THE NUCLEOLI IN MITOTIC DIVISIONS OF MAMMALIAN CELLS IN VITRO

In a number of mammalian cell strains nucleoli persisted through mitosis. This phenomenon was especially pronounced in several cell lines derived from Chinese hamster tissues. All the methods employed, including radioautography with tritiated uridine, cytochemical stains (methyl green-pyronin and azure B), fluorescent microscopy (coriphosphine O), ribonuclease digestion, and electron microscopy, demonstrated that the bodies identified as persistent nucleoli in the mitotic stages had the same characteristics as did the nucleoli in the interphase. Persistent nucleoli may attach to the chromosomes or may be free in the cytoplasm. In cells where no persistent nucleoli as such were noted, nucleolar material was observed to attach to the chromosomes in shapeless masses which moved with the chromosomes during anaphase. At least a portion of the nucleolar material was included in the daughter nuclei, presumably for immediate use for protein synthesis after cell division.     

Persistent Nucleoli in Early Postimplantation of Rat Embryos

Ultrastructural changes of the nucleolus in mitotic embryonic ectodermal cells of 7 1/2-day and 7 2/3-day rat embryos were examined. It was found that the nucleolus was broken down into small fragments during late prophase and metaphase, and that some of these fragments persisted in the cytoplasm of telophase cell (persistent nucleoli). No interphase embryonic ectodermal cells contained persistent nucleoli. Persistent nucleoli were also found in telophase cells of extraembryonic ectoderm, extraembryonic visceral endoderm and parietal endoderm of the embryos, but they disappeared in interphase cells. Persistent nucleoli in telophase cells tended to decrease in size with embryonic age, and they had almost completely disappeared in neuroectodermal cells of the telencephalon in 14 1/2-day embryos. They were concluded to be remnants of disappearing nucleoli in embryonic cells that were cycling too rapidly to permit their nucleoli to disappear completely.     

Localization of chromatin in "persistent" nucleoli in okadaic acid treated HeLa cells.

In okadaic acid treated HeLa cells, the chromosomes sometimes condense without being accompanied by nuclear envelope breakdown. These cells show "persistent" nucleoli. Within these "persistent" nucleoli the intranucleolar chromatin condenses and can be observed in the region of the dense nucleolar component (DNC) of the nucleoli. Other nucleolar components, namely the fibrillar centre (FC) and the granular component (GC) remain unchanged. These observations strongly speak for the localization of nucleolar chromatin (ribosomal cistrons) within the dense nucleolar component of the interphase nucleolus.     

Prorocentrum属涡鞭毛虫核仁的观察

迄今未能在光学显微镜下观察到Prerocentrum属的涡鞭毛虫有核仁。本文作者用伊红的酒精溶液和用甲基缘—派若宁法染色,也未能在Prerocentrum micans和Proro-centrum cassubica的细胞核中显示出核仁来。但是在用专门为显示单细胞生物的核仁组织者区（NOR）而改进了的Ag—1法进行染色时,这两种涡鞭毛虫的核仁都会被染作鲜明的深褐色或深黑色,而身体的所有其他部份,包括染色体,全都不着色。染色适当时可以看出,实际上只是核仁的中央部分被染上色。在电镜下可见,此时所有的银粒全部是集中在核仁的纤维区中。染色的结果表明,Prorocentrum cassbica只有一个扁园形的小核仁,后者是贴附在核膜上,其NOR通常是作O形或C形。与P.cassubica不同,P.micans的核仁的数量变化很大,可以有一个至七个;其核仁的大小与形状同样也变化很大;其NOR的形状也复杂多变。发现P.micans的核仁数量与个体的生活状况有一定的关联:向老的培养液中加入等量的新的培养液一天以后,具有三个核仁的个体是最多的（占三分之一）,具有4—6个核仁的个体占28.5％,只有一个核仁的个体只占8.6％;加入新培养液三天后,具两个核仁的个体变成是最多的（占38.8％）,具4—6个核仁的个体降为占18.4％;加入新培养液一个月以后,只有一个核仁的个体是最多的（占3     

On the nucleoli of the dinoflagellate Prorocentrum

To date no nucleolus had been observed in Prorocentrum under the light microscope. The author failed to show the nucleoli of P. micans and P. cassubica with eosin in 70% alc or with methyl green-pyronin. But when these dinoflagellates were treated with an Ag-1 technique which had been improved for demonstrating NORs in unicellular organisms, nucleoli were stained dark brown or black, while all other parts showed no colour. When the materials were stained well, only the central part of the nucleolus was stained. Under the electron microscope, it was observed that all the silver grains were concentrated in the pars fibrosa of the nucleolus. P. cassubica had only one small oblate nucleolus attached to the nuclear envelope, with NOR usually in the shape of the letters O or C. P. micans had 1–7 nucleoli of various sizes and shapes with NORs in various complicated forms. The number of nucleoli bore a certain relationship to the living state of the dinoflagellate. One day after fresh medium was added, cells with 3 nucleoli were most common, and 28.5% of the individuals had 4–6 nucleoli. Cells having only one nucleolus accounted for 8.6%. 3 days after, cells with 2 nucleoli became dominant, and those with 4–6 decreased to 18.4%. After a month, cells with 1 nucleolus became most abundant, cells having 4 nucleoli decreased to 2.4%, and no cells had 5 or 6 nucleoli.     

