Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.Abbreviations NAC nucleolus associated chromatin - CES centromeric structures - NOR nucleolar organizing region - NAB nucleolus associated body - IG interchromatin granules - RNP ribonucleoprotein - Mab monoclonal antibody by M.F. Trendelenburg     

Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles

Nucleoli from auxin-treated tissues (Glycine max L. var Wayne or Kaoshiung No. 3) were isolated and purified by Percoll density gradient centrifugation. There was a 2.1-fold increase in RNA and a 2.8-fold increase in protein after a 24-h auxin treatment per unit nucleolar DNA. More than 150 acid-soluble protein spots were associated with the auxin-treated nucleoli on two dimensional (2-D) gel electropherograms.

Nucleoli from auxin-treated tissue were fractionated by suspension in 20 millimolar dithiothreitol at room temperature for 20 minutes into two distinct fractions referred to as the nucleolar chromatin and preribosomal particle fractions. The DNA:RNA:protein ratio of the chromatin fraction was 1:2.5:14. Most of RNA polymerase 1 activity and nucleolar DNA recovered in this fraction. The acid-soluble proteins in the chromatin were resolved into 32 protein spots on 2-D gel electropherogram. The most abundant spots were identified as histones.

The nucleolar preribosomal particle fraction had a DNA:RNA:protein ratio of 1:24:102 and contained only trace amounts of RNA polymerase 1 activity and only 10 per cent of the nucleolar DNA. Acid-soluble proteins associated with these particles were resolved into 78 protein spots; 72 of these (acid-soluble) protein spots corresponded in 2-D gel electrophoresis to 80S cytoplasmic ribosomal proteins. Some 15 protein spots found in 80S ribosomal proteins were absent in the preribosomal particles. It seems reasonable, based on these data, that the enlargement of nucleoli after auxin treatment is primarily due to the large increase in ribosomal proteins and rRNA which accumulate and assemble in the nucleoli in the form of preribosomal particles.

     

Studies on chromatin organization in a nucleolus without fibrillar centres

In embryonic cell-line derivative KCo of Drosophila melanogaster, the nucleolus, like most nucleoli, contains a small proportion of ribosomal DNA (1-2% of the total nucleolar DNA). The ribosomal DNA is virtually the only active gene set in the nucleolus and is found among long stretches of inactive supercoiled heterochromatic segments. We have demonstrated by use of a Feulgen-like ammine-osmium staining procedure that, depending on the state of growth, more or less fibres of decondensed DNA emanating from the intra-nucleolar chromatin (which is in continuity with the nucleolus-associated chromatin) ramify and unravel within the central nucleolar core to be transcribed. The nucleolus expands or contracts with the variation of activity and could belong to a supramolecular matricial structure such as is shown after extraction of the nuclei. After a long period of exposure to high doses of actinomycin D, the central nucleolar core became an homogeneous fibrous structure that could be interpreted as an aggregate of protein skeletal elements. The mechanism of repression and derepression of the nucleolar chromatin could thus be explained by a mechanism involving in part a sub-nucleolar structure. We propose a schematic organization of the nucleolar chromatin in KCo cells of Drosophila and discuss it in relation with other nucleolar organizations.     

Nucleolar lesions induced by ribonuclease in living cultured cells

The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.     

Nucleolar lesions induced by ribonuclease in living cultured cells

The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.     

Effect of chromium(III) on nucleolar RNA synthesis

The enhancement of hepatic nucleolar RNA synthesis induced by Cr(III) in partially hepatectomized rats and its mechanisms are described. Cr(III)-administered (0.5 mg Cr/kg, ip) and then partially hepatectomized rats were significantly enhanced in the hepatic nucleolar RNA synthesis at the very early stage of liver regeneration. This enhancement was caused both by the induction of newly found nucleolar Cr-bound protein of 70 kD (Cr-p70) and by the activation of nucleolar chromatin, both of which arose from nuclear accumulation of Cr together with partial hepatectomy. Studies on the mechanism of this enhancement indicated that the Cr-p70 bound to the activated nucleolar chromatin and loosened its higher-order structure, resulting in an increase of the B-form fraction of chromatin DNA. The degree of this loosening well correlated with the amount of Cr-p70 bound to chromatin and also with the extent of elevation of RNA synthesis. Some molecular species of nonhistone proteins in chromatin were found to play an important role in the interaction to Cr-p70. These results suggest a possibility that the action of Cr is involved in cell proliferation process.     

Nucleolar DNA organization in the interphase nuclei of mouse fibroblasts.

The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.     

Actinomycin D binding in vitro: active chromatin preferred.

When [3H] Actinomycin D (Act. D) is used to interact with nuclei and nucleoli in vitro, it binds preferentially to nucleolar chromatin. The preferential binding is no longer detectable, when purified nuclear and nucleolar DNAs are used. In parallel, Act. D preferentially inhibits nucleolar over nuclear RNA synthesis when chromatin templates are used, and the preferential inhibition is lost when purified nuclear and nucleolar DNAs are used. It is concluded: 1) the preferential inhibition of nucleolar over nuclear RNA synthesis by Act. D is a direct reflection of the preferential binding of Act. D to the nucleolar chromatin; and 2) the nucleolar chromosomal proteins, not the nucleolar DNA, confer the preferential binding of Act. D.     

Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.     

Isolation of a chromatin fraction from calf endometrium highly enriched in estradiol binding sites.

The intranuclear distribution of [3H]-estradiol binding sites was studied in highly purified nuclei isolated from calf endometrial tissue pre-incubated with the labeled hormone. The major part (approximately 85%) of the receptor bound estradiol was found associated with the extranucleolar chromatin; only a negligible amount of [3H]-estradiol (approximately 8%) sedimented with the nucleolar fraction. [3H]-estradiol labeled chromatin was then fragmented by sonication and fractionated by sucrose density gradient sedimentation under different conditions of centrifugation. The vast majority of the [3H]-estradiol was invariably found to be associated with a fast sedimenting fraction which contained only 5 to 10% of the nuclear DNA. The concentration of estradiol receptors (per weight of DNA) in this fraction was 25- to 50-fold higher than that found in the slow sedimenting major chromatin component. Chemical analysis showed this fraction to have a high protein/DNA ratio but no phospholipids were detected.     

Genome organization in and around the nucleolus

The nucleolus is the largest compartment of the cell nucleus and is where ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. In addition to rRNA gene clusters that build the core of this subnuclear structure, nucleoli are associated with condensed chromatin. Although the higher order structures of rRNA genes and nucleolus-associated chromatin have been studied for decades, detailed molecular insights into the constituents and organization of the nucleolar genome are only beginning to emerge. Here, we summarize current views on the structural organization of nucleolar DNA and on the targeting and anchoring of chromatin domains to this subnuclear compartment.     

Studies on the solubilized ribonucleic acid polymerase from rat ventral prostate gland

1. By using ultrasonic treatment in media of high ionic strength, the RNA polymerase activities associated with prostatic nuclei and nucleoli can be completely solubilized. Such enzyme preparations are entirely dependent on the provision of added DNA for full activity. 2. The solubilized enzymes from the nucleolar and extranucleolar regions can be separated by ion-exchange chromatography. 3. Based on differences in the optimum DNA templates, pH optima and the effects of ammonium sulphate on the activities in vitro, Mn(2+)- and Mg(2+)-specific enzymes are associated with both the nucleolar and extranucleolar regions of prostatic nuclei. 4. Androgenic hormones administered in vivo have a particularly pronounced effect on the activity of Mg(2+)-dependent enzyme associated with the isolated prostatic nucleolus. 5. Time-course experiments in vivo show that androgens induce a rapid stimulation of the solubilized Mg(2+)-dependent nucleolar enzyme before a pronounced activation of nucleolar chromatin can be measured. 6. The implications of these findings to the mechanism of action of androgenic steroids are discussed.     

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.     

Ultrastructural studies of H-1 parvovirus replication : III. Intracellular localization of viral antigens with immunocytochrome c

Immunoelectron microscopy with cytochrome c conjugated anti-H-1 IgG was used to localize antigens of the parvovirus H-1 within synchronized human NB cells. Since glutaraldehyde destroyed H-1 antigenicity, a fixative containing formaldehyde was developed which preserved both cellular ultrastructure and antigenic function. The earliest H-1 specific staining occurred on the heterochromatin bordering the nuclear envelope at 8 h post infection (p.i.). At 10 h p.i., labeling was found on the chromatin associated with the nucleolar surface and tufts of heterochromatin distributed throughout the nucleoplasm. Except for this H-1 labeling, the chromatin appeared indistinguishable from that of uninfected cells. By 12 h p.i., however, coinciding with the abrupt rise in synthesis of H-1 hemagglutinin and infectious virus, H-1 labeled intranuclear chromatin had condensed and migrated toward the nuclear membrane. Also, trabeculae of intranuclear chromatin were tagged with anti-H-1 conjugate as contraction of nucleolar chromatin and disintegration of nucleolar ultrastructure began. Condensation of the nucleolar associated chromatin and nuclear heterochromatin appeared complete by 18–36 h p.i. when thick zones of this H-1 labeled material were observed at the nucleolar and nuclear periphery. Our results indicate that the binding of unassembled H-1 proteins to specific regions of chromatin is associated with their condensation and margination, resulting in early nucleolar destruction and subsequent nuclear damage during H-1 infection.     

Relationship of nucleolus-associated bodies with the nucleolar organizer tracks in plant interphase nuclei (Pisum sativum)

Nucleolus-associated bodies (NABs) have long been noted in interphase nuclei of a wide variety of plant species. We have recently shown that these bodies consist largely of snRNPs and that they are located on the nucleolar surface in the immediate vicinity of the nucleolar organizer tracks. The present study revealed that, following exposure of roots to KCN, an agent that induces nucleolar segregation, NABs were intimately associated with intranucleolar chromatin. Although immunocytochemical tests with anti-DNA indicated that NABs contained no demonstrable amounts of DNA, our observations nevertheless add further support to the notion that these bodies are somehow related to the nucleolar chromosomes.