rDNA Repeats Research Articles

Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis

Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5-Air1/Air2-Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5' region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5' region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5' region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.

Karyotype, chromosomal characteristics of multiple rDNA clusters and intragenomic variability of ribosomal ITS2 in Caryophyllaeides fennica (Cestoda)

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SWI/SNF Has Intrinsic Nucleosome Disassembly Activity that Is Dependent on Adjacent Nucleosomes

The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome.

DNA barcoding of arbuscular mycorrhizal fungi

*Currently, no official DNA barcode region is defined for the Fungi. The COX1 gene DNA barcode is difficult to apply. The internal transcribed spacer (ITS) region has been suggested as a primary barcode candidate, but for arbuscular mycorrhizal fungi (AMF; Glomeromycota) the region is exceptionably variable and does not resolve closely related species. *DNA barcoding analyses were performed with datasets from several phylogenetic lineages of the Glomeromycota. We tested a c. 1500 bp fragment spanning small subunit (SSU), ITS region, and large subunit (LSU) nuclear ribosomal DNA for species resolving power. Subfragments covering the complete ITS region, c. 800 bp of the LSU rDNA, and three c. 400 bp fragments spanning the ITS2, the LSU-D1 or LSU-D2 domains were also analysed. *Barcode gap analyses did not resolve all species, but neighbour joining analyses, using Kimura two-parameter (K2P) distances, resolved all species when based on the 1500 bp fragment. The shorter fragments failed to separate closely related species. *We recommend the complete 1500 bp fragment as a basis for AMF DNA barcoding. This will also allow future identification of AMF at species level based on 400 or 1000 bp amplicons in deep sequencing approaches.

Genetic identification of new biological species Saccharomyces arboricolus Wang et Bai

Direct genetic testing for hybrid sterility unambiguously showed that the newly described yeast Saccharomyces arboricolus Wang et Bai is reproductively isolated from Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces kudriavzevii, Saccharomyces mikatae and Saccharomyces paradoxus and, therefore, represents a new biological species of the genus Saccharomyces. Combined phylogenetic analysis of the rDNA repeat sequences (18S, 26S, ITS), nuclear ACT1 and mitochondrial ATP9 genes revealed that S. arboricolus, along with S. kudriavzevii and S. bayanus, is distantly related to the other four biological species.

Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a

RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.

Translocation of Y-Linked Genes to the Dot Chromosome in Drosophila pseudoobscura

One of the most striking cases of sex chromosome reorganization in Drosophila occurred in the lineage ancestral to Drosophila pseudoobscura, where there was a translocation of Y-linked genes to an autosome. These genes went from being present only in males, never recombining, and having an effective population size of 0.5N to a state of autosomal linkage, where they are passed through both sexes, may recombine, and their effective population size has quadrupled. These genes appear to be functional, and they underwent a drastic reduction in intron size after the translocation. A Y-autosome translocation may pose problems in meiosis if the rDNA locus responsible for X-Y pairing had also moved to an autosome. In this study, we demonstrate that the Y-autosome translocation moved Y-linked genes onto the dot chromosome, a small, mainly heterochromatic autosome with some sex chromosome-like properties. The rDNA repeats occur exclusively on the X chromosome in D. pseudoobscura, but we found that the new Y chromosome of this species harbors four clusters bearing only the intergenic spacer region (IGS) of the rDNA repeats. This arrangement appears analogous to the situation in Drosophila simulans, where X-rDNA to Y-IGS pairing could be responsible for X-Y chromosome pairing. We postulate that the nascent D. pseudoobscura Y chromosome acquired and amplified copies of the IGS, suggesting a potential mechanism for X-Y pairing in D. pseudoobscura.

Abundance of Ribosomal RNA Gene Copies Maintains Genome Integrity

The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large cluster(s) of tandemly repeated copies in the chromosome, with each species having a specific number of copies. Yeast has many untranscribed rDNA copies (extra copies), and we found that when they are lost, the cells become sensitive to DNA damage induced by mutagens. We show that this sensitivity is dependent on rDNA transcriptional activity, which interferes with cohesion between rDNA loci of sister chromatids. The extra rDNA copies facilitate condensin association and sister-chromatid cohesion, thereby facilitating recombinational repair. These results suggest that high concentrations of heavily transcribed genes are toxic to the cells, and therefore amplified genes, such as rDNA, have evolved.

