Active Nucleolar Organizer Regions Research Articles

RRNA Genes Are Not Fully Activated in Mouse Somatic Cell Nuclear Transfer Embryos

The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.

C-banding and Ag-NOR distribution patterns in Euphrates jerboa, Allactaga euphratica (Mammalia: Rodentia), from Turkey

A chromosomal study of Allactaga euphratica from the Şanlıurfa Province in Southeast Anatolia, Turkey, was performed. The diploid number of 48 chromosomes was found in all four specimens examined. The C-band-positive regions were distributed in centromeric areas of all the autosomal pairs and the X chromosome. Extensive C-positive heterochromatin intercalary blocks were observed in the largest pair of autosomes. The small Y chromosome was stained uniformly and C-negatively. The active nucleolus organizer regions (NORs) were localized in two pairs of small bi-armed autosomes. The C-heterochromatin distribution and localization of the secondary constrictions bearing NORs apparently differ between karyotypes of the related jerboa species A. euphratica and Allactaga williamsi. This cytogenetic difference may be implied as a suitable marker in further studies of the relationships in contact zones and possible hybridization between these two taxa.

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse ( Equus caballus)

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag–NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag–NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.

Ommexecha virens (Thunberg, 1824) and Descampsacris serrulatum (Serville, 1831) (Orthoptera, Ommexechidae): karyotypes, constitutive heterochromatin and nucleolar organizing regions

Chromosomes of Ommexecha virens and Descampsacris serrulatum (Ommexechidae) were analyzed through conventional staining, C-banding, base specific fluorochromes, silver nitrate impregnation (AgNO3), and fluorescent in situ hybridization (FISH) with probe for 45S rDNA. The two species presented diploid number 2n= 23,X0 in males and acrocentric autosomes, except the pair one that presented submetacentric morphology. The X chromosome has distinct morphology in the two analyzed species, being a medium acrocentric in Ommexecha virens and large submetacentric in Descampsacris serrulatum. The C-banding revealed pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of Descampsacris serrulatum. For Ommexecha virens it was evidenced that the blocks of CH are preferentially located in the pericentromeric area (however some bivalents presents additional blocks) or in different positions. The staining with CMA3/DA/DAPI showed GC rich CH blocks (CMA3+) in some chromosomes of the two species. The nucleolar organizer regions (NORs) were located in the bivalents L2, S9, S10 of Ommexecha virens and M5, M6, M7, S11 of Descampsacris serrulatum. The FISH for rDNA showed coincident results with the pattern of active NORs revealed by AgNO3. This work presents the first chromosomal data, obtained through differential cytogenetics techniques in Ommexechidae, contributing to a better characterization of karyotypic evolution for this grasshopper family.

Karyotype variability in neotropical catfishes of the family Pimelodidae (Teleostei: Siluriformes)

Karyotypic data are presented for four species of fish belonging to the Pimelodidae family. These species show a conserved diploid number, 2n = 56 chromosomes, with different karyotypic formulae. The analyzed species showed little amount of heterochromatin located preferentially in the centromeric and telomeric regions of some chromosomes. The nucleolus organizer regions activity (Ag-NORs) and the chromosomal location of ribosomal genes by fluorescent in situ hybridization (FISH), with 18S and 5S probes, showing only one chromosome pair marked bearer of ribosomal genes, the only exception was Pimelodus britskii that presented multiple NORs and syntenic location of the 18S and 5S probes. Non-Robertsonian events, as pericentric inversion and NORs duplication are requested to explain the karyotype diversification in Pseudoplatystoma from the rio Paraguay (MS), Pimelodus from the rio Iguaçu (PR), Sorubim from the rio Paraguay (MS) and Steindachneridion from the rio Paraíba do Sul (SP). The obtained data for the karyotype macrostructure of these species corroborates a conserved pattern observed in Pimelodidae. On the other hand, interspecific variations detected by molecular cytogenetics markers made possible cytotaxonomic inferences and differentiation of the species here analyzed.

Nucleolar Follistatin Promotes Cancer Cell Survival under Glucose-deprived Conditions through Inhibiting Cellular rRNA Synthesis

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

The structure of long telomeres in chromosomes of the Iberian shrew

It is shown that the size, localization, and structure of telomeres in the Iberian shrew (Sorex granarius) are not characteristic of mammals. In this species, long telomeres of an average size of 213 kb are localized on the short arms of all 32 acrocentrics; ribosomal blocks and active nucleolus-organizing regions (NORs) were also discovered there. At the remaining chromosome ends the average size of telomeres is 3.8 kb. However, in a closely related species, Sorex araneus, all telomeres have size similar to that of human telomeres, i.e., 6.8-15.2 kb. Despite the fact that some long telomeres contain ribosomal repeats in addition to telomeric ones, the long telomeres have preserved asymmetry of G- and C-rich strands as in functional telomeres. It is probable that long telomeres were formed in meiosis at the stage of chromosome bouquet as a result of global reorganization of the chromosome ends. The provoking factors for such reorganization might be the fission of several metacentrics and the necessity of telomerization of the resulting acrocentrics.

