Nucleolar Organizer Research Articles

Cytogenetic and Molecular Characterization of Eigenmannia aff. desantanai (Gymnotiformes: Sternopygidae): A First Report of System of Sex Chromosomes ZW1W2/ZZ in Gymnotiformes.

Gymnotiformes a monophyletic group of fish endemic to the Neotropics, represent an important component of the freshwater ichthyofauna that presents relevant taxonomic problems. Thus, in view of the morphological complexity involving Eigenmannia (Gymnotiformes) fish species, this study aimed to characterize Eigenmannia aff. desantanai of the upper Paraguay River basin through cytogenetic and molecular analyses, to help in the correct identification and delimitation of species. This study reports a multiple sex system of the type ZW1W2/ZZ, with 2n = 31 for females and 2n = 30 for males. A single pair of chromosomes carrying the nucleolar organizing regions (NORs) was detected. The heterochromatin was colocated in NOR sites and mainly located in the centromeric regions of chromosomes. Besides that, individual sequences COI from the specimens of E. aff. desantanai were obtained, totalizing three haplotypes. The distance p between the haplotypes in E. aff. desantanai, ranged from 0.2% to 7.1%. Species delimitation tests indicated the existence of two possible operational taxonomic units of E. aff. desantanai. Thus, this study reports a new multiple sex system in Gymnotiformes and these specimens previously identified as E. aff. desantanai may belong to two distinct species.

The p-Arms of Human Acrocentric Chromosomes Play by a Different Set of Rules.

The p-arms of the five human acrocentric chromosomes bear nucleolar organizer regions (NORs) comprising ribosomal gene (rDNA) repeats that are organized in a homogeneous tandem array and transcribed in a telomere-to-centromere direction. Precursor ribosomal RNA transcripts are processed and assembled into ribosomal subunits, the nucleolus being the physical manifestation of this process. I review current understanding of nucleolar chromosome biology and describe current exploration into a role for the NOR chromosomal context. Full DNA sequences for acrocentric p-arms are now emerging, aided by the current revolution in long-read sequencing and genome assembly. Acrocentric p-arms vary from 10.1 to 16.7 Mb, accounting for ∼2.2% of the genome. Bordering rDNA arrays, distal junctions, and proximal junctions are shared among the p-arms, with distal junctions showing evidence of functionality. The remaining p-arm sequences comprise multiple satellite DNA classes and segmental duplications that facilitate recombination between heterologous chromosomes, which is likely also involved in Robertsonian translocations. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 24 is August 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Chromosome and bands: comparative cytogenetics of the morphotypes of Ameerega trivittata (Anura: Dendrobatidae)

Abstract Ameerega trivittata is a widely distributed dendrobatid species with colour polymorphism for which only the diploid number, nucleolar organizer regions (NORs) and heterochromatic pattern are known. Here, we investigated the existence of cytogenetic structure in A. trivittata populations with different morphotypes. Four morphotypes and their geographical distributions were defined. All the individuals have 24 chromosomes and a similar heterochromatic pattern, the same location of telomeric regions and 5S rDNA. The NOR site was coincident with the 18S rDNA in pair 9, but some individuals presented an additional site in one of the homologues of pair 11. The highest variation was found in the microsatellite distribution patterns. Comparing the cytogenetic characters of the individuals, two groups were detected: (1) Tabatinga (morphotype B) and Benjamin Constant (morphotype A), and (2) Presidente Figueiredo and Tefé (morphotype C). The cytogenetic data appear to be correlated to the structure pattern of the morphotypes, and corroborate the dispersal routes proposed in the literature. This is the first study of population cytogenetics for the group. Our results provide a series of cytogenetic characters for A. trivittata and for Dendrobatidae for the first time and highlight the importance of considering cytogenetics in population research.

Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes.

One of the first biological machineries to be created seems to have been the ribosome. Since then, organisms have dedicated great efforts to optimize this apparatus. The ribosomal RNA (rRNA) contained within ribosomes is crucial for protein synthesis and maintenance of cellular function in all known organisms. In eukaryotic cells, rRNA is produced from ribosomal DNA clusters of tandem rRNA genes, whose organization in the nucleolus, maintenance and transcription are strictly regulated to satisfy the substantial demand for rRNA required for ribosome biogenesis. Recent studies have elucidated mechanisms underlying the integrity of ribosomal DNA and regulation of its transcription, including epigenetic mechanisms and a unique recombination and copy-number control system to stably maintain high rRNA gene copy number. In this Review, we disucss how the crucial maintenance of rRNA gene copy number through control of gene amplification and of rRNA production by RNA polymeraseI are orchestrated. We also discuss how liquid-liquid phase separation controls the architecture and function of the nucleolus and the relationship between rRNA production, cell senescence and disease.

