NUCLEOLAR AGING IN TETRAHYMENA DURING THE CULTURAL GROWTH CYCLE

Nucelolar morphology was studied by electron microscopy in control and actinomycin D-treated populations of Tetrahymena pyriformis (W) during the cultural growth cycle. Nucleoli exhibit an "aging" cycle concomitant with the cultural growth cycle, but independent of the individual cell cycle. Four different stages in the course of this aging process have been defined. Stage 1 occurs upon inoculation (low number of cells per milliliter) and lasts through lag and accelerating growth phases. In this stage, many small nucleoli are found at the nuclear periphery. In stages 2 and 3, nucleolar fusion begins. Stage 2 dominates the first half of logarithmic growth, and stage 3 dominates the second half. In late decelerating growth phase, the nucleoli enter stage 4. In this stage, only a few large nucleoli are present and these are apparently inactive in ribosome production. In stationary phase, where total RNA remains constant, only stage 4 nucleoli are present. The relative preponderance of granular vs. fibrous components in the nucleoli changes during this cycle, the granular component dominating stage 1 nucleoli and the fibrillar, stage 4 nucleoli. There is a shortening of the intermediate nucleolar stages in the treated cultures; fusion occurs early and is now pronounced. Not enough ribosomes accumulate to carry the treated cultures through the number of generations equivalent to those of the control, which produces a premature stationary phase.     

Isolation and DNA content of nuclei of Physarum polycephalum

Methods have been developed for isolation of nuclei from Physarum polycephalum at various stages of the life cycle and mitotic figures and nucleoli from the plasmodial stage. Organelles of growing plasmodia and myxamoebae were isolated by Waring blending in 0.25 M sucrose, 0.1% Triton X-100, 0.01 M CaCl2 (0.001 M for nucleoli) and 0.01 M Tris buffer, pH 7.2, and centrifuging through 1 M sucrose. The same procedure was used for starving cultures, except that before homogenization starving and sporulating plasmodia were washed with 0.01 M EDTA in 0.25 M sucrose, and spherules were washed with EDTA-sucrose and broken in the French pressure cell.     

Properties and Functions of a New Nucleolar Protein,Surf-6, in 3T3 Mouse Cells

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle.     

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.     

Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.     

Isolation of nucleoli from Tetrahymena pyriformis.

A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented.     

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.     

Structure of the nucleoli of developing microsporangiate strobili and root tips of scotch pine

Summary The characteristics of the nucleoli of the microsporangiate strobili and the root tips of Scotch pine (Pinus sylvestris L.) vary both during the course of the cellular cycle and, with regard to the pattern and stage of organ and tissue differentiation. Nucleologenesis takes place in interphase and the nucleoli last until prophase. Several types of nucleoli occur during the nucleolar cycle, the pattern and age of tissues determining which type or types dominate. In the strobilus primordia collected at the end of July and in August, the mitotic frequency is high. Nucleoli remain small throughout the nucleolar cycle, and at the electron microscopic level, they display intermingled fibrillar and fibrillogranular components. Strobilus primordia collected in September contain larger nucleoli in the sporogenous nuclei than in the nuclei of the tapetum or of the wall cells. Amongst the nucleoli with completely intermingled fibrous and granular material, nucleoli with nucleolonema or with vacuoles occur frequently. Small balls of fibrous material are seen on the nucleolar surface and in the nucleoplasm. In October, the mitotic frequency of strobilal cells is low. Nucleoli with completely intermingled fibrillar and granular components have vanished whereas a new, compact type of nucleolus with a dense fibrillogranular main portion and with nucleolonema, has developed. The nucleoli of the sporogenous cells have enlarged continuously whereas those of the wall cells are small. The nucleoli of the root tip cell resemble, to a certain extent, those of the strobilus primordia collected in September. In squashed preparations, the nucleoli of the strobilal cells bind the common nucleolar stains poorly whereas the nucleoli of the root cells can be stained with all the methods used. In certain cases, DNase treatment improves the stainability of the strobilal nucleoli. AgNO₃-staining is successful after acetic acid: alcohol fixation but not after formalin: hydrochinone fixation.     

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1–18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.     

Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.     

Human population, urban settlement patterns and their impact on Plasmodium falciparum malaria endemicity

Background

The length of the gonotrophic cycle varies the vectorial capacity of a mosquito vector and therefore its exact estimation is important in epidemiological modelling. Because the gonotrophic cycle length depends on temperature, its estimation can be satisfactorily computed by means of physiological time analysis.

Methods

A model of physiological time was developed and calibrated for Anopheles pseudopunctipennis, one of the main malaria vectors in South America, using data from laboratory temperature controlled experiments. The model was validated under varying temperatures and could predict the time elapsed from blood engorgement to oviposition according to the temperature.