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.

Karyotype Conservation in 2 Populations of the Parthenogenetic Scorpion Tityus serrulatus (Buthidae): rDNA and Its Associated Heterochromatin Are Concentrated on Only One Chromosome

Within the order Scorpiones, the parthenogenetic mode of reproduction has been described for 11 species, 6 of which belong to the genus Tityus. In this work, an investigation of the chromosome characteristics of 2 populations of Tityus serrulatus, the first scorpion species known to be thelytokously parthenogenetic, is described. An analysis of 40 individuals revealed holocentric chromosomes of large, medium, and small sizes and an invariable diploid number of 2n = 12. In addition to the conserved macrokaryotype structure, specific chromosome regions also appeared unchanged within and between the samples studied; that is, each sample displayed only one chromosome carrier of the active nucleolar organizer region containing ribosomal genes (5.8S, 18S, and 28S) and AT-rich heterochromatin associated with the ribosomal DNA. The high conservation of the chromosomal features observed in T. serrulatus differed from that verified in certain species of other groups of animals that possess both holocentric chromosomes and parthenogenetic reproduction. Moreover, the cytogenetic results obtained herein permit us to suggest how the eggs of T. serrulatus develop, whether by apomixis or automixis.

Population distribution of 45S and 5S rDNA in golden mahseer, Tor putitora: population-specific FISH marker

Chromosomal locations of major 45S and minor 5S ribosomal DNAs (rDNAs) and organization of 5S rRNA genes were analysed in five different populations of golden mahseers (Tor putitora) using fluorescence in situ hybridization (FISH) and Southern blot hybridization. All five populations of T. putitora (2n = 100) showed a similar type of macro-karyotype composed of 12 metacentric, 22 submetacentric, 14 subtelocentric and 52 telocentric chromosomes. Analysis of active nucleolar organizer regions (NORs) by silver staining did not show any differences in number and chromosomal position in different populations. But FISH data showed significant difference between the populations, four of the five populations showed six 18S (three pairs) and two 5S (one pair) signals with positional polymorphism, while one population showed eight 18S and four 5S signals, respectively. Southern blot data confirms that 5S rDNA clusters present on two different chromosome pairs in Kosi river population contain non-transcribed spacers (NTS) of same length. In the present study, simultaneous localization of 45S and 5S rDNA by in situ hybridization helped us to develop the discrete population-specific markers in different geographically isolated populations of T. putitora.

Nucleolar organizer regions, satellite associations and nucleoli of goat cells (&amp;lt;i&amp;gt;Capra hircus&amp;lt;/i&amp;gt;)

Abstract. The Polish White Improved goat karyotype consists of 29 pairs of acrocentric autosomes, a large acrocentric X chromosome and a metacentric Y chromosome which is the smallest in the karyotype. Staining of chromosomes with AgNO,sub&gt;3 solution has revealed active nucleolar organizer regions (NOR) in terminal parts of q arms of pair 2, 3, 4, 5, and 28 chromosomes. Out of the total of 100 analysed cells 736 active NORs have been found, on average 7.4±0.2 per cell. Active NORs were most frequently observed in pair 2, and 3 chromosomes, most rarely on pair 5 chromosomes. In all the analysed cells 141, satellite associations (SA) were observed, on average 1.4±0.2 per cell. SAs most often occurred in cells with seven active NORs, and least often in cells with three or four nucleolar organizer regions. Most frequently in SAs the presence of pair 2, 3 and 28 chromosomes was observed. On meiotic chromosomes staining with AgNO3 solution revealed two nucleoli stained with different intensity. Both nucleoli in the cell were of similar size.

Relationship between the Chromosome Nucleoli-Forming Regions and Somatometric Parameters in Humans

Relationship between activity of nucleolar organizer regions in chromosomes and somatometric parameters was studied in residents of the Kursk region. Significant differences in the somatometric parameters were revealed between the three groups differing by the number of nucleolar organizer regions, which can be explained by different proliferative activities in these groups. A greater number of nucleolar organizer regions was associated with a trend to greater body weight and body mass index, waist and hip circumferences, waist to hip circumference ratio, and shoulder width in men and increased body height, greater body weight and body mass index, hip circumference, and leg length in women.