Ultrastructural and proteomic evidence for the presence of a putative nucleolus in an Archaeon.

Nucleoli are subcellular compartments where transcription and maturation of pre-ribosomal RNAs occur. While the transcription of ribosomal RNAs is common to all living cells, the presence and ultrastructure of nucleoli has been only documented in eukaryotes. Asgard-Archaea, the closest prokaryotic relatives of eukaryotes, and their near relatives TACK-Archaea have homologs of nucleolar proteins and RNAs in their genome, but the cellular organization of both is largely unexplored. Here we provide ultrastructural and molecular evidence for the presence of putative nucleolus-like subcellular domains in the TACK crenarchaeon Saccharolobus solfataricus (formerly known as Sulfolobus solfataricus). Transmission electron microscopy (TEM) revealed consistent electron-dense fibro-granular compartments, also positive to the specific silver staining for nucleolar organizer regions (AgNOR). TEM also confirmed that ribosomal DNA (rDNA) is spatially distributed in non-random, clustered arrays underlying fine structures, as observed by ultrastructural in situ hybridization (UISH). To further explore these observations, proteomic sequencing of isolated bands from AgNOR-stained protein gels was conducted and compared against a compiled inventory of putative nucleolar homologs from the S. solfataricus P1 genome. Sequenced AgNOR-sensitive peptides encoded homologs of eukaryotic nucleoli proteins, enriched for nucleolus-related functions. Our results provide first evidence that subcellular domains of nucleolar-like nature are not exclusive to eukaryotes. Based on our data, we propose a model for a putative nucleolus in S. solfataricus. Whereas technical limitations and further aspects remain a matter for future functional studies, our data supports the origin of nucleoli within the common ancestor of Eukarya and TACK-Archaea, based on a two-domain tree of life.

Genetic Differentiation of the Fourhorn Sculpin <i>Myoxocephalus quadricornis</i> (Linnaeus, 1758) and Its Position in the Tribe Myoxocephalini Taranetz (Cottidae: Myoxocephalinae)

A molecular genetic and karyological study of the fourhorn sculpin Myoxocephalus quadricornis (Linnaeus, 1758) was carried out. The karyotype was studied for the first time and the results of Ag-staining of the chromosomes of specimens of the species from the East Siberian Sea are presented. The karyotype of M. quadricornis is stable, contains 32 (10 metacentrics, 2 submetacentrics, 20 subtelocentrics) chromosomes, the number of chromosome arms is 44. Nucleolar organizers were found in the telomeric regions of the short arms of two homologous medium-sized subtelocentrics. Variability in the number of nucleolarforming chromosomes and stained blocks of nucleolar organizers was found. Common features in the chromosome sets of M. quadricornis and Megalocottus platycephalus indicate their greater similarity compared to species of the genus Myoxocephalus. The results of the karyological analysis are consistent with the molecular genetic data about the sister relationships between M. quadricornis and Megalocottus platycephalus. A complex of karyological and molecular-genetic features has been identified that allow one to reliably distinguish M. quadricornis from species of the tribe Myoxocephalini and indicate the need for its removal from the genus Myoxocephalus.

Nucleolus Organizer Region Functioning in Chicken Growing Oocytes: A Revision of the Existing Views

Nucleolus Organizer Region Functioning in Chicken Growing Oocytes: A Revision of the Existing Views

Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.

