Chromosomal Nucleolar Organizer Research Articles

Cytogenetic instability of chromosomal nucleolar organizer regions (NORs) in cloned mouse L929 fibroblasts.

Ribosomal DNA (rDNA) gene codes for 18S, 5.8S, and 28S rRNA form tandem repetitive clusters, which occupy distinct chromosomal loci called nucleolar organizer regions (NORs). The number and position of NORs on chromosomes are genetic characteristics of the species although within a cell, the NOR sizes can significantly vary due to loss or multiplication of rDNA copies. In the current study, we used mouse L929 fibroblasts, the aneuploid cells which differ in the FISH- and Ag-NOR numbers, to examine whether the parental NOR variability is inherited in clones. By statistical analysis, we showed that the cloned fibroblasts were able to restore the NOR numerical characteristics of the parental cells after long-term culturing. These results support the idea that mammalian cells may have mechanisms which control the number and activity of NORs at the population level. In L929 fibroblasts, we also regularly observed laterally asymmetry of FISH-NORs that evidenced in an unequal distribution of the mother rDNA copies between the daughter cells in mitosis.

The Karyotype of the flathead sculpin Megalocottus platycephalus taeniopterus (Kner, 1868) (Pisces: Cottidae) from Vostok Bay, Sea of Japan

For the first time, we studied the karyotype of the flathead sculpin Megalocottus platycephalus taeniopterus (Kner, 1868) from Vostok Bay of the Sea of Japan. The karyotype is stable: 2n = 42 (2 metacentric, 2 submeta-subtelocentric, 30 subtelocentric, and 8 acrocentric chromosomes), NF = 44 + 2. The nucleolar organizers (NOs) were identified using Ag banding in two pairs of chromosomes: in the telomeric parts of the short arm of the medium-size subtelocentric chromosome and the long arm of the large acrocentric chromosome. Variations in the number of nucleolar organizer chromosomes and in the number of NO staining blocks were found. Comparison of the karyotypes of M. p. taeniopterus and previously studied M. p. platycephalus (Pallas, 1814) from the northern Sea of Okhotsk revealed their similarity in the number and morphology of chromosomes and the number of chromosome arms and difference between the subspecies in the number and location of NO, which allows their discrimination.

The karyotype of the flathead sculpin Megalocottus platycephalus platycephalus (Pallas, 1814) (Pisces: Cottidae) from Odyan Bay, Sea of Okhotsk

This study deals with the karyotype of the flathead sculpin Megalocottus platycephalus platycephalus (Pallas, 1814) (family Cottidae) from Odyan Bay of the Sea of Okhotsk. The karyotype is stable: 2n = 42 (2 metacentric, 2 submeta-subtelocentric, 30 subtelocentric, and 8 acrocentric chromosomes), NF = 44 + 2. The nucleolar organizers (NOs) were identified using Ag-banding in the telomere parts of the short arms of two large subtelocentric chromosomes. Variations in the number of nucleolar organizer chromosomes and in the number of NO-staining blocks were found. Comparison of the karyotype of M. p. platycephalus with the karyotypes of previously studied species of the genus Myoxocephalus from the Sea of Japan and the Sea of Okhotsk revealed their significant similarity in the main features and difference in the number and localization of NORs.

Relationship between the Argyrophilic Proteins of the Nucleolar Organizer Region and Stage of Squamous-Cell Carcinoma of the Lung

Argyrophilic proteins associated with chromosome nucleolar organizer regions (Ag-NOR proteins) were studied in lung squamous-cell carcinoma cells. After routine staining with silver nitrate, the area index and coefficient of variations of Ag-NOR protein were estimated by computer-aided image analysis. The Ag-NOR protein area indexes of tumor cells differed in clinical morphological variants of squamous-cell cancer (differing by T and N parameters, size of the primary node <3 cm or larger, by disease stage, and differentiation degree). Ag-NOR protein variation coefficient can be used for discriminating the tumors by size (<3 cm and larger), presence/absence of metastases, and evaluation of squamous-cell cancer differentiation degree. Ag-NOR protein area index and variation coefficient can be used as additional objective differential diagnostic criteria of clinical and morphological forms of squamous-cell carcinoma of the lung.

Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs)

Raptor is the key scaffolding protein that recruits mTOR substrates to rapamycin-sensitive mTOR complex 1 (mTORC1), a molecular integrator of mitogenic and nutrient/energy environmental inputs into protein translation and cell growth. Although Raptor phosphorylation on various sites is pivotal in the regulation of mTORC1 activity, it remains to be elucidated whether site-specific phosphorylation differentially distributes Raptor to unique subcellular compartments. When exploring the spatiotemporal cell cycle dynamics of six different phospho (P)-Raptor isoforms (Thr706, Ser722, Ser863, Ser792 and Ser877), a number of remarkable events differentially defined a topological resetting of P-RaptorThr706 on interphasic and mitotic chromosomes. In interphase nuclei, P-RaptorThr706 co-localized with fibrillarin, a component of the nucleolar small nuclear ribonucleoprotein particle, as well as with RNA polymerase I, the enzyme that transcribes nucleolar rRNA. Upon Actinomycin D-induced nucleolar segregation and disaggregation, P-RaptorThr706 was excluded from the nucleolus to accumulate at discrete nucleoplasmic bodies. During mitosis, CDK1 inhibition-induced premature assembly of nucleoli relocated fibrillarin to the surrounding regions of chromosomal-associated P-RaptorThr706, suggesting that a subpopulation of mitotic P-RaptorThr706 remained targeted at chromosomal loops of rDNA or nuclear organizer regions (NORs). At the end of mitosis and cytokinesis, when reassembly of incipient nucleoli begins upon NORs activation of rDNA transcription, fibrillarin spatially reorganized with P-RaptorThr706 to give rise to daughter nucleoli. Treatment with IGF1 exclusively hyperactivated nuclear P-RaptorSer706 and concomitantly promoted Ser2481 autophosphorylation of mTOR, which monitors mTORC1-associated catalytic activity. Nucleolar- and NOR-associated P-RaptorSer706 may physically link mTORC1 signaling to ever-growing nucleolus plurifunctionality including ribosome biogenesis, cell stress sensor and cell cycle/aging control.

Quantitative analysis of NOR expression in a B chromosome of the grasshopper Eyprepocnemis plorans

The B24 chromosome in the Torrox population of the grasshopper Eyprepocnemis plorans is recurrently attached to a nucleolus in diplotene cells, indicating the activity of its distally located ribosomal DNA (rDNA). The frequency of males expressing the B chromosome nucleolus organizer region (B-NOR) almost doubled in 4 years. The likelihood of expressing the B-NOR increased with the B number and, in males expressing it, about 20% of their cells showed a nucleolus attached to the B. When active, the B-NOR contributed more than 25% of total cell nucleolar area (NA). Within males expressing the B-NOR, total cell NA did not differ between cells showing the active or inactive B-NOR, suggesting that total cell NA is tightly regulated in this species. However, this parameter tended to increase in this population from 1999 to 2004, in parallel to the neutralization process which is taking place in this population. Finally, an analysis of A chromosome NOR interdependence for activity revealed a positive correlation among autosomes but a negative correlation between autosomes and the X chromosome, the manifestation of which depends on B-NOR activity. These results are discussed in the context of the nucleolus as a sensor of the stress caused by parasitic B chromosomes.

THE ANALYSIS OF NUCLEOLAR ORGANIZER REGIONS IN Chalcalburnus mossulensis (Pisces: Cyprinidae)

In this study, nucleolar organizer regions of Chalcalburnus mossulensis were investigated. Analysis of karyotypes revealed that the diploid numbers of chromosomes was 2n=50. There were six pairs of metacentric, eight pairs of submetacentric, five pairs of subtelocentric and six pairs of acrocentric chromosomes in the karyotype. It was shown that C. mossulensis possessed two pairs of nucleolar organizer regions-bearing chromosomes. One nucleolar organizer region was located terminally on the longer arm of a larger sized submetacentric chromosome, whereas the other nucleolar organizer region was located terminally on the short arm of a medium sized submetacentric chromosome. There was no significant difference in chromosomal nucleolar organizer region phenotypes and in number of nucleolar organizer regions among localities. Our results were compared with previous results of other researchers.

Disposition of ribosomal DNAs in the chromosomes of perennial oats (Poaceae: Aveneae)