PERSISTENCE OF NUCLEOLI IN SHORT TERM AND LONG TERM CELL CULTURES AND IN DIRECT BONE MARROW PREPARATIONS IN MAMMALIAN MATERIALS

Persistent nucleoli were studied in Chinese hamster and human long term cultures, human peripheral blood short term cultures, as well as direct bone marrow preparations. No colchicine or hypotonic treatments were applied and the cells were differentially stained with the Feulgen method and light green. Nucleoli were found to persist in the three systems studied, although to a much greater extent in the long term culture. The persistent nucleolar materials were usually in the form of individualized nucleoli mainly at chromosome ends. They also sometimes existed in a fluidlike or dropletlike condition around the chromosomes. Association of acrocentrics in humans and end-to-end associations in hamsters are likely to result from persistence of nucleoli and the possible effects of colchicine and hypotonic treatments that are usually applied. Other phenomena, such as stickiness at metaphase and separation difficulties and fragmentation at anaphase, may result from persistence of nucleoli. Nucleoli were often associated with large chromosomes and sometimes at sites exhibiting faint or clear constrictions. The possibilities of a partial correspondence between sites of persistence and sites of organization, as well as of the organization of nucleolar materials at sites other than the main organizers, are discussed. The persistent nucleoli were not included in daughter nuclei. They either degenerated in the cytoplasm or were eliminated from the cell. The three systems used may represent different intensities of metabolism reflected in the amounts of nucleolar materials built up and the amount that persists.     

Tissue and sex differences in the expression of nucleoli in mouse somatic cells.

In Mus musculus, the nucleolus organizer regions (NORs), or sites of ribosomal RNA-encoding genes, map at three chromosomal pairs. A silver procedure was modified to stain nucleoli in interphasic somatic cells of mice. The number of nucleoli per cell nucleus was determined in squashed cells of kidney, liver and pancreas obtained from male and female mice. In liver and pancreas cells the average number of nucleoli per cell was 4.84 and 4.66, respectively, and only 2.83 in kidney cells (p < 0.001). Less than 8% of pancreas cells and about 15% of liver cells contained more than 6 nucleoli per cell, which was the maximum expected number. In addition, the number of nucleoli per cell was significatively different (p < 0.01) when male and female liver or pancreas cells (not kidney cells) were compared. In both cases, female cells presented more nucleoli than the respective male cells. Assuming that the available NORs are the same, the variable number of nucleoli in the examined cell types would be the consequence of a tissue specific NOR regulation. The apparent influence of sex on this regulation is noted.     

Cytogenetic responses of birch to stress factors

The following range of changes in response to anthropogenic stress has been revealed in experimental birch seedlings relative to control: increased mitotic index, the range and frequency of abnormal mitoses, number of cells with persistent nucleoli, and number of cells in prophase. Cells with vacuolated cytoplasm were also observed. The mutagenic pressure on the organisms in the industrial areas of Voronezh demonstrated a trend to increase, which points to their high environmental pollution.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

Translocation of nucleolar phosphoprotein B23 (37 kDa/pI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of alpha-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of alpha-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribonucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Translocation of nucleolar phosphoprotein B23 ( ) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

BEHAVIOR OF NUCLEOLI AND CONTRACTING NUCLEOLAR VACUOLES IN TOBACCO CELLS GROWING IN MICROCULTURE

The ability to observe for extended periods of time individual tobacco cells growing in microculture has made it possible to describe the behavior of their nucleoli and contracting nucleolar vacuoles. Nucleoli typically disappeared in prophase and reappeared in telophase. If several nucleoli were present in telophase they generally fused to form only one or two during interphase. In one instance a nucleolus was seen to separate into two nucleoli prior to disappearance in late prophase. In aging and senescent cells the number of nucleoli or bodies similar to normal nucleoli often increased, and occasionally fragmentation of nucleoli was noted prior to death of cells. Budding of solid material from the nucleolus was also observed. The amount of nucleolar material decreased rapidly prior to death of tobacco cells. Nucleolar vacuoles were found to be a general and consistent component of tobacco cells in microculture. Nucleolar vacuoles typically formed and contracted repeatedly in interphase nuclei and apparently released a fluid material into the nucleus. Associated with the contraction of the nucleolar vacuoles was a corresponding decrease in diameter of the nucleolus. Nucleolar vacuoles were observed to occur in about 70% of the actively growing cells examined, whereas they were present in only 33% of the senescent or weakened cells. These data indicate a relationship between nucleolar vacuoles and the morphogenic status of the cells. Since it has been shown by others that the nucleolus is an active site of RNA metabolism, it is suggested that the contracting nucleolar vacuoles may be involved in the controlled release of a soluble product associated with RNA metabolism.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Isolation and characterization of different-sized nucleoli from Ehrlich ascites tumor cells : Evidence of different nucleolar ribosomal cistrons