Intergenic transcripts originating from a subclass of ribosomal DNA repeats silence ribosomal RNA genes in trans

Epigenetic silencing of a fraction of ribosomal DNA (rDNA) requires association of the nucleolar chromatin-remodelling complex NoRC to 150-250 nucleotide RNAs (pRNA) that originate from an RNA polymerase I promoter located in the intergenic spacer separating rDNA repeats. Here, we show that NoRC-associated pRNA is transcribed from a sub-fraction of hypomethylated rRNA genes during mid S phase, acting in trans to inherit DNA methylation and transcriptional repression of late-replicating silent rDNA copies. The results reveal variability between individual rDNA clusters with distinct functional consequences.

The SNF2-Family Member Fun30 Promotes Gene Silencing in Heterochromatic Loci

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.

The FEAR network

Mitosis is governed by the oscillation of cyclin dependent kinase (CDK) activity and ubiquitin-dependent proteolysis. Entry into mitosis is initiated by mitotic cyclin-CDK activation. Anaphase onset occurs upon activation of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that promotes the destruction of the anaphase inhibitor Securin. Destruction of Securin initiates chromosome segregation by activation of the protease Separase, allowing it to cleave a subunit of the cohesin complexes that hold the duplicated sister chromatids together. Upon completion of nuclear division cells exit from mitosis, a process defined by the inactivation of CDKs, disassembly of the mitotic spindle, and cytokinesis. In the budding yeast S. cerevisiae, a signaling network known as the FEAR network is critical to ensure accurate anaphase chromosome segregation and the integration of this process with other anaphase events. Here, we summarize what is known about the regulation and function of the FEAR network in budding yeast and discuss the potential for conserved FEAR network functions in other eukaryotes.

Phylogenetic Analysis of Subgenus Vigna Species Using Nuclear Ribosomal RNA ITS: Evidence of Hybridization among Vigna unguiculata Subspecies

Molecular phylogeny among species belonging to subgenus Vigna (genus Vigna) was inferred based on internal transcribed spacer (ITS) sequences of 18S-5.8S-26S ribosomal RNA gene unit. Analysis showed a total of 356 polymorphic sites of which approximately 80% were parsimony informative. Phylogenetic reconstruction by neighbor joining and maximum parsimony methods placed the 57 Vigna accessions (belonging to 15 species) into 5 major clades. Five species viz. Vigna heterophylla, Vigna pubigera, Vigna parkeri, Vigna laurentii, and Vigna gracilis whose position in the subgenus was previously not known were placed in the section Vigna. A single accession (Vigna unguiculata ssp. tenuis, NI 1637) harbored 2 intragenomic ITS variants, indicative of 2 different types of ribosomal DNA (rDNA) repeat units. ITS variant type-I was close to ITS from V. unguiculata ssp. pubescens, whereas type-II was close to V. unguiculata ssp. tenuis. Transcript analysis clearly demonstrates that in accession NI 1637, rDNA repeat units with only type-II ITS variants are transcriptionally active. Evidence from sequence analysis (of 5.8S, ITS1, and ITS2) and secondary structure analysis (of ITS1 and ITS2) indicates that the type-I ITS variant probably does not belong to the pseudogenic rDNA repeat units. The results from phylogenetic and transcript analysis suggest that the rDNA units with the type-I ITS may have introgressed as a result of hybridization (between ssp. tenuis and ssp. pubescens); however, it has been epigenetically silenced. The results also demonstrate differential evolution of ITS sequence among wild and cultivated forms of V. unguiculata.

Multiple patterns of rDNA evolution following polyploidy in Oryza

Ribosomal ITS sequences are commonly used for phylogenetic reconstruction because they are included in rDNA repeats, and hence are ubiquitous and present in high copy number. Ribosomal rDNA repeats often undergo rapid concerted evolution within and between arrays. Interspecific hybridization merges divergent repeat types in a single nucleus, setting in motion evolutionary processes leading to coexistence, maintenance of paralogs, origin of novel sequence variants, loss of arrays, or inter-array sequence homogenization via concerted evolution. Here we examined ITS polymorphism within and among six Oryza tetraploids of varying genomic composition to infer the extent and direction of concerted evolution following allopolyploid speciation. We demonstrate that different polyploids have experienced varying fates, including maintenance or homogenization of divergent arrays, even among allopolyploids having the same genomic origins but in different geographic locations. Bidirectional concerted evolution, in which arrays become homogenized to alternative progenitor diploid types in different allopolyploid derivatives, is evident among species in one clade. Our results exemplify the panoply of outcomes for ribosomal DNA evolution following allopolyploid speciation.

