Ribosomal Gene Repeat Research Articles

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.

Nucleolar Organizer Regions as Transcription-Based Scaffolds of Nucleolar Structure and Function.

Eukaryotic genomes maintain multiple copies of ribosomal DNA gene repeats in tandem arrays to provide sufficient ribosomal RNAs to make ribosomes. These DNA repeats are the most highly transcribed regions of the genome, with dedicated transcriptional machinery to manage the enormous task of producing more than 50% of the total RNA in a proliferating cell. The arrays are called nucleolar organizer regions (NORs) and constitute the scaffold of the nucleolar compartment, where ribosome biogenesis occurs. Advances in molecular and cellular biology have brought great insights into how these arrays are transcribed and organized within genomes. Much of their biology is driven by their high transcription level, which has also driven the development of unique methods to understand rDNA gene activity, beginning with classic techniques such as silver staining and Miller spreads. However, the application of modern methodologies such as CRISPR gene editing, super-resolution microscopy, and long-read sequencing has enabled recent advances described herein, with many more discoveries possible soon. This chapter highlights what is known about NOR transcription and organization and the techniques applied historically and currently. Given the potential for NORs to impact organismal health and disease, as highlighted at the end of the chapter, the field must continue to develop and apply innovative analysis to understand genetic, epigenetic, and organizer properties of the ribosomal DNA repeats.

Antagonising Chromatin Remodelling Activities in the Regulation of Mammalian Ribosomal Transcription.

Ribosomal transcription constitutes the major energy consuming process in cells and is regulated in response to proliferation, differentiation and metabolic conditions by several signalling pathways. These act on the transcription machinery but also on chromatin factors and ncRNA. The many ribosomal gene repeats are organised in a number of different chromatin states; active, poised, pseudosilent and repressed gene repeats. Some of these chromatin states are unique to the 47rRNA gene repeat and do not occur at other locations in the genome, such as the active state organised with the HMG protein UBF whereas other chromatin state are nucleosomal, harbouring both active and inactive histone marks. The number of repeats in a certain state varies on developmental stage and cell type; embryonic cells have more rRNA gene repeats organised in an open chromatin state, which is replaced by heterochromatin during differentiation, establishing different states depending on cell type. The 47S rRNA gene transcription is regulated in different ways depending on stimulus and chromatin state of individual gene repeats. This review will discuss the present knowledge about factors involved, such as chromatin remodelling factors NuRD, NoRC, CSB, B-WICH, histone modifying enzymes and histone chaperones, in altering gene expression and switching chromatin states in proliferation, differentiation, metabolic changes and stress responses.

DNA Damage Response in Nucleoli

Nucleoli, the largest subnuclear compartments, are formed around arrays of ribosomal gene repeats transcribed by RNA polymerase I. The primary function of nucleoli is ribosome biogenesis. Specific DNA damage response mechanisms exist to maintain the genomic stability of ribosomal repeats. Here, we provide a snapshot of our current understanding of processes involved in nucleolar DNA damage response. We discuss structure and function of ribosomal repeats, techniques developed for studying DNA damage response in nucleoli, as well as molecular mechanisms of DNA damage-induced repression of nucleolar transcription and nucleoli reorganization.

Cytogenetic characterization of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902) through analysis of mitotic chromosomes from early larvae

This paper describes the cytogenetic features of the Antarctic silverfish Pleuragramma antarctica (Boulenger 1902), a keystone species of the Antarctic coastal marine ecosystem. Conventional cytogenetic analyses and physical mapping of repetitive DNA sequences were performed on metaphase plates obtained through direct chromosome preparation from P. antarctica early larvae.The Antarctic silverfish have a diploid number (2n) = 48, and a karyotype made up of a majority of two-armed chromosomes (karyotype formula36m/sm + 10st + 2a, fundamental number = 94). Major ribosomal gene repeats were detected on three chromosome pairs (20, 21, and 23), in correspondence of dim DAPI stained regions. Long Interspersed Nuclear Elements (LINEs) were abundant and wide spread over all chromosomes.Overall, the cytogenetic data presented herein are consistent with a long independent cytogenetic and evolutionary history for the species. The large number of two-armed chromosomes, indicative of highly-rearranged karyotype, coupled with a diploid number of 48, a presumed primitive character for this fish group, and the spread of the major ribosomal genes on three chromosome pairs, make the Antarctic silverfish distinct from all other notothenioid species.

Integrating the genomic architecture of human nucleolar organizer regions with the biophysical properties of nucleoli.

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells.

Nucleolar organizer regions: genomic 'dark matter' requiring illumination.