Mapping nucleolus-associated chromatin interactions using nucleolus Hi-C reveals pattern of heterochromatin interactions

As the largest substructures in the nucleus, nucleoli are the sites of ribosome biogenesis. Increasing evidence indicates that nucleoli play a key role in the organization of 3D genome architecture, but systematic studies of nucleolus-associated chromatin interactions are lacking. Here, we developed a nucleolus Hi-C (nHi-C) experimental technique to enrich nucleolus-associated chromatin interactions. Using the nHi-C experiment, we identify 264 high-confidence nucleolus-associated domains (hNADs) that form strong heterochromatin interactions associated with the nucleolus and consist of 24% of the whole genome in HeLa cells. Based on the global hNAD inter-chromosomal interactions, we find five nucleolar organizer region (NOR)-bearing chromosomes formed into two clusters that show different interaction patterns, which is concordant with their epigenetic states and gene expression levels. hNADs can be divided into three groups that display distinct cis/trans interaction signals, interaction frequencies associated with nucleoli, distance from the centromeres, and overlap percentage with lamina-associated domains (LADs). Nucleolus disassembly caused by Actinomycin D (ActD) significantly decreases the strength of hNADs and affects compartment/TAD strength genome-wide. In summary, our results provide a global view of heterochromatin interactions organized around nucleoli and demonstrate that nucleoli act as an inactive inter-chromosomal hub to shape both compartments and TADs.

The influence of biotic factors on the parameters of the nucleolus

Relevance. The nucleolus regulates many aspects of cell physiology, including genome organization, stress response, aging, and lifespan. The number of visible nucleoli per cell varies depending on the cell cycle, cellular activity, or differentiation status. The aim of the study was to study the influence of abiotic factors on the parameters of animal nucleoli depending on their species (genus Ovis and genus Capra) and type of crossing (interspecific hybrids). Materials and methods. The studies were carried out at the Federal Research Center for Animal Husbandry named after Academy Member L. K. Ernst. The object of the study were domestic sheep (Romanov breed) and its hybrids (½ Romanov sheep x ½ mouflon), as well as goats of the Karachay breed and their hybrids (¾ Karachay goat x ¼ caucasian tur). The material of the study was preparations obtained from venous blood and prepared according to the Havell – Blake method. For microscopy and visualization of preparations, a “Nikon Eclipse Ni” microscope equipped with a “DS-Qi2” digital video camera was used. The processing of photo- and videomaterials was carried out using the “NIS-Elements BR4.30” and “Image Scope 1.0” software. Results. It has been established that in interspecific hybrids of goats (¾ Karachai goat x ¼ caucasian tur) the number of nucleolar-organizing regions (NOR) is 17 % more than in purebred individuals, while individual of interspecific hybrids of sheep (½ Romanov sheep x ½ mouflon) exceeded their purebred counterparts in this indicator by 47 %. The kurtosis and asymmetry of NOR in the population of purebred goats and their hybrids with the caucasian tur have the same directions. The results of the analysis of variance indicate that the effect of species on the size of the nucleoli in the studied populations is not statistically significant. The average value of the nucleolus diameter in representatives of the genus Capra was 6.17 ± 0.45 μm, genus Ovis – 6.63 ± 0.32 μm.

Nucleolus Organizer Region Functioning in Chicken Growing Oocytes: A Revision of the Existing Views

It is believed that the nucleolus organizer region (NOR) in the ovary of adult birds is inactivated at all stages of oocyte growth. However, in the ovary of juvenile chicken (Gallus g. domesticus) one or two nucleoli were observed in 50% of early diplotene oocytes. These nucleoli were functioning up to the lampbrush stage. It was assumed that diplotene oocytes with inactivated NOR are stored as a pool for development in the ovary of adult hens. Such a contradiction in the NOR functional activity in the oocytes of juvenile and adult hens required special studies. Here we represent new results of a comparative study of the nucleoli in oocytes of juvenile and adult hens. We show for the first time that in the oocytes of laying hens the nucleolus appears at the early diplotene stage and the pre-rRNA synthesis continues up to the lampbrush stage. At the lampbrush stage, the NOR is getting inactivated. The nucleolus disintegrates into large fragments containing both pre-rRNA and fibrillarin. Then these larger fragments break down into small granules that do not contain pre-rRNA but contain fibrillarin. The results resolve the above contradiction and demonstrate a similar pattern of NOR functioning in oocytes of adult and juvenile birds.

A comparative cytogenetic study of Hypsibarbusmalcolmi and H.wetmorei (Cyprinidae, Poropuntiini).

