Nucleolar Activity Research Articles

Nucleolar Activity in the Fusiform Cambial Cells of Abies balsamea (Pinaceae): Effect of Season and Age

Nucleolar involvement in the regulation of the activity-rest-quiescence cycle of the vascular cambium was assessed by determining the seasonal variation in number, diameter, and volume of nucleoli in fusiform cells of Abies balsamea (L.) Mill. The cells were isolated from 1- and 19-yr-old cambia and stained with either silver nitrate or Feulgen + naphthol yellow-S. The ability of fusiform cells to incorporate [5-3H]-uridine into nuclei and nucleoli was also determined. In the 1-yr-old cambium, the activity of the nucleoli, as evidenced by their diameter, total volume per cell, and intensity of staining with silver nitrate, exhibited two maxima during the year—a large one during cambial reactivation in April-May and a small one during the rest-quiescence transition in October. Incorporation of radiolabeled uridine at 20 C was low at the end of the active period and increased during the rest–quiescence transition, suggesting that the quiescent, but not the resting, cambium can rapidly resume nucleolar activity when the temperature is permissive. The number of nucleoli per cell varied between two and eight, and was higher during the dormant than the active period. The increase in number took place during the autumnal activity–rest–quiescence transition, when cambial cells were arrested in the G1 phase of the cell cycle. Similar seasonal changes in nucleolar morphology were observed in the 19-yr-old cambium. Nucleolar diameter and total nucleolar volume were larger in the 19-yr-old cambium than in the 1-yr-old cambium, whereas nucleolar number was lower. Th results suggest that repression of rRNA genes underlies the development of rest when the cambium will not produce new cells.

Nucleolar organizer regions in carcinomas of the oropharynx and hypopharynx.

It is supposed that the nucleolar organizer regions (NORs), genes on which ribosomal RNA is coded, may contain information on the structure of the nucleolus and on nucleolar activity which permits discrimination between normal and neoplastic cells and their proliferation activity. In the present study, the histological sections of 144 patients with carcinomas of the oropharynx and hypopharynx were studied in a retrospective approach to determine the absolute NOR counts per 100 nuclei and investigate their influence on the prognosis and extent of lymphogenic formation of metastases. An absolute NOR count of 681 per 100 nuclei was found to give reliable discrimination between stage N0 or N1 carcinomas and stage N2 or N3 disease.

Different patterns of rDNA distribution in Pisum sativum nucleoli correlate with different levels of nucleolar activity

ABSTRACT We have used in situ hybridization with probes to rDNA, labelled either with digoxygenin or directly with fluorescein, to determine the arrangement of these genes within the nucleoli of Pisum sativum L. root cells. Confocal laser scanning microscopy was used to image the three-dimensional structures revealed, but we have also compared this technique with deconvolution of conventional (wide-field) fluorescence images measured with a cooled CCD camera, and have shown that the results are remarkably similar. When the deconvolution technique was applied to the confocal data it gave clearer images than could be achieved by confocal microscopy alone. We have analysed the distribution of rDNA in the different cell types observable in root tips: the quiescent centre; active meristematic cells; and relatively differentiated root cap, epidermal and cortical cells. In addi-tion to four perinucleolar knobs of condensed, inactive rDNA genes, corresponding to the four nucleolar organizers in P. sativum, which were the most brightly labelled structures, several characteristic patterns of intranucleolar labelling were apparent, including bright foci, large central chromatin masses, and fine, decondensed interconnecting fibres. The larger and more active the nucleolus, the smaller the proportion of condensed perinucleolar rDNA. In some large and active meristematic nucleoli, all the internal rDNA is decondensed, showing that transcription cannot be restricted to the bright foci, and is most likely to occur on the decondensed fibres.

Cell cycle regulated expression of nucleolar antigen P120 in normal and transformed human fibroblasts

Normal and SV40 virus-transformed WI-38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [3H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non-synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G1-phase cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G1 and P120 protein synthesis initiated in middle G1. A dramatic increase of P120 protein level occurred in S-phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI-38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G1 to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation.