The chromosomal loci of 5S and 45S ribosomal DNAs (rDNAs) and the activity of nucleolar-organizing regions (NORs) were analysed in perennial oats of the genera Ammophila, Amphibromus, Arrhenatherum, Avena, Deschampsia, and Helictotrichon s.l. (Poaceae: Aveneae) using fluorescence in situ hybridization, staining with chromomycin/4′,6-diamidino-2-phenylindole (DAPI), and silver impregnation. All chromosomes with a secondary constriction were nucleolar active. In chromosomes without a secondary constriction, NORs corresponded exclusively to broad bands of 45S rDNA with chromomycin-positive, DAPI-negative, and silver-positive stainability. Additional minor bands of 45S rDNA showed no nucleolar activity. 5S rDNA was localized mostly in loci different from the nucleolar-active 45S rDNA. If both rDNAs occurred within the same chromosome, they were at largely corresponding distances from the centromere, irrespective of their particular localization in either the same chromosome arm or in opposite arms. In the latter case, 5S rDNA was never more distal to the centromere than 45S rDNA. A new model was devised to explain this non-random distribution of both rDNAs in nucleolar-organizing chromosomes, which identified the Rabl orientation of chromosomes as ensuring a spatial proximity of 5S to 45S rDNA in interphase nuclei, even if they were localized in opposite arms. The possible role of the Rabl orientation in determining the spread and accumulation of 5S rDNA sequences in further chromosomes of the genome was discussed. B chromosomes were devoid of 5S rDNA, but most contained 45S rDNA and were nucleolar active. In some large groups of species, the number and arrangement of 5S and 45S rDNA sites in the chromosomes were remarkably uniform, especially in Helictotrichon subgenus Helictotrichon and Helictotrichon subgenus Pratavenastrum. Such distribution patterns have survived many speciation processes and have also remained widely unchanged in polyploids.

Cytogenetics Studies in Two Populations of Astyanax scabripinnis with 2n=48 Chromosomes(Teleostei, Characidae).

Cytogenetic analysis in 2 populations of Astyanax scabripinnis from Sao Domingos and Tamboara streams, affluents of the Ivai river basin, was carried out. Both populations presented same diploid of 2n=48 chromosomes, although they might be differentiated by karyotypic formulae (10M, 20SM, 8ST, 10A and 10M, 24SM, 6ST, 8A, respectively) and number of nucleolar organizer chromosomes (3 and 7 NORs, respectively). Besides the population from Sao Domingos showed 1 metacentric B microchromosome restricted to females. Low frequency of this B microchromosome suggests a recent origin in populations of Astyanax scabripinnis and its adaptive function remains unknown. Aspects related to karyotype diversity of this species complex are discussed, contributing to the understanding of the mechanisms involved in the diversity and speciation on the group.

Activation Patterns of the Nucleolar Organizer Region in Astyanax scabripinnis Populations (Pisces, Characidae).

The analysis of the nucleolar organizer regions (NORs) in 4 Astyanax scabripinnis populations collected in the Yukatan stream (population A), Ligeiro stream (population B), Sarandi river (population C) and Agua do Rancho river (population D) indicated the existence of inter-and intrapopulation variations in the number of NORs. The present study also showed a probable mechanism of preferential activation of nucleolar chromosomes, suggesting the presence of main activators as well as different activation patterns in each population. In population A, the large metacentric chromosome was the main nucleolar organizer occurring in about 67% of the cells analyzed, with this population showing the most characteristic NOR pattern compared to the other populations. In populations B, C and D, the main nucleolar organizer chromosomes were a large acrocentric chromosome (65%), a large submetacentric (70%) and a medium-sized submetacentric (71%), respectively.

Relative distribution of rDNA and proteins of the RNA polymerase I transcription machinery at chromosomal NORs.

Using confocal and immunofluorescence microscopy the relative distribution of the ribosomal chromatin and some proteins of the RNA polymerase I transcription machinery such as upstream binding factor (UBF), RNA polymerase I and DNA topoisomerase I was analyzed on chromosomal nucleolus organizer regions (NORs) of PtK1 cells. Staining with various DNA fluorochromes revealed that the ribosomal chromatin may be found at the axial region of the NOR and also at lateral expansions around the axis that can also be detected by in situ hybridization. It was observed that the transcription machinery shows a crescent-shaped distribution around the axial ribosomal chromatin at the NOR of metaphase and anaphase chromatids. An ultrastructural analysis of serially sectioned NORs supports this crescent-shape organization. Taking into account previous and present results and the loop/scaffold model of chromosome structure, we propose a model of NOR organization. The model proposes that ribosomal genes that were inactive in the preceding interphase would be present as condensed short Q-loops occupying the axial region of the NOR. Ribosomal genes previously active during interphase would be undercondensed as large R-loops associated with the transcription machinery, which is distributed in a crescent-shaped fashion around the previously active ribosomal DNA.