The Epigenetics of rRNA Genes: From Molecular to Chromosome Biology

In eukaryotes, the genes encoding ribosomal RNAs (rDNA) exist in two distinct epigenetic states that can be distinguished by a specific chromatin structure that is maintained throughout the cell cycle and is inherited from one cell to another. The fact that even in proliferating cells with a high demand of protein synthesis a fraction of rDNA is silenced provides a unique possibility to decipher the mechanism underlying epigenetic regulation of rDNA. This chapter summarizes our knowledge of the molecular mechanisms that establish and propagate the epigenetic state of rRNA genes, unraveling a complex interplay of DNA methyltransferases and histone-modifying enzymes that act in concert with chromatin remodeling complexes and RNA-guided mechanisms to define the transcriptional state of rDNA. We also review the critical role of the RNA polymerase I transcription factor UBF in the formation of active nucleolar organizer regions (NORs) and maintenance of the euchromatic state of rRNA genes.

Oxidant-induced decrease of the expression of nucleolar organizer regions in pig lymphocytes can be useful for monitoring the cellular effects of oxidative stress

The technique of silver staining of nucleolar organizer regions (AgNORs) was chosen to estimate the transcriptionally active metaphase and interphase nucleolar organizer regions (NORs) in pig peripheral blood lymphocytes exposed to oxidative agents in vitro. The quantitative analysis of AgNORs was performed by using the counting method and the morphometric method. We found that hydrogen peroxide and 2,2′-azobis-(2-amidinopropane)-dihydrochloride (AAPH) induced a concentration-dependent decrease in NORs activity – in the case of metaphase the NORs activity was exclusively seen on chromosome pairs 8 – which can be considered as another estimate of cellular oxidative stress. Moreover, biomarkers of cyto- and genotoxicity, such as the percentage of apoptotic and necrotic cells, the nuclear division index (NDI) and formation of micronuclei (MN) were used to measure harmful effects provoked by the agents tested.

Cytogenetic studies in three species of Lutjanus (Perciformes: Lutjanidae: Lutjaninae) from the Isla Margarita, Venezuela

In the present study, three species of Lutjaninae, Lutjanus analis, L. griseus and L. synagris, were analyzed by conventional Giemsa staining, C-banding and silver staining, to reveal active Nucleolus Organizer Regions (NORs). Fluorescent in situ hybridization (FISH) was also applied to establish the number and location of the ribosomal gene clusters (18S and 5S rRNA genes). Counts of diploid metaphasic cells revealed a diploid modal chromosome complement composed of 48 acrocentric chromosomes in both L. analis and L. griseus. Two cytotypes were observed in L. synagris: cytotype I, with 2n=48 acrocentric chromosomes, found in 19 specimens, and cytotype II, with 46 acrocentric chromosomes and one large metacentric, found in two specimens. The large metacentric, which possibly originated from a Robertsonian rearrangement, was not found to be sex-related. In the three species, constitutive heterochromatin is located in the centromeres of all chromosomes. NORs were detected on the short arms of a single chromosome pair, number 24 in L. analis and number 6 in both cytotypes of L. synagris. In L. griseus, a polymorphism of the NORs number was detected, by both Ag-staining and FISH, as females show a maximum of three NORs, and males a maximum of six NORs. In all species, minor ribosomal genes were found located on a single chromosome pair. The obtained data, along with those previously reported for other five Lutjanidae species, show that a general chromosome homogeneity occurs within the family, but that derived karyotypes based on Robertsonian rearrangements as well as multiple and variable NORs sites can also be found.

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).

Effects of Aging on Mouse Tongue Epithelium Focusing on Cell Proliferation Rate and Morphological Aspects

The aim of this study was to investigate cell proliferation rate and certain morphological features of mouse epithelium as aging progresses. Tongue biopsies were performed on female mice (Mus domesticus domesticus) at 2, 8, 14 and 20 months of age as indicative of adolescence, adulthood, early senescence and senescence, respectively. Histological sections of tongue were stained with hematoxylin-eosin and subjected to silver staining for active nucleolar organizer region counting. Cell proliferation rate and epithelial thickness analysis were carried out. Analysis of variance detected no differences between the groups in terms of numbers of silver-stained dots associated with nucleolar proteins. There was an increase in mean epithelial thickness in adult animals, followed by a gradual reduction until senescence. Mean keratin thickness presented an increase at 8 and 20 months of age. This difference is probably related to puberty, growth or dietary habits. Aging has no influence on oral epithelial proliferation rate in mice. A gradual reduction in epithelial thickness is a feature of aging in mammals. A conspicuous increase in the keratin layer was observed in senescence as an adaptative response to the reduction in epithelial thickness. These results suggest that aging affects the oral epithelium maturation process through a mechanism that is not related to cell proliferation.