Sequenced BAC anchored reference genetic map that reconciles the ten individual chromosomes of Brassica rapa

BackgroundIn view of the immense value of Brassica rapa in the fields of agriculture and molecular biology, the multinational Brassica rapa Genome Sequencing Project (BrGSP) was launched in 2003 by five countries. The developing BrGSP has valuable resources for the community, including a reference genetic map and seed BAC sequences. Although the initial B. rapa linkage map served as a reference for the BrGSP, there was ambiguity in reconciling the linkage groups with the ten chromosomes of B. rapa. Consequently, the BrGSP assigned each of the linkage groups to the project members as chromosome substitutes for sequencing.ResultsWe identified simple sequence repeat (SSR) motifs in the B. rapa genome with the sequences of seed BACs used for the BrGSP. By testing 749 amplicons containing SSR motifs, we identified polymorphisms that enabled the anchoring of 188 BACs onto the B. rapa reference linkage map consisting of 719 loci in the 10 linkage groups with an average distance of 1.6 cM between adjacent loci. The anchored BAC sequences enabled the identification of 30 blocks of conserved synteny, totaling 534.9 cM in length, between the genomes of B. rapa and Arabidopsis thaliana. Most of these were consistent with previously reported duplication and rearrangement events that differentiate these genomes. However, we were able to identify the collinear regions for seven additional previously uncharacterized sections of the A genome. Integration of the linkage map with the B. rapa cytogenetic map was accomplished by FISH with probes representing 20 BAC clones, along with probes for rDNA and centromeric repeat sequences. This integration enabled unambiguous alignment and orientation of the maps representing the 10 B. rapa chromosomes.ConclusionWe developed a second generation reference linkage map for B. rapa, which was aligned unambiguously to the B. rapa cytogenetic map. Furthermore, using our data, we confirmed and extended the comparative genome analysis between B. rapa and A. thaliana. This work will serve as a basis for integrating the genetic, physical, and chromosome maps of the BrGSP, as well as for studies on polyploidization, speciation, and genome duplication in the genus Brassica.

Fission yeast Ku protein is required for recovery from DNA replication stress

The fundamental function of the conserved Ku70-Ku80 heterodimer is to promote the non-homologous end-joining (NHEJ) pathway in double-strand break repair. Although it is thought that Ku plays several roles other than NHEJ in maintaining chromosomal integrity including telomere protection, these precise functions remain unclear. In this study, we describe a novel role of fission yeast Ku proteins encoded by pku70(+) and pku80(+) genes in dealing with DNA replication stress. In the absence of Rqh1, the fission yeast RecQ helicase, the cells are sensitive to reagents inducing replication stress. pkuDeltarqh1Delta double mutant showed synergistic sensitivities to these reagents. However, this synthetic phenotype was not observed when rqh1Delta mutant was coupled with the deletion of lig4(+) that encodes a ligase essential for NHEJ, indicating that the role of Ku in replication stress is NHEJ independent. pkuDeltarqh1Delta double mutant also showed highly variable copy numbers of rDNA repeats even under unstressed condition. Furthermore, the double mutant exhibited inefficient replication resumption after transient replication stalling. These results suggest the possibility that Ku proteins play an important role in genome integrity recovering replication stress.

Allopolyploid origin of Mediterranean species in Helictotrichon (Poaceae) and its consequences for karyotype repatterning and homogenisation of rDNA repeat units

Molecular and molecular‐cytogenetic studies reveal that allopolyploidisation can dramatically impact genome organisation from sequence level to chromosome structure. To clarify the origin of polyploids in the Mediterranean Helictotrichon bromoides species complex (Poaceae, grass family) detailed chromosome studies were carried out including fluorescence in situ hybridisation with different repetitive DNA probes and various types of chromosome staining. Cloned nuclear ribosomal ITS1–5.8S‐ITS2 repeats were sequenced to estimate their molecular diversity in diploids and polyploids. The results concordantly suggest that 2× H. bromoides is one genome donor of all polyploids. Two further genomes were involved in their origin. The polyploids show unidirectional homogenisation of rDNA consistently at the cost of repeats from the H. bromoides genome. Frequent occurrence of mosaic and chimerical sequences points to slow sequence homogenisation. Parental chromosomal sites of nucleolar organisers are maintained despite molecular change of their rDNA repeat units. Major chromosomal re‐arrangements are confined to the 6x‐8x higher polyploids of H. gervaisii and accompanied by marked amplification of 5S rDNA and subtelomeric heterochromatin. The polyploids have narrower overall geographical distributions than the diploids, but are more successful in dry areas. This ecological pattern is discussed in relation to their allopolyploid origins.