Characterization of mitotic chromosomes of four species of the genusDiplodus: karyotypes and chromosomal nucleolar organizer region phenotypes

Characterization of mitotic chromosomes of four species of the genusDiplodus: karyotypes and chromosomal nucleolar organizer region phenotypes

Characterization of mitotic chromosomes of four species of the genus Diplodus: karyotypes and chromosomal nucleolar organizer region phenotypes

Karyotypes have been described in four Mediterranean species of the genus Diplodus (Teleostei, Sparidae), D. vulgaris, D. puntazzo, D. sargus and D. annularis. Chromosomes were mainly acrocentric in all but D. vulgaris, where certain chromosome pairs were subtelocentric. A remarkable intraspecific heteromorphism in the number of NOR‐bearing chromosomes along with a substantial interspecific variability in position of chromosomal Ag signals have been encountered. The presumed origin of multiple NOR‐bearing chromosomes in Diplodus species and variation of the NOR location is discussed.

Chromosomal nucleolar organizer region (NOR) phenotypes in nine species of the genus Ophryotrocha (Polychaeta: Dorvilleidae)

Chromosomal nucleolar organizer region (NOR) phenotypes have been characterized in nine species of the genus Ophryotrocha (Polychaeta: Dorvilleidae), namely O. notoglandulata, O. sp. macrovifera, O. sp. labronica pacifica, O. labronica labronica, O. puerilis puerilis, O. diadema, O. sp. robusta, O. gracills and O. hartmanni. Irrespective of chromosome number and morphology, Ag positive regions were terminally located in all but one species, O. diadema, where the NORs were pericentromerical in a metacentric pair. The presence of a single chromosome pair bearing NOR in invertebrates is considered an ancestral trait. According to this assumption, O. sp. robusta, O. dialema, and perhaps O. p. puerilis appear to be more ancestral than the other species. On the contrary, O. notoglandulaia, O. sp. macrovifera, O. sp. labronica oacifica, with two chromosomal pairs bearing NOR sites, seem to represent examples of further evolution within the genus Ophryotrocha.

Prognosis of Canine Mast Cell Tumors: A Comparison of Three Methods

In this study, age, sex, recurrence, metastasis, death rate, and histologic patterns were in agreement with those of previous reports on canine mast cell tumors. Histologic grading, mitotic index, chromosome nucleolar organizer regions stained with silver (AgNORs), and anti-proliferating cell nuclear antigen (PCNA) were evaluated as indicators of prognosis. Histologic grading, AgNORs estimated in 100 cells, and PCNA-labeled fraction estimated in five high power fields (HPFs) were significantly different between recurring and nonrecurring tumors. Those prognostic factors were also significantly different between tumors that metastasized and those that did not. The survival time was lower in dogs with mast cell tumors with histologic grade 3 (Patnaik's), AgNOR counts higher than 2.25, and PCNA count in five HPFs higher than 261. The significance of these factors as markers for prognosis determined by logistic regression analysis differed with the time period considered. By combining the three most significant prognostic factors in a prognostic index, three models were obtained to determine the probability of nonrecurrence at 3, 6, and 9 months after surgery. The models were accurate in the prediction of the outcome of up to 80% of mast cell tumors. The use of these models provides a less subjective means of prognosticating mast cell tumors than the use of any one component alone.

Chromosomal nucleolar organizer regions in four sturgeon species as markers of karyotype evolution in Acipenseriformes (Pisces)

Nucleolar organizer regions in Acipenser ruthenus (2n = 118 +/- 4) are localized on the telomeric regions of two morphologically different pairs of chromosomes. In A. baeri (2n = 250 +/- 8), A. transmontanus (2n = 248 +/- 8), and A. naccarii (2n = 246 +/- 8) they are localized on eight chromosomes arranged in two quadruplets. Contrary to what is commonly accepted, such distribution suggests that species with 120 chromosomes should be considered diploids and species with 240-250 chromosomes should be considered tetraploid.

Immunodetection of the ribosomal transcription factor UBF at the nucleolus organizer regions of fish cells.

A human autoimmune serum to nucleolus organizer regions (NORs) has been used to localize these structures at the light microscopic level in carp and trout tissue culture cells. In interphase cells, the immunofluorescence pattern indicates that the NORs autoantigens are contained exclusively within the nucleolus of carp epithelial (EPC) and trout gonad (RTG) cells. This fluorescence is punctuate rather than uniform, and presumably represents transcriptional complexes of ribosomal DNA. During mitosis, the autoantigens are detected by immunofluorescence microscopy at the chromosomal nucleolus organizer regions of condensed chromosomes, indicating that a considerable quantity of the molecule(s) remains bound to the ribosomal RNA genes. The major nucleolus autoantigen, defined in mammals as the upstream ribosomal binding factor (UBF), has been identified on immunoblots as a 90 kDa protein in extracts from fish cell lines and tissues. Thus, NORs appear to function as nucleation centers for ribosomal RNA together with a complex set of well-conserved protein factors, such as UBF. Our results suggest evolutionary conservation from fish to mammals with respect to ribosomal RNA biosynthesis driven by RNA polymerase I.

