suppression was evident when the proportion of TPA-pretreated T lymphocytes exceeded 50%. TPA-induced suppressor cells were present in both OKT8+ (suppressor/cytotoxic) and OKT4+ ("helper/inducer") subpopulations. The suppression was diminished but not abolished by the irradiation of T lymphocytes. In addition, TPA treatment modulated the expression of OKT4 antigen, whereas the expression of OKT8, 9.6 (sheep erythrocyte receptors) and surface Ig remained unchanged. Modulation of OKT4 was energy dependent and was not blocked by a maximal saturation of TPA receptors at 4 degrees C. We postulate that TPA-induced suppression of Ig secretion is T cell dependent and is likely to be associated with proliferation and activation of OKT8+ and OKT4+ lymphocytes and the induction of OKT4+ suppressor cells.     

A further contribution on nucleoli of human lymphocytes.

Cultured human lymphocytes were investigated by means of light as well as electron microscopic procedures to provide more information on the structural organization of their nucleoli. The transformation of ring shaped nucleoli to nucleoli with less or more distinct nucleolonemata in PHA stimulated cells was characterized by a marked increase of granular RNP components in number indicating the activation of their production. This phenomenon seems to be related not only to the activation of the ribosomal RNA synthesis but also to its processing. The appearance of fibrillar RNP components in the central area of the ring shaped nucleoli apparently represents the first sign of the nucleolar RNA synthesis in these cells. The proportion of fibrillar and granular nucleolar RNP comonents in PHA inresponsive lymphocytes was similar to that in lymphocytes from patients with the usual type of lymphocytic leukemia. The intranucleolar chromatin areas appeared to be larger in PHA stimulated lymphocytes but the proportion of these areas to the nucleolar body did not show substantial difference as compared to the resting cells.     

Development of activity of lymphocytes estimated from nucleolar morphology in infants

Changes in nucleolar morphology of the lymphocytes were studied in peripheral blood smears of infants from birth up to one year of life. A marked proportional increase of lymphocytes with active nucleoli was found during the first month.     

Morphology and index of nucleoli in malignant lymphomas

The significance of morphology and the number of lymphocyte nucleoli was evaluated by electronmicroscopic sections of malignant lymphomas. It was not only the number and the size of nucleoli which characterized malignant lymphomas differing in histological respect, but above all the different morphology of the nucleoli. The prognostic significance of the nucleolus can be illustrated, if malignant lymphomas of a low grade malignancy with a preponderance of micronucleoli and a low nucleolar index are compared with a significantly higher nucleolar index and markedly increased macronucleoli. These differences are further supplemented by additional morphological properties of nucleoli in malignant lymphomas.     

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.