Cyprininae are a highly diversified but demonstrably monophyletic lineage of cypriniform fishes. Here, the karyotype and chromosomal characteristics of Hypsibarbusmalcolmi (Smith, 1945) and H.wetmorei (Smith, 1931) were examined using conventional, nucleolus organizing regions (NORs) and molecular cytogenetic protocols. The diploid chromosome number (2n) of H.malcolmi was 50, the fundamental number (FN) was equal to 62, and the karyotype displayed 8m + 4sm + 38a with NORs located at the centromeric and telomeric positions of the short arms of chromosome pairs 1 and 2, respectively. 2n of H.wetmorei was 50, FN 78, karyotype 14m + 14sm + 22a with the NORs at the telomeric position of the short arm of chromosome pair 2. 2n and FN in males and females were identical. Fluorescence in situ hybridization using different microsatellite motifs as probes also showed substantial genomic divergence between both studied species. In H.wetmorei, (CAG)n and (CAC)n microsatellites accumulated in the telomeric regions of all chromosomes, while in H.malcolmi, they had scattered signals on all chromosomes. Besides, the (GAA)n microsatellites were distributed along all chromosomes of H.malcolmi, but there was a strong hybridization pattern in the centromeric region of a single pair in H.wetmorei. These cytogenomic difference across the genomes of these Hypsibarbus Rainboth, 1996 species are markers for specific evolutionary differentiation within these two species.

Chromosomal characterization of three species of Serrasalmini (Serrasalmidae: Characiformes).

The tribe Serrasalmini is a diverse group with paraphyletic genera and taxonomic uncertainties. Several studies have been carried out in this group of fish in order to understand this problem, including the cytogenetic approach. In this study, three species of a clade of Serrasalmini were characterized cytogenetically - Pristobrycon striolatus, Catoprion absconditus and Pygopristis denticulatus. The three species presented diploid number (2n) equal to 62 chromosomes, of one and two arms, with karyotypic formulas and species-specific fundamental numbers. Heterochromatin is centromeric and terminal (bi-telomeric) in most chromosomes, with a conspicuous interstitial block at pair 1 (m) in all three species. The nucleolar organizer regions were multiple and C-band positive, and their location was confirmed via 18S ribosomal DNA mapping; however, with additional sites. The 5S rDNA was located in interstitial region of long arm of pair 1 (m), in the three species (homeologous). Moreover, we observed synteny between 18S and 5S in the species C. absconditus and P. denticulatus, which, according to fiber-FISH, are interspersed. Thus, the maintenance of 2n (62) evidences the diversification of chromosomal formulas within the clade by non-Robertsonian rearrangements and reflects the paraphyly of the related species.

A Comparison of Proliferative Capacity of Reticular and Erosive Variants of Oral Lichen Planus by Argyrophilic Nucleolar Organizer Regions Method.

Oral lichen planus (OLP), an immune mediated disorder, has been recognized since 1869 and is presented as any one of the six variants. Reticular and erosive are the most frequently encountered. Its proliferative capacity can give some information regarding its progression. We adopted the argyrophilic nucleolar organizer regions (AgNORs) method because of its simplicity to use and dependable results. We evaluated AgNORs in basal, suprabasal, and squamous cell layers. We also compared these three layers within two variants, reticular, and erosive. Thirty clinically diagnosed patients of OLP were included in the study. Reticular and erosive variants were included in our study. This was followed by hematoxylin and eosin staining and later by the AgNOR method. The mean number of AgNORs per nucleus was calculated. Thirteen males and 17 females were the gender distribution. Twenty-three (76.67%) had reticular pattern and seven (23.33%) had erosive pattern. The basal cell layer had the highest mean AgNOR compared to suprabasal and squamous layers. Even among, erosive and reticular variants, the former had higher mean AgNOR counts. Our results suggest that the inflammatory infiltrate close to the epithelial cells can alter the proliferation index for the pattern of protein synthesis of these cells. Moreover, the high proliferative index in OLP can be related to a specific immunologic response. We conclude that AgNOR can be used as a proliferative marker in earlier lesions to detect the severity.

Silver Nitrate Staining of Nucleolar Organizer Regions (Ag-NORs) in Plant Chromosomes.

Silver nitrate staining to evidence the location of nucleolar organizer regions (Ag-NORs) in chromosomes is widely used as a classical method in plant cytogenetics. Here, we present the most used procedures and highlight some aspects in terms of their replicability by plant cytogeneticists. Some technical features described are materials and methods used, procedures, protocol modifications, and precautions in order to obtain positive signals. The methods to obtain Ag-NOR signals have different degrees of replicability, but do not require any sophisticated technology or equipment for their application.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.).