COMPARATIVE ANALYSIS OF KARYOTYPES AND NUCLEOLAR ORGANIZER REGIONS IN DIFFERENT POPULATIONS OF MYTILUS TROSSULUS, MYTILUS EDULIS AND MYTILUS GALLOPROVINCIALIS

The cylotaxonomical characteristics of Mytilus tros-sulus, M. edulis and M. galloprovincialis were studied using karyometric analysis and silver staining. The karyotype of M. trossulus, reported here for the first time, consists of six metacentric and eight submetacentric-subtelocentric chromosome pairs. The occurrence of five metacentric chromosome pairs in M. galloprovincialis differentiates this taxon from M. trossulus and M. edulis which both have six meta-centrics. The number of submetacentric to subtelo-centric chromosome pairs was variable between populations and between taxa. We suggest that this variability has arisen from differential chromosome condensation rather than from structural change. Intercomparison of chromosome relative length showed strong similarity among the three taxa. Chromosomal nucleolar organizer regions (NORs) were identically located on two submetacentric-subtelocentric chromosome pairs in all three taxa. However, M. trossulus showed a characteristic difference in the occurrence of NORs on one metacentric pair, which was absent in M. galloprovincialis and M. edulis. Variability in the number of NORs per cell was observed in all populations studied. We conclude that the three taxa of Mytilus studied here cannot be differentiated by asingle karyological character, but that a combination of karyological characters is virtually diagnostic

Loss of nucleolar-organizer regions during polyploid evolution in Scilla autumnalis

The organization and behaviour of nucleolar-organizer regions (NOR) of Scilla autumnalis were studied in two diploid races, their allotetraploid and autoallohexaploid derivatives and hybrids between them. The diploids of genome constitution AA and B7B7 each have a pair of nucleolar-organizer chromosomes while the polyploids AAB7B7 and AAB7B7B7B7 only carry visible organizers on the B7 chromosomes. In situ hybridization using ribosomal probe pTA71 was used to localize ribosomal cistrons. In polyploids the sites of hybridization are limited to the B7-derived nucleolar-organizer chromosomes and ribosomal cistrons have thus been deleted from the A genome. A NOR-associated C-band has also been deleted.

The nucleolus.

Nucleoli are the sites of biosynthesis of the ribosomal precursors. They contain may copies of the genes for the main rRNAs (18S- and 28 S-rRNA) in the form of tandemly arranged repeats at the chromosomal nucleolar organizer regions (NORs). They also contain the small rRNA (5S-rRNA) that is synthesized outside the nucleolus, specific nucleolar proteins, among them the factors and enzymes necessary for transcription and transcript processing, and the precursor units of the ribosomes. In man as in may vertebrate species, three main components of nucleoli, besides chromatin, can be detected: fibrillar centres (FC), dense fibrillar component (DCF), and granular component (GC). Within a nucleolus the FCs are in many cases situated in its central region. The DFc forms a network of strands surrounding the FCs, but may sometimes reach for out towards the periphery of the nucleolus. The GC is usually situated in the peripheral regions of the nucleolus. In cells with a low level of ribosomal biosynthesis the nucleoli are small, usually with a single FC and little surrounding DFC and GC ("ring-shaped nucleolus"). In active cells the DFC forms a large network enclosing several, sometimes up to hundreds of FCs, and the GC covers a large area in the periphery ("compact nucleoli"). In cells at the onset of a new stimulation, the DFC is very prominent whereas the FCs are few and small, and the GC is also not very extensive ("reticulate nucleoli"). In some special cell types that are very active other arrangements of the structural components are found. In Sertoli cells, for instance, only one nucleolus is found, or occasionally two, each with a single large FC and a distinct area of GC, both areas being engulfed by DFC intermingled with some peripheral GC. Immunocytological and in situ hybridization studies to localize the rRNA genes within the nucleolus have so far led to divergent results. Both fibrillar components, the FCs and the DFC, have been claimed as the most probable candidates. Transcription of rDNA and the subsequent early steps of ribosome biosynthesis are localized in the DFC, whereas later steps (mature rRNA, preribosomes) are localized in the GC. The FCs may also serve as sites for the preparation of the rDNA for transcription, and as a store for certain nucleolar proteins. During mitosis, parts of the nucleolar proteins remain at the NORs. A direct contact between the nucleolus and the nuclear envelope is frequently observed but is not dependent on nucleolar activity.(ABSTRACT TRUNCATED AT 400 WORDS)