Chromosomal Nucleolar Organizer Regions Research Articles

On the Origin of Neo-Sex Chromosomes in the Neotropical Dragonflies Rhionaeschna bonariensis and R. planaltica (Aeshnidae, Odonata).

Odonata have holokinetic chromosomes. About 95% of species have an XX/X0 sex chromosome system, with heterogametic males. There are species with neo-XX/neo-XY sex chromosomes resulting from an X chromosome/autosome fusion. The genus Rhionaeschna includes 42 species found in the Americas. We analyzed the distribution of the nucleolar organizer region (NOR) using FISH with rDNA probes in Rhionaeschna bonariensis (n = 12 + neo-XY), R. planaltica (n = 7 + neo-XY), and Aeshna cyanea (n = 13 + X0). In R. bonariensis and A. cyanea, the NOR is located on a large pair of autosomes, which have a secondary constriction in the latter species. In R. planaltica, the NOR is located on the ancestral part of the neo-X chromosome. Meiotic analysis and FISH results in R. planaltica led to the conclusion that the neo-XY system arose by insertion of the ancestral X chromosome into an autosome. Genomic in situ hybridization, performed for the first time in Odonata, highlighted the entire neo-Y chromosome in meiosis of R. bonariensis, suggesting that it consists mainly of repetitive DNA. This feature and the terminal chiasma localization suggest an ancient origin of the neo-XY system. Our study provides new information on the origin and evolution of neo-sex chromosomes in Odonata, including new types of chromosomal rearrangements, NOR transposition, and heterochromatin accumulation.

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.

Polymorphism of interphase nucleolus organizer regions in imported pigs and their offspring

Data are given on polymorphism of interphase nucleolus organizer regions of chromosomes in Large White piglets, replacement gilts, sows obtained from PIC boars and replacement boars of this breed in different sex and age groups. An association between the frequency of interphase nucleolus organizer regions and adaptive response of the animals to new environmental conditions is shown.

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.

Nucleolar behavior during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.

Evaluation of the Relationship between Chromosome Aberrations and Transcription Activity of Nucleolus Organizer Regions in Indigenous Population of the Kursk Region

The relationship between activity of chromosomal nucleolus-organizer regions and levels of chromosome aberrations was studied in 215 residents of the Kursk region by visual semiquantitative method (silver staining of the nucleolus-organizer regions, NOR) in chromosomes of peripheral blood lymphocytes. The levels of chromosome aberrations differed significantly in three groups differing by the levels of 10AgNOR, which can be explained by different proliferative activity in these groups. The lowest level of chromosome aberrations was found in subjects with high transcription activity of NOR, presumably due to high proliferative activity in this group and more intensive synthesis of proteins, including the reparation enzymes. The highest level of chromosome aberrations was detected in the group with the medium level of 10AgNOR.

Evaluation of the dependence of mutagenesis intensity on activity of nucleolus organizer regions of chromosomes in aboriginal population of Kursk region

Visual semiquantitative method of silver staining of nucleolus organizer regions in peripheral blood leukocytes was applied for evaluation of the correlation between activity of nucleolus organizer regions and the level of chromosome aberrations in 241 residents of Kursk region. Significant differences in the percent of chromosome aberrations were revealed between three groups differing by the number of active nucleolus organizer regions, which can be explained by different proliferative activity in these groups. The lowest level of chromosome aberrations was found in individuals with high transcription activity of the nucleolus organizer regions (due to high proliferative activity in this group and due to more intensive protein biosynthesis, e.g., synthesis of reparation enzymes). The maximum level of chromosome aberrations was observed in the group with medium number of active nucleolus organizer regions (adaptive norm).

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.

Chromosomal NOR activity in mice selected for high and low swim stress-induced analgesia.

Metaphase nucleolar organizer region (NOR) activity was studied in mouse lines selectively bred for high analgesia (HA) and for low analgesia (LA) induced by 3-min swimming in 20 degrees C water. Apart from pain-related traits, HA mice also manifest, as compared to the LA line, higher emotionality in certain behavioral tests and are less capable of coping with the hypothermic challenge of swimming in cold water, in addition to being more susceptible to the mutagenic effect of whole-body gamma-radiation and mitomycin C injection. We here compared NOR activity in HA and LA mice. A statistically significant difference (p < or =.01) was detected in mean silver-stained nucleolar number/cell between the HA and LA lines, being lower for HA mice. We propose that the breeding strategy, along with the differentiation of stress-related phenomena, has altered the activity of genes coding rRNA and that this activity is important in controlling DNA repair in each line. It is concluded that quantitative studies of nucleolar changes may be useful in evaluating the biological reactivity to stress stimuli.

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.

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells.