Variation in the restriction fragments of 18S–26S rRNA loci in South American Elymus (Triticeae)

The repeat units of the 18S–5.8S–26S ribosomal genes (rDNA) of 15 South American and 6 Asiatic and North American species of Elymus were shown to vary between 8.7 and 11.3 kb among species. Interspecific variation in the EcoRI and BamHI cleavage sites was observed. BamHI restriction sites were present in the subrepeats in the intergenic spacers in rDNA from diploid Hordeum (H genome) and absent in the diploid Pseudoroegneria (S genome) and in nearly all the tetraploid species of Elymus (SH genomes). Tetraploid Elymus species have only two pairs of chromosomes with secondary constrictions similar to those found in Pseudoroegneria and a maximum of two different repeat unit length classes. These results suggest that the rDNA in the H genome was lost during the evolution of the tetraploid Elymus. However, the additional H genome present in the hexaploid Elymus with genome formula SSHHH′H′ still show active ribosomal genes. Nucleolar organizer activity was also observed in rDNA from both the S and H genomes in artificial SS × HH amphiploids, suggesting no complete nucleolar dominance (amphiplasty) between the S and H genomes.Key words: Elymus, ribosomal genes, RFLP, nucleolus.

Quantitative analysis of nucleoli and nucleolar organizer regions in cultured primary human normal, reactive and malignant mesothelial cells

The number and the size of silver-stained intranuclear granules, which correspond to the nucleolus and nucleolar organizer regions, have been determined by means of quantitative methods in cultured primary human mesothelial cells obtained from normal, reactive and malignant mesothelium. The mean values per nucleus of the number, the total area, the average area, and the relative area of the silver-stained granules and the mean nuclear area were determined for each of the three conditions. Normal, reactive and malignant mesothelial cells differed significantly in all the features. These findings at the optical level reflect the differing rate of the nucleolar biosynthetic activity related to the different biological properties of the three cell types, and the features can be useful morphometric descriptors in the diagnostic pathology of the mesothelium.

Identification of C-banded chromosomes in meiosis and the analysis of nucleolar activity in Avena byzantina C. Koch cv ‘Kanota’

The Giemsa C-banding technique was used to identify individual meiotic and somatic chromosomes in 21 monosomic lines of Avena byzantina C. Koch cv 'Kanota' (genome designation AACCDD). The hexaploid complement is composed of three sets of seven chromosome pairs. The heterochromatin in the putative diploid progenitors is located at the telomeres (genome A), at the centromeric and interstitial regions (genome C), or more evenly spread throughout the set (genome D). Comparisons based on C-banding between A. byzantina and its diploid progenitor species allowed us to allocate individual chromosomes into specific genomes. The C-banding technique may be useful for interspecific chromosome pairing analyses. Nucleolar activity and competition were studied using a silver-staining procedure. Only three chromosome pairs showed nucleolar organizer regions, thus indicating that nucleolar competition occurs naturally in hexaploid oats.

Benzyladenine‐controlled protein synthesis and growth in apple cell suspensions

Prolific growth in cytokinin‐requiring apple cell suspensions was achieved during 10 days of culture with appropriate concentrations of benzyladenine (BA). Unlike the controls, BA‐treated cells showed a well developed sub‐cellular organization and protein synthesis. Upon transfer to fresh cytokinin‐free medium, however, these cells exhibited a rapid decrease in polyribosome formation, but with unchanged nuclear and nucleolar activities. Cells lacking added cytokinin showed a reduced metabolic activity and failed to divide. Deficiency in the translation process of cytokinin‐deprived cells was overcome by exogenous BA‐treatment. In cells maintained on a cytokinin‐free medium for 5 days, BA‐treatment enhanced protein synthesis from pre‐existing mRNA in a specific way.

Changes in nucleolar transcriptional activity in hepatitis B virus-associated chronic liver diseases: Preliminary results from a quantitative study of silver-stained nucleoli

Modifications of gene expression may occur in hepatitis B virus (HBV)-related chronic liver diseases, possibly also involving ribosomal RNA (rRNA) genes contained in the nucleolus. Changes in the level of transcriptional activity of rRNA genes are reflected by variations in the number and/or size of the nucleoli. Therefore a quantitative analysis of the silver-stained nucleoli (AgNus) was performed in a small series of liver needle biopsies from patients with HBV+ chronic persistent hepatitis (CPH) (n = 3), HBV+ chronic active hepatitis (CAH) (n = 3) and HBV+ cirrhosis (CIR) (n = 3). In each case, 100 hepatocytes were selected. The number of the nucleoli (AgNuN), their total area (tAgNuA), the average area of each nucleolus (xAgNuA), the nuclear area (NA) and the percentage ratio of tAgNuA related to NA (rAgNuA) were determined for each hepatocyte nucleus. The pooled mean values of all the features were significantly different (p less than 0.001) among the case groups. The results point towards a remarkable increase of nucleolar activity in CAH in comparison with CPH, whereas an additional increment of this activity is associated with the progress from CAH to CIR.