Opposing Role of Condensin and Radiation-sensitive Gene RAD52 in Ribosomal DNA Stability Regulation

Blocking target of rapamycin signaling by starvation or rapamycin inhibits ribosomal DNA (rDNA) transcription and causes condensin-mediated rDNA condensation and nucleolar contraction. In the absence of condensin, however, repression of rDNA transcription leads to rDNA instability and elevated level of extrachromosomal rDNA circles and nucleolar fragmentation. Here, we show that mutations in the Rad52 homologous recombination machinery block rDNA instability. Rad52 is normally excluded from the nucleolus. In the absence of condensin, however, repression of rDNA transcription results in Rad52 localization to the nucleolus, association with rDNA and subsequent formation of extrachromosomal rDNA circles, and reduced cell survival. In contrast, deletion of RAD52 restores cell viability under the same conditions. These results reveal an opposing role of condensin and Rad52 in the control of rDNA stability under nutrient starvation conditions.

Going, Going, Not Quite Gone: Nucleomorphs as a Case Study in Nuclear Genome Reduction

Nucleomorphs are the relic nuclei of algal endosymbionts that became permanent fixtures inside nonphotosynthetic eukaryotic host cells. These unusual organelles exist in only 2 lineages, the cryptophytes, which possess nucleomorphs and plastids (chloroplasts) derived from the uptake of a red algal endosymbiont, and the chlorarachniophytes, which harbor green algal derived nucleomorphs and plastids. Despite having evolved independently of one another, the nucleomorph genomes of cryptophytes and chlorarachniophytes are strikingly similar in size and basic structure. Both are <1 Mbp in size-the smallest nuclear genomes known-and are composed of only 3 chromosomes, each with its own subtelomeric rDNA repeats. Nucleomorph-containing algae thus represent an interesting system in which to study genome and chromosome evolution in eukaryotes. Here, we provide an overview of nucleomorph genome biology and focus on new information gleaned from comparisons of complete nucleomorph genome sequences, both within and between cryptophytes and chlorarachniophytes. Such comparisons provide fascinating insight into the evolution of these highly derived organelles and, more generally, the potential causes and consequences of genome reduction in eukaryotes.

A bicontinental origin of polyploid Australian/New Zealand Lepidium species (Brassicaceae)? Evidence from genomic in situ hybridization

Incongruence between chloroplast and nuclear DNA phylogenies, and single additive nucleotide positions in internal transcribed spacer (ITS) sequences of polyploid Australian/New Zealand (NZ) Lepidium species have been used to suggest a bicontinental hybrid origin. This pattern was explained by two trans-oceanic dispersals of Lepidium species from California and Africa and subsequent hybridization followed by homogenization of the ribosomal DNA sequence either to the Californian (C-clade) or to the African ITS-type (A-clade) in two different ITS-lineages of Australian/NZ Lepidium polyploids. Genomic in situ hybridization (GISH) was used to unravel the genomic origin of polyploid Australian/NZ Lepidium species. Fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA) probes was applied to test the purported ITS evolution, and to facilitate chromosome counting in high-numbered polyploids. In Australian/NZ A-clade Lepidium polyploids, GISH identified African and Australian/NZ C-clade species as putative ancestral genomes. Neither the African nor the Californian genome were detected in Australian/NZ C-clade species and the Californian genome was not detected in Australian/NZ A-clade species. Five of the eight polyploid species (from 7x to 11x) displayed a diploid-like set of rDNA loci. Even the undecaploid species Lepidium muelleriferdinandi (2n = 11x = 88) showed only one pair of each rDNA repeat. In A-clade allopolyploids, in situ rDNA localization combined with GISH corroborated the presence of the African ITS-type. The nuclear genomes of African and Australian/NZ C-clade species were detected by GISH in allopolyploid Australian/NZ Lepidium species of the A-clade, supporting their hybrid origin. The presumed hybrid origin of Australian/NZ C-clade taxa could not be confirmed. Hence, it is assumed that Californian ancestral taxa experienced rapid radiation in Australia/NZ into extant C-clade polyploid taxa followed by hybridization with African species. As a result, A-clade allopolyploid Lepidium species share the Californian chloroplast type and the African ITS-type with the C-clade Australian/NZ polyploid and African diploid species, respectively.

Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III-transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1-v allele. In W303 cells that carry the SSD1-d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm-to-nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm-to-nucleolus translocation. Together, these observations provide new insights into the chromatin-dependent mechanism by which TORC1 controls transcription by Pol III.