Results

In laboratory experiments, a batch of An. pseudopunctipennis fed at the same time may lay eggs during several consecutive nights (2–3 at high temperature and > 10 at low temperature). The model took into account such pattern and was used to predict the range of the gonotrophic cycle duration of An. pseudopunctipennis in four characteristic sites of Bolivia. It showed that the predicted cycle duration for An. pseudopunctipennis exhibited a seasonal pattern, with higher variances where climatic conditions were less stable. Predicted mean values of the (minimum) duration ranged from 3.3 days up to > 10 days, depending on the season and the geographical location. The analysis of ovaries development stages of field collected biting mosquitoes indicated that the phase 1 of Beklemishev might be of significant duration for An. pseudopunctipennis. The gonotrophic cycle length of An. pseudopunctipennis correlates with malaria transmission patterns observed in Bolivia which depend on locations and seasons.

Conclusion

A new presentation of cycle length results taking into account the number of ovipositing nights and the proportion of mosquitoes laying eggs is suggested. The present approach using physiological time analysis might serve as an outline to other similar studies and allows the inclusion of temperature effects on the gonotrophic cycle in transmission models. However, to better explore the effects of temperature on malaria transmission, the others parameters of the vectorial capacity should be included in the analysis and modelled accordingly.     

Silver staining of the nucleoli of pig embryonic kidney cells during the hyperactivation and inhibition of the synthesis of nucleolar RNA by UV microirradiation

Silver staining of the nucleoli in pig embryo kidney cells (PK) was studied during the cell cycle and also upon mature nucleoli modifications induced by UV microirradiation. During anaphase only four silver-stained granules were revealed in each daughter set of chromosomes in the four nucleolus-organizing regions (NORs). In the following 1-2 hours, the number of granules in the NORs rapidly increased up to 25-30 per nucleus. During the next 20-25 hours of the cell cycle, the number of silver-stained granules was slowly doubling as the nucleoli grew in size. UV microirradiation of one nucleolus in the nucleus with two nucleoli induced a profound degradation of the injured nucleolus and a compensatory hypertrophy of the intact one. Such nucleolar modifications were accompanied by redistribution of the silver-stained granules between the injured and non-injured nucleoli and by alterations in the levels of nucleolar RNA synthesis in the NORs. These data support a hypothesis that silver-stained proteins may be involved in the regulation of the nucleolar activity.     

ISOLATION AND PROPERTIES OF LIVER CELL NUCLEOLI

1. The significance of the term nucleolus has been discussed. 2. A detailed method for the isolation of nucleoli from already isolated rat or cat liver nuclei has been presented. 3. The presence of DNA in isolated liver cell nucleoli has been indicated by histochemical methods. 4. The percentages of DNA and RNA in the isolated nucleoli have been determined by chemical analysis. 5. The specific activities of aldolase, arginase, and catalase have been determined for two subnuclear fractions and for the isolated nucleoli of rat and cat liver, and the relative amounts of these enzymes in the same subnuclear fractions and nucleoli of rat liver have been measured. 6. The significance of the above findings has been discussed and consideration has been given to what types of isolated nuclei might best serve as starting material for the isolation of nucleoli. 7. A new hypothesis has been presented that nucleoli of the liver cell type may function primarily in furnishing (directly or indirectly) templates for the synthesis of the particular enzymes that must govern the chemistry of mitosis.     

Stimulation of RNA synthesis in nucleoli by inorganic phosphate in vitro

The biological function of a phosphoprotein with a molecular weight of 120 000 daltons localized in the nucleoli of mouse ascites sarcoma cells was studied by examining the effect of the phosphoprotein on RNA synthesis in the nucleoli in vitro. The phosphoprotein did not stimulate ribosomal RNA synthesis in vitro. During this study, it was observed that inorganic phosphate enhanced RNA synthesis in the nucleoli in vitro in the presence of either Mn²⁺ or Mn²⁺ plus Mg²⁺ as divalent cations. Inorganic phosphate stimulated the rate of the chain elongation reaction in RNA synthesis.     

Morphological and Histochemical Changes Occurring during the Life-span of Root-tip Galls on Lolium perenne Induced by Longidorus elongatus

The RNA and protein content of perennial ryegrass root-tip galls induced by Longidorus elongatus were measured from transverse sections and the morphology described. Galls progressed through five distinct stages and were viable for only 10-12 days at 18 C, after which they collapsed and became necrotic. In the initial stage hypertrophy occurred and cells contained enlarged nuclei and nucleoli, a greater proportion of cytoplasm, and increased concentrations of protein. This was followed by hyperplasia; cells divided to give two or four daughter cells, accompanied by a proportionate reduction in volumes of cytoplasm, nuclei, and nucleoli and reduced concentrations of RNA and protein. The third stage was secondary hypertrophy with enlarged, amoeboid nuclei and nucleoli and a significant increase in concentration of RNA and protein. In the final two stages, as feeding by L. elongatus progressively removed cell contents, most cells were devoid of inclusions and galls collapsed and were invaded by soil bacteria. This ordered development and exploitation of galls suggests that L. elongatus may have two phases in its feeding.     

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.     

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation cells.