Nucleoli form around tandem arrays of a ribosomal gene repeat, termed nucleolar organizer regions (NORs). During metaphase, active NORs adopt a characteristic undercondensed morphology. Recent evidence indicates that the HMG-box-containing DNA-binding protein UBF (upstream binding factor) is directly responsible for this morphology and provides a mitotic bookmark to ensure rapid nucleolar formation beginning in telophase in human cells. This is likely to be a widely employed strategy, as UBF is present throughout metazoans. In higher eukaryotes, NORs are typically located within regions of chromosomes that form perinucleolar heterochromatin during interphase. Typically, the genomic architecture of NORs and the chromosomal regions within which they lie is very poorly described, yet recent evidence points to a role for context in their function. In Arabidopsis, NOR silencing appears to be controlled by sequences outside the rDNA (ribosomal DNA) array. Translocations reveal a role for context in the expression of the NOR on the X chromosome in Drosophila Recent work has begun on characterizing the genomic architecture of human NORs. A role for distal sequences located in perinucleolar heterochromatin has been inferred, as they exhibit a complex transcriptionally active chromatin structure. Links between rDNA genomic stability and aging in Saccharomyces cerevisiae are now well established, and indications are emerging that this is important in aging and replicative senescence in higher eukaryotes. This, combined with the fact that rDNA arrays are recombinational hot spots in cancer cells, has focused attention on DNA damage responses in NORs. The introduction of DNA double-strand breaks into rDNA arrays leads to a dramatic reorganization of nucleolar structure. Damaged rDNA repeats move from the nucleolar interior to form caps at the nucleolar periphery, presumably to facilitate repair, suggesting that the chromosomal context of human NORs contributes to their genomic stability. The inclusion of NORs and their surrounding chromosomal environments in future genome drafts now becomes a priority.

Ribosomal DNA and cellular senescence: new evidence supporting the connection between rDNA and aging

The yeast Saccharomyces cerevisiae is a powerful model system to understand the molecular basis of aging. It has been known for over 50years that yeast cells have a finite replicative capacity and develop an aging phenotype, and much recent research has focused on the molecular changes that underlie this replicative senescence. A common thread in many yeast replicative aging studies is the involvement of the ribosomal DNA gene repeats (rDNA), beginning with the discovery that the rDNA silencing gene, SIR2, regulates life span. In 2008, a novel aging hypothesis, termed the rDNA theory of aging, was presented where the high level of genomic instability at the rDNA repeats was proposed to dominate global genome stability and determine the life span. Here, we review the rDNA theory of aging and discuss a number of recent studies that provide important new data on the roles of the rDNA in yeast replicative aging. Based on these recent results, we propose an integrative model of the rDNA theory of aging that encompasses genomic instability, chromatin relocalization following DNA repair, and replication stress in a self-reinforcing cyclical pathway that is primarily manifested at the rDNA repeats and results in the aging phenotype.

The shared genomic architecture of human nucleolar organizer regions

The short arms of the five acrocentric human chromosomes harbor sequences that direct the assembly and function of the nucleolus, one of the key functional domains of the nucleus, yet they are absent from the current human genome assembly. Here we describe the genomic architecture of these human nucleolar organizers. Sequences distal and proximal to ribosomal gene arrays are conserved among the acrocentric chromosomes, suggesting they are sites of frequent recombination. Although previously believed to be heterochromatic, characterization of these two flanking regions reveals that they share a complex genomic architecture similar to other euchromatic regions of the genome, but they have distinct genomic characteristics. Proximal sequences are almost entirely segmentally duplicated, similar to the regions bordering centromeres. In contrast, the distal sequence is predominantly unique to the acrocentric short arms and is dominated by a very large inverted repeat. We show that the distal element is localized to the periphery of the nucleolus, where it appears to anchor the ribosomal gene repeats. This, combined with its complex chromatin structure and transcriptional activity, suggests that this region is involved in nucleolar organization. Our results provide a platform for investigating the role of NORs in nucleolar formation and function, and open the door for determining the role of these regions in the well-known empirical association of nucleoli with pathology.

A Phylogenetic Analysis of the Genus Dahlia (Asteraceae) Based on Internal and External Transcribed Spacer Regions of Nuclear Ribosomal DNA

The genus Dahlia presently consists of 35 species, primarily from Mexico. Species are usually placed in four sections: Pseudodendron, Epiphytum, Entemophyllon, and Dahlia, based largely on morphological characters, supplemented with cytological, geographical, and biochemical data. Combined molecular sequence data from both the internal and external transcribed spacer regions (ITS and ETS), located within the nuclear ribosomal gene repeat unit, are used to infer a phylogeny of the genus. Section Entemophyllon forms a very well-defined clade based on these data. Dahlia merckii and D. tubulata are positioned between sect. Entemophyllon the remaining taxa. Sections Pseudodendron and Epiphytum are closely allied with each other and a few species from sect. Dahlia to form the variable root clade (VRC), which incorporates all species with unusual underground structures, along with some species exhibiting the more typical tuberous type. The remaining species of sect. Dahlia form a well-defined clade, the core Dahlia clade (CDC).