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Relationship Between Argyrophilic Nucleolar Organizer Region Proteins of Pediatric Epilepsy and T Lymphocyte Subpopulation

Relationship Between Argyrophilic Nucleolar Organizer Region Proteins of Pediatric Epilepsy and T Lymphocyte Subpopulation

Evaluation of copper salivary level in oral squamous cell carcinoma, occupationally copper exposed, and its normal population and its association with cytomorphologic changes of oral mucosa

Background: Oral squamous cell carcinoma (SCC) is one of the most common malignancies in oral cavity. Hence, presenting methods for early diagnosis and find the etiologic factors of oral SCC are important. Saliva analysis can be used to discover various conditions because of its noninvasive methods. Copper as a useful metal has been used by men since ancient times. The level of copper increases when the cancerous changes occur in addition to biopsy, an alternative method for examining oral lesions is exfoliative cytology. The primary objective of this study was to determine the salivary copper level and cytomorphologic changes of oral mucosa among three study groups. Materials and Methods: This cross-sectional study included 15 individuals with oral SCC, 15 workers exposed to copper, and 15 healthy individuals. Saliva samples were collected and analyzed by atomic absorption spectrophotometer. The exfoliative smears were prepared by brush biopsy and stained by Papanicolaou and argyrophilic nucleolar organizer region (AgNOR) staining methods. Data analysis using one-way ANOVA and Kruskal–Wallis test. P < 0.05 was considered significant. Results: There was a significant difference in mean salivary copper (P = 0.008), cytomorphology of oral mucosa, and AgNOR among the three groups (P < 0.001). Conclusion: The results suggested that occupational exposure to copper increases the salivary levels of this element and causes changes in mucosal cells. Since this increase was very high and evidence of nuclear activity was seen in this group and in oral SCC patients, exposure to copper should be considered an important risk factor for oral mucosal changes.

Prognostic Potential of Argyrophillic Nucleolar Organizer Regions (Agnors) in Oral Lesions: A Systematic Review

Early detection of oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMD) is important for dental professionals to improve patient survival rates. More than half of patients with oral squamous cell cancer had advanced disease at the time of diagnosis, indicating a lack of early detection and risk assessment biomarkers. The development of new protein biomarkers will help in early diagnosis and treatment.The argyrophilic nucleolar organiser regions (AgNORs) staining technique is simple and cost-effective. These are replicatory markers that identify epithelial dysplasia. And it also plays a very important role in differentiating the benign, pre-malignant, and malignant lesions of the oral cavity. The number of AgNORs per cell has been considered an indicator of cellular proliferative activity. Microscopically, NORs can be identified as well-defined black dots located throughout the cell nucleus. The agNOR quantity is strictly proportional to the proliferative activity of the cell. AgNOR quantification helps in the determination of the degree of epithelial dysplasia and, consequently, in the analysis of its potential for malignant transformation. AgNOR qualitative characteristics help in differentiating hyperplastic, premalignant, and malignant oral lesions. The sliver staining technique is useful for studying the structure of the nucleolus as well as the variations in its activity. AgNORs are a valuable parameter in tumour pathology.

Cytogenetic Analysis of the Bimodal Karyotype of the Common European Adder, Vipera berus (Viperidae).

Vipera berus is the species with the largest range of snakes on Earth and one of the largest among reptiles in general. It is also the only snake species found in the Arctic Circle. Vipera berus is the most involved species of the genus Vipera in the process of interspecific hybridization in nature. The taxonomy of the genus Vipera is based on molecular markers and morphology and requires clarification using SC-karyotyping. This work is a detailed comparative study of the somatic and meiotic karyotypes of V. berus, with special attention to DNA and protein markers associated with synaptonemal complexes. The karyotype of V. berus is a remarkable example of a bimodal karyotype containing both 16 large macrochromosomes and 20 microchromosomes. We traced the stages of the asynchronous assembly of both types of bivalents. The number of crossing-over sites per pachytene nucleus, the localization of the nucleolar organizer, and the unique heterochromatin block on the autosomal bivalent 6-an important marker-were determined. Our results show that the average number of crossing-over sites per pachytene nucleus is 49.5, and the number of MLH1 sites per bivalent 1 reached 11, which is comparable to several species of agamas.