Indirect immunolabeling with anti-UBF antibodies, in situ hybridization with an rDNA probe, and confocal scanning laser microscopy were used to study nucleolar organizer regions (NORs) during the cell cycle in pig embryonic kidney (PK) cells. The chromosomal distribution of the polymerase I transcription factor UBF and rDNA was compared with the number of silver-stained NORs (Ag-NORs) present and nucleolar size. It was shown, both at interphase and mitosis, that the majority of UBF and rDNA signals were located at the same foci and that the amounts of UBF and rDNA at any given site were in a striking positive correlation. At mitosis, only the NORs were labeled; at interphase, the signals for both UBF and rDNA were arranged in necklace-like structures around the nucleoli. No chromosomal NORs without Ag-proteins or UBF were present, indicating that all NORs in PK cells are active at interphase. It was concluded that (1) UBF and rDNA co-localize throughout the cell cycle in PK cells; (2) their association with mitotic NORs is determined by the number of rDNA repeats, rather than by any differential ability of NORs to recruit the transcription factor; and (3) the amount of UBF can be correlated with the size and activity of the nucleoli at interphase.

A new method for detecting nucleolus organizer regions in fish chromosomes using denaturation and propidium iodide staining.

A rapid method for detecting nucleolus organizer regions (NORs) in fish chromosomes based on thermal denaturation and staining with propidium iodide is described. Under epifluorescence, the NORs of 15 fish species from six families could be detected. This protocol differentiates constitutive heterochromatin in mammalian and avian chromosomes, and in some cases, heterochromatic blocks in fish chromosomes. The staining of NORs of fish chromosomes with propidium iodide following denaturation with formalin is likely the result of differential denaturation of the rDNA due to the thermal characteristics of AT- and GC-rich domains of the rDNA cistron. This technique provides a new useful marker for descriptive fish cytogenetic studies.

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.

Chromosomal NOR phenotypes in North American pikes and pickerels, genus Esox, with notes on the Umbridae (Euteleostei: Esocae)

A comparison was made of the localization of nucleolar organizer region (NOR) sites in the karyotypes of the North American pikes and pickerels Esox lucius, E. masquinongy, E. niger, and the two subspecies of E. americanus (Esocidae). NORs were located by silver staining or chromomycin A3 fluorescence. The study revealed that the position of chromosomal NORs in all Esox taxa examined was the same: the pericentromeric region of one middle-sized chromosome pair. The same NOR phenotype was found previously in a related European species, Umbra krameri (Umbridae), and in U. pygmaea, one of the two North American representatives of the genus. The NOR phenotype of the remaining North American species (U. limi) is different. It is hypothesized that this kind of NOR phenotype, shared by two phyletic lineages in the Esocae, represents an ancestral character for the members of that group.

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.

Chromosomal NOR Karyotypes and Genome Size Variation among Squawfishes of the Genus Ptychocheilus (Teleostei: Cyprinidae)

Nucleolus organizer region (NOR) chromosomes from species of the North American cyprinid genera Ptychocheilus, Mylopharodon, and Lavinia were documented and tested for presumed homology using trypsin G-banding. Four species of Ptychocheilus (P. lucius, P. grandis, P. umpquae, and P. oregonensis), M. conocephalus, and L. exilicauda possessed two pair of NOR-bearing chromosomes with the NORs situated terminally on the short arms of medium-sized acrocentric chromosomes (NOR phenotype A). Both pair of NOR chromosomes in all three genera were identical in trypsin G-banding pattern and hence were uninformative for inference of phylogenetic relationships within Ptychocheilus. The apparent conservatism of chromosomal NOR evolution in these cyprinid genera from western North America is in striking contrast to that documented among cyprinids from eastern North America. Genome sizes (DNA contents) of the six species also were documented using flow cytometry. Heterogeneity tests revealed three homogeneous groups of samples: (1) L. exilicauda, P. lucius, P. grandis, and P. umpquae from the Siuslaw River with low genome sizes; (2) M. conocephalus with an intermediate genome size; and (3) P. oregonensis and P. umpquae from the Umpqua River drainage with high genome sizes. The difference in genome size between P. umpquae from the Siuslaw and Umpqua river drainages is greater than that found, on average, between any two North American cyprinid species drawn at random. The difference in genome size, coupled with morphological differences documented previously, suggests that P. umpquae from the two drainages may represent distinct species. Using an optimization procedure, genome sizes were partitioned among branches of two contrasting hypotheses of relationships in Ptychocheilus. The hypothesis of {P. lucius [P. grandis (P. umpquae, P. oregonensis)]} required about 20% less change in genome size (i.e., was more parsimonious) than the hypothesis of {P. oregonensis [P. umpquae (P. grandis, P. lucius)]}.

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

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.