Ancient divergence of Paragonimus westermani in Sri Lanka

Adult flukes of Paragonimus species were obtained from lungs of experimental animals infected with metacercariae found in field-collected freshwater crabs in Sri Lanka. Morphological studies of adult worms under a scanning electron microscope as well as a ordinary microscope were performed in the present study. All of morphological features observed clearly indicated that this species is P. westermani. On the other hand, the shapes of metacercariae were found to be mainly oval, but semioval and spherical ones also coexisted. In spite of the variety of their morphology of the metacercariae, there is no correlation between their shapes of metacercariae and the deoxyribonucleic acid (DNA) sequences. Molecular phylogenetic analyses using two DNA regions (partial mitochondrial cytochrome c oxidase subunit 1 and second internal transcribed spacer of the nuclear ribosomal gene repeat) placed the adult flukes of P. westermani from Sri Lanka basal in a tree including all specimens of P. westermani from various areas in Asia and P. siamensis from Thailand. The present study showed that P. westermani from Sri Lanka is an ancestral form.

A role for upstream binding factor in organizing ribosomal gene chromatin

Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus, and during metaphase form novel silver-stainable secondary constrictions, termed pseudo-NORs, that are morphologically similar to NORs.

Systematics of Hypocrea citrina and related taxa

Morphological studies and phylogenetic analyses of DNA sequences from three genomic regions – the internal transcribed spacer (ITS) regions of the nuclear ribosomal gene repeat, a partial sequence of RNA polymerase II subunit (rpb2), and a partial sequence of translation elongation factor (tef1) – were used to investigate the systematics of Hypocrea citrina and related species. A neotype specimen is designated for H. citrina that conforms to Persoon's description of a yellow effuse fungus occurring on leaf litter. Historical information and results obtained in this study provide the foundation for selection of a lectotype specimen from Fries's herbarium for H. lactea. The results indicate that (1) Hypocrea citrina and H. pulvinata are distinct species; (2) H. lactea sensu Fries is a synonym of the older name H. citrina; (3) H. pulvinata, H. protopulvinata, and H. americana are phylogenetically distinct species that form a well-supported polyporicolous clade; (4) H. citrina is situated in a clade closely related to H. pulvinata; and (5) H. microcitrina and H. pseudostraminea reside in a highly supported clade phylogenetically distinct from H. citrina. Hypocrea protopulvinata, H. microcitrina, H. megalocitrina, H. pseudostraminea, and a new species, H. aurantiistroma, are reported and described from North America. Variation in rpb2 and tef1 gene sequences suggests geographical subgroupings between European and North American isolates of H. pulvinata. The phylogenies inferred from ITS, rpb2, and tef1 gene sequences are concordant. Hypocrea citrina var. americana is elevated to species status, Hypocrea americana.

Taxonomy and phylogenetic relationships of nine species of Hypocrea with anamorphs assignable to Trichoderma section Hypocreanum

Morphological studies and phylogenetic analyses of DNA sequences from the internal transcribed spacer (ITS) regions of the nuclear ribosomal gene repeat, a partial sequence of RNA polymerase II subunit (rpb2), and a partial sequence of the large exon of tef1 (LEtef1) were used to investigate the taxonomy and systematics of nine Hypocrea species with anamorphs assignable to Trichoderma sect. Hypocreanum. Hypocrea corticioides and H. sulphurea are reevaluated. Their Trichoderma anamorphs are described and the phylogenetic positions of these species are determined. Hypocrea sulphurea and H. subcitrina are distinct species based on studies of the type specimens. Hypocrea egmontensis is a facultative synonym of the older name H. subcitrina. Hypocrea with anamorphs assignable to Trichoderma sect. Hypocreanum formed a well-supported clade. Five species with anamorphs morphologically similar to sect. Hypocreanum, H. avellanea, H. parmastoi, H. megalocitrina, H. alcalifuscescens, and H. pezizoides, are not located in this clade. Protocrea farinosa belongs to Hypocrea s.s.

Nucleolar biogenesis: the first small steps

The nucleolus is the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus assembles around clusters of ribosomal gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. The initial step in nucleolar formation is ribosomal gene transcription, which is mediated by Pol I (RNA polymerase I) and its associated transcription factors: UBF (upstream-binding factor), SL1 (selectivity factor) and TIF-IA (transcription initiation factor IA)/Rrn3. Ribosomal gene clusters, termed NORs (nucleolar organizer regions), are found on each of the five human acrocentric chromosomes. Though transcription is repressed during metaphase, NORs that were active in the previous interphase form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under-condensation in comparison with the rest of the chromosome. Extensive binding of UBF over the ribosomal gene repeat is responsible for the formation of this chromosomal feature. During interphase, the majority of the Pol I transcription machinery, though present in nucleoli, is not actively engaged in transcription. Interaction with UBF bound across the gene repeat provides an explanation for how this non-engaged Pol I machinery is sequestered by nucleoli.