A procedure was developed for isolation of variously sized nucleoli in order to study the mechanism of nucleolar formation from multiple nucleolar organizers and to compare the compositions of different-sized nucleoli from Ehrlich ascites tumor cells. Relatively small nucleoli and large nucleoli from Ehrlich ascites tumor cells were separated by centrifugation at 400 g for 5 min in a layer of 0.34 M sucrose over 0.88 M sucrose. Small nucleoli remained in the 0.34 M sucrose layer while the large nucleoli accumulated in the 0.88 M sucrose.Three fractions, provisionally named small, intermediate and large nucleoli, containing 0.33, 0.41 and 0.84 pg DNA/nucleolus, respectively, were separated. Unfractionated nucleoli contained 0.59 pg DNA/nucleolus. The RNA content also increased with the size of the nucleolus and no significant difference was observed in the RNA/DNA ratios in the three fractions. Large nucleoli incorporated more [³H]uridine and [³²P]orthophosphate into RNA than did small nucleoli, but the base compositions of the RNAs extracted from the different-sized nucleoli were similar. No significant fragmentation occurred on sonication of large nucleoli for 3 min, so the observed difference in the DNA contents was not due to mechanical damage of the nucleoli.The DNAs of these different-sized nucleoli were analysed on CsCl gradients. The nucleoli contained similar percentages of satellite DNA (20–22%) which were also similar to those of total, unfractionated nucleoli. Approx. 10% of the extranucleolar DNA is satellite DNA—thus the nucleolar fractions were probably not appreciably contaminated with extranucleolar DNA. The DNA of small nucleoli contained a slightly lower percentage (0.058%) of ribosomal cistrons than large nucleoli (0.081%). This means that the higher content of DNA in the large nucleoli is not merely due to longer sized chromatin with extra regions of the vicinity of nucleolar organizers. Thus these results suggest that the total content of ribosomal cistrons/nucleolus is roughly proportional to the DNA content of the nucleoli, at least in Ehrlich ascites tumor cells. Namely, the number of ribosomal cistrons per nucleolus for small, intermediate and large nucleoli is 40, 60 and 130, respectively.     

Accessory nucleoli in microsporocytes of hybrid Phlox

Studies were conducted on the number of nucleoli present during diplotene and diakinesis in plants adjacent to, at the edge, and at the center of a zone of secondary intergradation between Phlox pilosa subsp. pilosa and P. pilosa subsp. fulgida. Assessory nucleoli were present in some cells of about 75% of the plants examined. Nucleolar numbers varied from 1 to 5. Where 2 or more nucleoli were present they were usually attached to different chromosomes. As the number of nucleoli increased, the volume of single nucleoli decreased so that volume of all nucleoli was roughly that of a normal single nucleolus. — Only 1% of the microsporocytes from populations adjacent to the zone had accessory nucleoli as compared to 4.6% of the cells in populations at the edge of the zone, and 7.7% in populations in the center. The correlation between population hybridity and incidence of accessory nucleoli is r = 0.80.     

Nucleolus and large nucleolar aggregates of condensed chromatin in interphase nuclei of L 929 cells

Summary Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase.In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.     

马缨丹乳油及其混剂对黄曲条跳甲的拒食活性

测试了自制的12.5%马缨丹乳油对黄曲条跳甲成虫拒食活性、持效性及其与7.5%鱼藤酮乳油、1.5%除虫菊水乳剂、0.3%印楝乳油混配的联合活性。该乳油1000倍液处理菜心叶片后48h对黄曲条跳甲成虫具有40%的拒食率,随着浓度升高,拒食活性增强;200、400和600倍液处理120h后对黄曲条跳甲成虫的累计拒食率分别达到76%、68%和43%。12.5%马缨丹乳油与7.5%鱼藤酮乳油以5∶5比例混配使用时,增效作用最为明显,拒食中浓度AFC50为5.25μg.ml-1,共毒系数CTC为213;与1.5%除虫菊水乳剂以1∶9比例混配使用时的增效作用次之,共毒系数CTC为149;与0.3%印楝乳油以9∶1、7∶3、5∶5、3∶7和1∶9比例混配使用时,其共毒系数CTC均100,表现为相互拮抗作用。结果表明:12.5%马缨丹乳油对黄曲条跳甲成虫具有强烈的拒食活性,其200倍液和400倍液拒食持效性好,且该乳油可与7.5%鱼藤酮乳油混配使用。