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.     

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.     

Nucleolar DNA in oocytes of crickets: representatives of the subfamilies Oecanthinae and Gryllotalpinae (Orthoptera: Gryllidae)

A large extrachromosomal mass of Feulgen positive material, the DNA body, has been visualized in early prophase oocytes of crickets (Orthoptera: Gryllidae) representative of the closely related subfamilies Gryllinae and Nemobiinae. A similar structure is present in oocytes of representatives of two subfamilies of crickets (subfamilies Oecanthinae and Gryllotalpinae) which taxonomically and phylogenetically are quite separate from those mentioned previously. In situ hybridization demonstrates that the body contains amplified copies of genes coding for ribosomal RNA. Unlike the DNA body in early diplotene oocytes of representatives of the subfamily Gryllinae, which is closely associated with the developing nucleolar apparatus, the DNA body in oocytes of the Oecanthinae and Gryllotalpinae cannot be demonstrated during diplotene. In the Oecanthinae, the nucleolar apparatus of early diplotene stage oocytes is composed of four to seven separate structures, the ribonucleoprotein of which has a characteristically lamellated appearance. During late diplotene, these nucleoli give rise to many smaller structures which are distributed throughout the germinal vesicle. In early diplotene stage oocytes of Scapteriscus acletus (Subfamily: Gryllotalpinae), the nucleolar apparatus consists of a single compact mass of ribonucleoprotein. In contrast to the oocytes of all other crickets that have been studied, the nucleolus of S. acletus remains single throughout diplotene. In situ hybridization analysis indicates that the amplified genes coding for rRNA which are localized in the DNA body of early prophase oocytes become incorporated into this compact nucleolar mass. Differences in nucleolar structure appear to reflect differences in the organization of amplified genes coding for rRNA.     

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.     

Chromatin organization in Paracentrotus lividus eggs

After purification by buoyant density centrifugation in ethidium bromide - CsCl gradient and electrophoretic fractionation, the DNA fragments isolated from P. lividus egg nuclei incubated with micrococcal nuclease exhibit a typical oligomeric pattern. Analysis of chromatin samples digested to an increasing extent by micrococcal nuclease reveals that the structural organization of egg chromatin is heterogeneous, both in terms of repeat size and degree of sensitivity to nuclease attack. The nucleosomal repeats of P. lividus sperms and embryos up to the mesenchyme blastula stage have also been determined, for comparison.     

Development of the large nucleolus in the oocytes of the copepod Acanthocyclops vernalis: an electron microscope study

A central feature of oogenesis in the copepod crustacean, Acanthocyclops vernalis, is the development of a very large nucleolus in the oocytes. This nucleolus appears to be the only source of rRNA for the oocyte, as no helper cells are present. Previous work has suggested that ribosomal DNA sequences other than those found at the morphological nucleolar organizers are participating in the elaboration of this nucleolus. It has been hypothesized that chromatin diminution, which occurs during early embryonic development, may involve the loss of these rDNA sequences, which are needed only for the production of ribosomes during oogenesis. The present study examines the development of the large oocyte nucleolus at the electron microscopic level. Nucleologenesis in A. vernalis was found to proceed through 5 stages. During the first 3 stages nucleolar morphology resembled that described in other organisms. In the last 2, however, nucleolar morphology changed radically and the nucleolus was seen to increase greatly in size while breaking up into multiple subunits. The subunits initially resemble active nucleoli, although in the last stage, synthesis appears to stop, as the nucleolus was found to consist only of dense areas containing ribosome-like particles. These observations are consistent with the hypothesis that diminuted DNA contains ribosomal RNA genes.     

Nucleolar organization in oocytes of gryllid crickets: subfamilies Gryllinae and Nemobiinae

A cytological and cytochemical survey was made of nucleolar changes during oocyte development in several different species of crickets (Gryllidae) representing the subfamilies Gryllinae and Nemobiinae. A large mass of extrachromosomal DNA is characteristic of the pachytene stage nuclei of all species examined. Nucleolar material accumulates at the periphery of the DNA body as the cells proceed into the diplotene stage of development. As the oocytes proceed through diplotene, the nucleoli reorganize into many small masses which eventually disperse in the nucleoplasm. These changes reflect both an increase in number and in size of the nucleolar material during the diplotene stage and the mode by which dispersal of nucleolar material is accomplished. These differences probably reflect differences in the organization of extrachromosomal nucleolar DNA.     

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.     

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     

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.     

Structural organization of chromatin in nucleolar organizer regions of nucleoli with a nucleolonema-like and compact ribonucleoprotein distribution

We have studied the relationship between the structural organization of intranucleolar chromatin and fibrillar nucleolar structures, fibrillar centers, and RNP fibrillar component, which are the interphase counterpart of metaphase nucleolar organizer regions (NORs), in regenerating rat hepatocytes and in a human tumor cell line (TG cells). These two cell types were characterized by a nucleolonema-like and compact nucleolar RNP distribution, respectively. We found that, in sections selectively stained for DNA, the intranucleolar chromatin composed of extended, nonnucleosomal DNA filaments formed roundish agglomerates with a spatial distribution which was superimposable on that of the fibrillar centers and the RNP fibrillar component around them and on sites of the silver reaction in samples selectively stained for Ag-NOR proteins. The agglomerates of extended nonnucleosomal DNA filaments were small and numerous in regenerating hepatocyte nucleoli, in which the RNP components had a nucleolonema-like distribution, whereas they were large and few in TG cell nucleoli, in which the RNP components showed a compact organization. Since the pattern of ribosomal RNA synthesis and processing was similar in the two cell types, a model was proposed in which the difference in size and shape of the agglomerates of extended DNA might be responsible for the different structural organization of the RNP components.