Nucleolar biogenesis: the first small steps.

The nucleolus is the site of rRNA transcription, pre-rRNA processing and ribosome subunit assembly. The nucleolus assembles around clusters of ribosomal gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. The initial step in nucleolar formation is ribosomal gene transcription, which is mediated by Pol I (RNA polymerase I) and its associated transcription factors: UBF (upstream-binding factor), SL1 (selectivity factor) and TIF-IA (transcription initiation factor IA)/Rrn3. Ribosomal gene clusters, termed NORs (nucleolar organizer regions), are found on each of the five human acrocentric chromosomes. Though transcription is repressed during metaphase, NORs that were active in the previous interphase form prominent cytogenetic features, namely secondary constrictions. The main defining characteristic of these constrictions is under-condensation in comparison with the rest of the chromosome. Extensive binding of UBF over the ribosomal gene repeat is responsible for the formation of this chromosomal feature. During interphase, the majority of the Pol I transcription machinery, though present in nucleoli, is not actively engaged in transcription. Interaction with UBF bound across the gene repeat provides an explanation for how this non-engaged Pol I machinery is sequestered by nucleoli.

Molecular Cytogenetic Characterization of Ciona intestinalis Chromosomes

The compact genome of the ascidian Ciona intestinalis has been sequenced. Chromosome karyotype and mapping of the genome sequence information on each of the 14 pairs of chromosomes are essential for genome-wide studies of gene expression and function in this basal chordate. Although the small chromosome size (most pairs measuring less than 2 mum) complicates accurate chromosome pairing based on morphology alone, the present results suggest that 20 chromosomes are metacentric and 8 are submetacentric or subtelocentric, and two pairs of large chromosomes (#1 and #2) were defined. The characterization of chromosomes by FISH and staining with propidium iodide indicated that 18S/28S ribosomal gene repeats are present in the short arms of three pairs of chromosomes and that the short arms of these pairs show remarkable size polymorphism. In addition, each chromosome was characterized molecular cytogenetically by mapping representative BAC clones with FISH. The present study is therefore a first step in expanding the karyotype analysis and entire physical mapping of the genome sequence of Ciona intestinalis.

Upstream binding factor association induces large-scale chromatin decondensation.

The function of upstream binding factor (UBF), an essential component of the RNA polymerase (pol) I preinitiation complex, is unclear. Recently, UBF was found distributed throughout ribosomal gene repeats rather than being restricted to promoter regions. This observation has led to the speculation that one role of UBF binding may be to induce chromatin remodeling. To directly evaluate the impact of UBF on chromatin structure, we used an in vivo assay in which UBF is targeted via a lac repressor fusion protein to a heterochromatic, amplified chromosome region containing lac operator repeats. We show that the association of UBF with this locus induces large-scale chromatin decondensation. This process does not appear to involve common remodeling complexes, including SWI/SNF and histone acetyltransferases, and is independent of histone H3 lysine 9 acetylation. However, UBF recruits the pol I-specific, TATA box-binding protein containing complex SL1 and pol I subunits. Our results suggest a working hypothesis in which the dynamic association of UBF with ribosomal DNA clusters recruits the pol I transcription machinery and maintains these loci in a transcriptionally competent configuration. These studies also provide an in vivo model simulating ribosomal DNA transactivation outside the nucleolus, allowing temporal and spatial analyses of chromatin remodeling and assembly of the pol I transcription machinery.

Molecular taxonomic position of the elephant schistosome, Bivitellobilharzia nairi, newly discovered in Sri Lanka

Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotyes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed.

Identities of two Paragonimus species from Sri Lanka inferred from molecular sequences.

Metacercariae of Paragonimus spp. were obtained from field-collected freshwater crabs in Sri Lanka. Genomic DNA was extracted from single metacercariae. Two gene regions (partial mitochondrial cytochrome c oxidase subunit 1 (CO1) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) were amplified using the polymerase chain reaction. Two differing sequences were obtained for each of these gene regions. Phylogenetic analyses placed the type 1 sequences as sister to a clade containing P. westermani and P. siamensis whereas the type 2 sequences were close to published sequences of P. siamensis from Thailand. The possible taxonomic status of these two types are discussed. This is the first report of molecular data about Paragonimus from Sri Lanka.