Nucleolar transcriptional activity in human round spermatids

Nucleolar transcriptional activity in human round spermatids

Ultrastructural changes in the nucleolus of facial motor neurons following axotomy during an early critical period in development

In this study, the effects of axotomy on the ultrastructure of the nucleolus and associated organelles were examined in fetal, newborn, and early postnatal facial motoneurons of the hamster. Golden hamsters used for this study were the 14-day fetus, newborn (0 days; less than 6 hr) and 2, 4, 7, and 9 days postnatal ages, with 3 animals per group. For prenatal surgeries, pregnant hamsters were anesthetized and the facial nerves severed in the fetuses via electrocautery through the uterine wall and amniotic membrane. For postnatal surgeries, the animals were anesthetized and the right facial nerve exposed and severed at its exit from the stylomastoid foramen. At the appropriate postoperative times, the animals were reanesthetized and perfused-fixed. The facial nuclear groups were dissected and processed for routine electron microscopy. Microbody and coiled body frequencies were determined from the number of neurons containing these structures per number of neurons sampled per animal in each experimental or control group and subjected to statistical analysis. Nucleolar reactive changes that occurred during this developmental sequence fell into two major categories. The first category displayed by most injured cells consisted of an initial compacting of fibrillar material and reduction in vacuolar space. The second category appeared to represent a progression from this first stage of nucleolar reactivity into degenerative changes involving a striking segregation of nucleolar components into five distinct regions. The incidence of microbodies increased as a result of axotomy, whereas the presence of coiled bodies decreased at the later postoperative stages in the older animals. With increasing age and nucleolar maturation, the nucleolar reactive pattern became less pronounced and severe, and neuronal survival predominated. It appears, therefore, that the two categories of nucleolar changes following axotomy during early development correlate with changes observed in nucleoli under conditions of rRNA downregulation. It is hypothesized from these results that a key step in the ability of neurons to survive axotomy and successfully regenerate at these early developmental stages occurs at some point in ribosomal RNA transcription and/or processing. Complementary information at the molecular level concerning changes in nucleolar synthetic activity and ribosome production will be necessary to test this hypothesis.

Role of polyamine in the regulation of RNA synthesis in uterine nucleoli

Administration of estradiol (E 2) to ovariectomized mature rats has been shown to result in synthesis of uterine polyamines in the same temporal manner as E 2 regulation of nucleolar transcription. Data is presented on the in vivo and in vitro effects of polyamines on uterine nucleolar RNA synthesis. Transcervical intrauterine administration of putrescine (100 μg), spermidine (100 μg), or spermine (100 μg) resulted in an increased transcriptional activity of 93 and 82% in uterine nucleoli isolated from putrescine and spermidine treated animals, respectively. Spermine administration was without effect on uterine nucleolar transcription. The polyamine-induced increase in transcription was totally accounted for by an increased rate of elongation of previously initiated RNA chains. No effect on the number of nucleolar RNA chains in the act of synthesis was observed. Preincubation of uterine nucleoli, isolated from control animals (no E 2) with putrescine, spermidine, or spermine in the presence, but not in the absence of ATP, resulted in 44, 83 and 31% increased nucleolar RNA synthesis, respectively. In vitro polyamine-induced nucleolar RNA synthesis was correlated with a polyamine activated phosphorylation of nucleolar proteins of 110,000 24,000, 18,000 and 14,000 Da. Results suggest that early E 2 action may result in activation of the polyamine pathway which modulates nucleolar protein kinase activity; initiating an increase in nucleolar transcription.

Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis

The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Biochemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural immunolocalization of these two proteins by means of specific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogenesis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermatogenesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene spermatocytes, and in Golgi phase spermatids, B23 and nucleolin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fibrillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactivation, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular component of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the presence of B23 or nucleolin are not directly related to each other.

Quantitative determination of rDNA transcription units in vertebrate cells

The adenosine analogue 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) unravels the compact nucleoli to necklace-like structures when applied to living cells. The nucleolar beads contain RNA polymerase I (RPI) and argyrophilic proteins, both properties considered to be characteristic of ribosomal gene activity. Each granule is supposed to represent a single transcription unit consisting of an actively transcribing gene and its RPI complex. Indirect immunofluorescence with anti-RPI antibodies was used to determine the number of transcription units in DRB-treated cells of some representative mammals, marsupials, birds, and amphibians. We estimate that 45 to 145 rRNA genes are transcriptionally active in vertebrate fibroblasts, depending on the species. Nucleolar transcriptional activity does not correlate with the total number of rRNA genes. During in vitro aging of fibroblasts, the number of transcription units appears to remain unchanged. Different cell types of one same organism show varying numbers of transcription units, reflecting their differential metabolic activity. A particular situation exists in phytohemagglutinin-stimulated lymphocytes. In the course of nucleolar activation, the number of transcription units is increased considerably, implying that formerly inactive rRNA genes are recruited for transcription. The opposite phenomenon is observed during spermatogenesis. With the diploid spermatocytes developing into haploid spermatids, the transcriptionally active rRNA genes decrease in number until rRNA synthesis is completely blocked.

