The Structural Transformation of Nucleolar DNA and Its Arrangement in Nucleolus of Allium cepa Cells

By using the DNA specific cytochemical staining method (NAMA-Ur) and conventional electron microscopic technique, the authors examined the configuration of intranucleolar DNA in Allium cepa L. cells and found that nucleolar DNA within the fibrillar center (FC) underwent a structural transformation process from condensed to extended state. The authors' observations also displayed a continuous arrangement process of nucleolar DNA, i.e., the extranucleolar DNA entered FC through the nucleolar organizer region (NOR) channel, then extended to the periphery of FC or to the border between FC and dense fibrillar component (DFC), and distributed along the periphery of FC. Thence, by passing through the NOR channel between FCs, the nucleolar DNA continued to transfer to other FCs and arranged in the same above-mentioned forms.     

洋葱细胞核仁内DNA的结构变化及排布过程(英文)

以洋葱 (AlliumcepaL .)细胞为研究材料 ,应用DNA细胞化学特异染色方法 (NAMA_Ur)及常规电子显微镜技术 ,观察了洋葱细胞核仁FC(纤维中心 )内DNA的超微结构 ,发现FC内DNA存在着一个介于集缩到解集缩之间的变化过程 ,并揭示了DNA在核仁内的连续排布过程 ,即核仁外DNA经过核仁通道进入到FC后 ,继续沿FC的边缘或DFC(致密纤维成分 )与FC的交界处环绕FC而排布 ,再经FC之间的核仁通道 ,延伸到另外的FC区域     

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.     

Phosphoprotein pp135 is an essential component of the nucleolus organizer region (NOR)

The association of phosphoproteins pp135 and pp105 with distinct substructures of the nucleolus was studied by cytochemical and immunological methods at the light microscopic and electron microscopic level. Both phosphoproteins exhibited a very high affinity for silver and Giemsa staining compared to other nucleolar proteins. Immunolocalization of pp135 and pp105 during mitosis by light microscopy revealed a tight association of pp135 with the silver staining nucleolus organizer region (NOR), whereas pp105 (cross-reacting with C23) appeared to be only partially associated with the NOR, exclusively at telophase. At the immunoelectron microscopic level the distribution of pp135 and pp105 was investigated in interphase nucleoli. Phosphoprotein pp135 was located in the fibrillar shell and pp105 in the fibrillar shell and the granular zone. The fibrillar centers were essentially free of both phosphoproteins..     

Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle of Physarum polycephalum

Monoclonal antibodies raised against the 34-kD nucleolar protein, B-36, from the slime mold Physarum polycephalum have been used to examine the electron microscopic localization of B-36 during the cell cycle in Physarum. During interphase, B-36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B-36, fibrillarin. During mitosis, B-36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA-specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that in Physarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the "NOR" equivalents do not separate during mitosis as in mammalian cells. In addition, the B-36-enriched nucleolar remnants appear to be recycled from one cell cycle to the next.     

Subnucleolar distribution and organization of Vicia faba L. rDNA in situ

The distribution and organization of nucleolar DNA in Vicia faba L. was analyzed by specific cytochemical staining using NAMA-Ur. The results showed that nucleolar DNA was distributed in the FCs and at the FC/DFC junctions. Statistical analysis showed that the rRNA genes occupied about one-third of the total dense fibrillar component region. The rDNA was condensed in some regions and uncondensed in others. Nucleolus-associated chromatin extended from outside the nucleolus to the periphery of the FCs via nucleolar channels, suggesting a possible origin for nucleolar DNA.     

Silver staining,immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.     

多头绒泡菌SC35类蛋白的免疫电镜研究

经抗SC35单克隆抗体标记后,在电子显微镜下观察到多头绒泡菌S、G2、前期、中期和后末期细胞核中存在大量金颗粒,说明多头绒泡菌细胞核含有SC35类蛋白。在G2期和前期时,SC35类蛋白主要分布在细胞核的核仁区域和非核仁区域的染色质间区域;中期和后-末期时,SC35类蛋白主要分布在细胞核内染色体间区域;说明染色质(体)间区域和核仁区域是富含SC35类蛋白的区域。对核仁的进一步观察指出,在核仁中金颗粒主要分布在DFC,FC中的金颗粒很少,说明在核仁中SC35类蛋白主要存在于DFC组分中。     

Studies on changes in nucleolar organizer region of human promyelocytic leukemia cells (HL-60) treated with retinoic acid

Changes of nucleolar organizer region in HL-60 cells after treated with retinoic acid (RA) were studied with techniques of silver-staining nucleolar organizer region (Ag-NOR) in metaphase karyotypes, Brachet's reaction and with our improved TEM techniques for studying silver-stained active nucleolar organizer region (Ag-aNOR) in interphase nucleoli. Number of Ag-NOR in HL-60 cells is 4.5/cell on average. The Ag-NOR number of cells treated with RA showed no remarkable difference from that of control group. Ag-aNOR number treated with RA was reduced obviously as compared with that of control group. Meanwhile, the changes of nucleolus number showed by Brachet's reaction were in accordance with those of Ag-aNOR. Therefore, it may be concluded: (1). Though the number of active rRNA genes did not changed after the differentiation of HL-60 cells induced by RA, their expression was clearly inhibited: (2). The relationship between the changes of Brachet-No and Ag-aNOR is in positive correlation (r = 0.98, p less than 0.01). EM examination of Ag-aNOR of HL-60 cells reveals that Ag-protein (RNA polymerase I) only presented in fibrillar centers (FC) and the dense fibrillar components (DFC) of nucleolus. In addition, in control group, large amount of Ag-protein, FC, DFC and granular components (GC) were observed, and there were many large nucleoli in a nucleus, meanwhile, the cells of the treated group tended to be mature, with a decrease in the amount of Ag-protein, FC, DFC and GC accordingly, and the nucleoli reduced both in size and number significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochemical variations in the nucleolus during spermiogenesis in man and monkey

Summary The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate; EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15–20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material. In mature spermatids granular material revealed by PTA and silver staining methods was found in the nuclear pockets bounded by the redundant nuclear envelope.     

Fine structure of nucleoli in micronucleated cells

The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate 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.     

豌豆细胞核仁中rDNA的超微定位和排布构型

利用细胞化学DNA特异染色法——NAMA-Ur特异染色法对豌豆细胞核仁中rDNA的位置及其排布构型进行了原位观察。结果表明,核仁中的rDNA位于纤维中心(FC)以及FC与致密纤维组分的交界处,以环绕FC的形式排布。不同位置的rDNA成分都具有集缩和解集缩两种形态结构,核仁外的核仁伴随染色质经过核仁通道进入核仁,沿FC周边排列,与其中的DNA相连。