Evolutionary cytogenetics of the Hoplias lacerdae, Miranda Ribeiro, 1908 group: a particular pathway concerning the other Erythrinidae fish

The taxonomy/systematics of the Erythrinidae fish is still imprecise, with several doubts on their relationships. Karyotypes and chromosomal characteristics of some species of the Hoplias lacerdae group (Erythrinidae), from different Brazilian hydrographic basins and pisciculture stations, were analyzed in the present study, using conventional Giemsa staining, C-banding, silver staining, Mithramycin and Distamycin/DAPI fluorochromes, and fluorescent in situ hybridization (FISH). A diploid chromosome number of 2n = 50 and karyotypes composed of meta- and submetacentric chromosomes without sex-related differences were found. Only one active NOR (Nucleolar Organizer Region) site was found, which was identified by silver staining (Ag-NOR) and FISH, located on the chromosome pair 11, although additional 45S rDNA sites were also mapped on other chromosome pairs only by FISH. The Ag-NOR of the chromosome pair 11 was found to be GC-rich, appearing positive after Mithramycin staining. Mithramycin-positive/DAPI-negative sites were also observed in the centromeric/pericentomeric regions of the chromosome pairs 4, 6, 15, and 19, which have also affinity to silver nitrate. However, these four sites were not detected by FISH with the rDNA probe, indicating to be only argentophilic GC-rich heterochromatic regions. Chromosome data show that the karyotype evolution in Hoplias lacerdae group is relatively conserved and follows a particular pathway concerning the other Erythrinidae fishes, such as Hoplias malabaricus, Hoplerythrinus unitaeniatus, and Erythrinus erythrinus, in which polytypic karyotypes are found. Thus, the H. lacerdae group shows chromosome features that are not closely related to those of the congeneric H. malabaricus group. These finds, together with genetic and morphologic data, are important tools to be considered in a major revision of the Erythrinidae family, as well as for conservation programs.

Recruitment of factors linking transcription and processing of pre-rRNA to NOR chromatin is UBF-dependent and occurs independent of transcription in human cells.

Efficient ribosome biogenesis requires coordination of a highly complex series of events. Early events include pre-RNA transcription, processing, and modification. Analysis in yeast has demonstrated that t-UTPs, components of the U3 snoRNA-containing pre-rRNA processing complex, are required for efficient transcription of ribosomal genes (rDNA) by RNA polymerase I (pol I). Here, we characterize human t-UTPs and establish that their ability to link transcription and pre-rRNA processing is evolutionarily conserved. The pol I transcription factor UBF binds extensively across rDNA throughout the cell cycle, resulting in a specialized form of chromatin that is the hallmark of active nucleolar organizer regions (NORs). Transcriptionally silent pseudo-NORs are ectopic, chromosomally integrated, artificial arrays that mimic this specialized chromatin structure. Pseudo-NORs sequester t-UTPs and factors linking transcription with pre-rRNA modification (Nopp140 and Treacle). Recruitment is independent of transcription, the underlying DNA sequence, and location within the nucleolus. Previously, we have demonstrated that pseudo-NORs sequester every component of the pol I transcription machinery. Taken together, these results highlight the importance of the specialized chromatin structure at active NORs in coordinating early events in ribosome biogenesis and nucleolar formation.

Extensive ribosomal DNA (18S-5.8S-26S and 5S) colocalization in the North American endemic sagebrushes (subgenus Tridentatae, Artemisia, Asteraceae) revealed by FISH

Chromomycin A3 banding and fluorescent in situ hybridization (FISH) have been performed for six Artemisia species with special emphasis on subgenus Tridentatae. Morphometrical data on karyotype characters were calculated and idiograms with the position of GC-rich regions and 18S-5.8S-26S and 5S sites of ribosomal DNA were constructed. These sites were all colocalized. To our knowledge, this is the first time in the large family Asteraceae, indeed in angiosperms in general, that colocalization of the two rDNA regions studied is found at every single marked locus. In addition, transcriptionally active nucleolar organizer regions were detected after silver nitrate staining. Tridentatae is a cytogenetically homogeneous subgenus, which suggests that evolution of these species has not been coupled with important karyotypic reorganization. However, a few species are taxonomically difficult and show substantial differences. A loss of rDNA loci has been detected in a tetraploid taxon with respect to the diploids studied. These data provide clarifying insight into interspecific relationships between the studied taxa and overall evolutionary and systematic relationships of the Tridentatae.