Nucleolar Activity in Differentiating Cotton Fibres is Related to the Position of the Boll on the Plant

Analysis of the regulation of cotton fibre growth in perspective of the relation gibberellin-nucleolar activity fibre growth which as been proposed by De Langhe et al.

Effect of magnetic field on the living cells of Allium cepa L.

The present investigation deals with the effect of static magnetic field of 2.25×103 Gauss on the mitotic and meiotic divisions and nucleoli of root meristematic cells of Alllum cepa L. In the present effort the effect of static magnetic field was compared with that of electro-magnetic field (He-Ne Laser beam of 2 mW) also.A significant fall in mitotic indices was noticed on static magnetic field whereas such fall was found much sharply in case of electrimagnetic field. Such treatment was also associated with various anomalies e. g. stickiness, clumping of chromosomes, anaphase chromosome bridge and disturbed spindles.Although a significant increase in meiotic indices was noticed due to static magnetic field after 30 minutes compared to the control, a considerable decrease in the frequency of cells at metaphase stage was recorded indicating the role of spindle abnormality due to magnetic field. Various meiotic abnormalities were also recorded.Average nuclear and nucleoar size was found decreased significantly due to magnetic field treatment with the increase in time of treatment. Since the nucleolar size is a reliable reflector of nucleolar activity, such decrease may be correlated to the mitostatic activity of the cells.

Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy

The ultrastructural distribution of proteins B23 and nucleolin in the nucleolus of mouse embryos from the zygote to the early blastocyst has been analyzed by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. In parallel, silver staining of nucleoli was carried out on ultrathin sections. Our results show that the compact prenucleolar bodies at 1- and 2-cell stage as well as the compact residual fibrillar masses observed up to the morula stage, are labelled with the two antibodies. These masses, however, are not stained with silver up to the 4-cell stage. In well-developed nucleoli, the two antibodies co-localize in the dense fibrillar component (DFC) and the granular component (GC) while fibrillar centers (FCs) are devoid of label. On the contrary, silver staining occurs in the FCs and DFC but not in the GC. Our observations suggest that there is no direct relationship between the occurrence of silver staining and the distribution of protein B23 or nucleolin. Moreover, neither the localization of the two above proteins nor silver staining are unequivocally related to the nucleolar activity.

Nucleolar transcriptional activity in mouse Sertoli cells is dependent on centromere arrangement

Experimental evidence suggests that centromere arrangement is relevant to the expression of ribosomal genes in murine Sertoli cells. Nuclei endowed with a nucleolus inactive in rRNA synthesis presented several clusters, each containing a bunch of individual centromeres. RNA polymerase I was not cytochemically detected in the nucleolar structure, which contained only small amounts of fibrillarin. In the course of nucleolar activation, the centromeres within the separate clusters became fused into larger centromeric bodies. Synthesis of precursor rRNAs and their processing were visualized by strong nucleolar fluorescence signals using antibodies to RNA polymerase I and fibrillarin.

1R chromosome nucleolus organizer region activation by 5-azacytidine in wheat × rye hybrids

Nucleolar activity was studied in several lines of Triticum aestivum cv. Chinese Spring, Triticum turgidum cv. Durum, and F1 hybrids from euploid and aneuploid lines of T. aestivum and Secale cereale cv. Centeio do Alto, in cells from root tips of seeds germinated in water or in 5-azacytidine. 5-Azacytidine, an analog of cytidine modified in the 5 position of the pyrimidine ring, inhibits DNA methylation. By using silver staining to determine the number of nucleolus organizer regions and the average number of nucleoli per root-tip cell from seeds germinated in both situations, it became apparent that the presence of 5-azacytidine during germination allowed for the expression of the nucleolus organizer region locus belonging to the rye genome, in contrast to the usual observed cytological absence of the rye nucleolus organizer region in wheat–rye hybrids. It is suggested that wheat nucleolar dominance in wheat–rye hybrids is mainly a consequence of methylation of rRNA genes or its regulators located on the 1R chromosome of rye.Key words: 1R nucleolar organizer, wheat–rye hybrids, methylation, Ag-NOR.

Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.