Ribosomal RNA Gene Repeats Research Articles

Visualization of the dynamic behavior of ribosomal RNA gene repeats in living yeast cells

The ribosomal RNA (rRNA) gene forms an extremely large repeat (rDNA) in the chromosome. In budding yeast, Saccharomyces cerevisiae, the rDNA is located on chromosome XII and occupies approximately 60% (1.5 Mb) of the chromosome and 10% of the total genome. The rDNA is composed of approximately 150 copies and produces rRNA that accounts for approximately 80% of the total RNA. Owing to its repetitive structure and high transcription activity, rDNA is recombinogenic. Thus, the repeat is a distinctive and unique region in the genome. To investigate the dynamic nature of the repeat during the cell division cycle, we developed yeast strains in which every rDNA repeat unit has lacO or tetO arrays that associate with LacI-GFP or TetR-mRFP proteins, respectively. In these strains, the entire repeat can be visualized in a living cell without the need for any special treatment. Analyses of these strains showed that the rDNA actively moved and changed shape at the boundary between the nucleolus and the nucleoplasm. At the G2/M phase, the associated sister chromatids of the rDNA formed a ring structure in the nucleolus that gave the appearance of 'rotating'. We also found that the length of the rDNA repeat affected the time taken for segregation during the M phase of cell cycle.

Complete Sructure of Nuclear rDNA of the Obligate Plant Parasite Plasmodiophora brassicae: Intraspecific Polymorphisms in the Exon and Group I Intron of the Large Subunit rDNA

Plasmodiophora brassicae is a soil-borne obligate intracellular parasite in the phylum Cercozoa of the Rhizaria that causes clubroot disease of crucifer crops. To control the disease, understanding the distribution and infection routes of the pathogen is essential, and thus development of reliable molecular markers to discriminate geographic populations is required. In this study, the nuclear ribosomal RNA gene (rDNA) repeat unit of P. brassicae was determined, with particular emphasis on the structure of large subunit (LSU) rDNA, in which polymorphic regions were expected to be present. The complete rDNA complex was 9513bp long, which included the small subunit, 5.8S and LSU rDNAs as well as the internal transcribed spacer and intergenic spacer regions. Among eight field populations collected from throughout Honshu Island, Japan, a 1.1 kbp region of the LSU rDNA, including the divergent 8 domain, exhibited intraspecific polymorphisms that reflected geographic isolation of the populations. Two new group I introns were found in this region in six out of the eight populations, and the sequences also reflected their geographic isolation. The polymorphic region found in this study may have potential for the development of molecular markers for discrimination of field populations/isolates of this organism.

A six locus phylogeny reveals high species diversity in Botryosphaeriaceae from California almond

Botryosphaeriaceae are important pathogens on a variety of woody hosts, including almond, a major crop in California. Almond is susceptible to Botryosphaeria dothidea that forms band cankers on almond trunks, and the same fungus was also isolated from cankers of the canopy. To study the diversity and host range of B. dothidea and allied species from almond we used 132 isolates from 36 plant hosts from five continents, including 45 strains from almond in California. Species were identified by comparison to 13 ex-type strains with phylogenetic analyses based on six loci, including the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene repeat and portions of the coding genes elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, heat shock protein, histone-3 and beta-tubulin. Seven species were found from almond: Botryosphaeria dothidea, Neofusicoccum parvum, Neof. mediterraneum, Neof. nonquaesitum, Diplodia seriata and Macrophomina phaseolina were identified from band cankers, and B. dothidea, Neof. mediterraneum, Neof. parvum and Dothiorella sarmentorum from canopy cankers. All were capable of inducing cankers on inoculated almond branches in the field. All species found on almond also occurred on other hosts, suggesting that infected vegetation adjacent to almond orchards could serve as source of inoculum of virulent almond strains. Of the 19 monophyletic groups obtained at the species level, 13 contained ex-type strains, five were morphologically similar to established species and one was morphologically distinct from its closest relatives, Neof. andinum and Neof. arbuti, as well as from the more than 190 described species of Fusicoccum and Neofusicoccum, and thus was described as the new species, Neof. nonquaesitum. Evidence for cryptic speciation was found in B. dothidea, Neof. ribis and Spencermartinsia viticola. Botryosphaeria dothidea and Neof. ribis comprised lineages that formed the morphologically distinct Dichomera anamorph not found in any other isolates recognized as B. dothidea and Neof. ribis. An S. viticola isolate from California was phylogenetically divergent and had conidia that differed morphologically from the type. Neofusicoccum parvum was diverse but lacked any morphological features correlating with molecular diversity. Phylogenetic analyses of combinations of datasets showed that pooled analyses of all six datasets resulted in the highest number of supported branches, suggesting that addition of more data might yet improve phylogenetic resolution.

Multilocus phylogenetics show high levels of endemic fusaria inhabiting Sardinian soils (Tyrrhenian Islands)

The Mediterranean island of Sardinia is well known for high levels of vascular plant diversity and endemism, but little is known about its microbial diversity. Under the hypothesis that Fusarium species would show similarly high diversity, we estimated variability in Fusarium species composition among 10 sites around the island. Markers previously adopted for multilocus sequence typing (MLST) were used to determine multilocus DNA sequence haplotypes for 263 Fusarium isolates. In addition portions of the translation elongation factor 1-alpha and second largest RNA polymerase subunit genes were sequenced for all isolates. The intergenic spacer (IGS) region of the nuclear ribosomal RNA gene repeat was sequenced for members of the F. oxysporum species complex (FOSC), and a portion of the nuclear ribosomal RNA gene repeat comprising the internal transcribed spacer (ITS) and part of the large nuclear ribosomal RNA subunit was sequenced for members of the F. solani species complex (FSSC). Seventy-three multilocus haplotypes were identified among the 263 isolates typed, of which 48 represented FOSC and FSSC. Thirty-seven of 48 FOSC two-locus and FSSC three-locus haplotypes had not been observed previously. The 38 non-FOSC/FSSC fusaria comprised 25 haplotypes distributed among 10 species, five of which appear to represent novel, phylogenetically distinct species. In general newly discovered haplotypes were restricted to one or a few sites. All FSSC isolates represented new haplotypes in phylogenetic species FSSC 5 and 9, which differ from the phylogenetic species dominant in soils worldwide. No obvious correlations were found between haplotype diversity and geospatial or habitat distribution. Overall these results indicate a high degree of Fusarium genetic diversity on multiple geographic scales within Sardinia. These results contrast with recent work showing that common, cosmopolitan species dominate Sardinia’s Trichoderma biodiversity. All data are available for access and viewing from the FUSARIUM-ID database.

Morphological and molecular characterization of Paragonimus westermani in northeastern India

Evidence for the presence of lung flukes of the Paragonimus westermani in India remains scant. In particular, evidence based on morphology of adult worms is lacking. Metacercariae of the genus Paragonimus, recovered from crabs in two regions of northeastern India, were raised to adulthood in laboratory rats. Morphologically, these worms appear to be P. westermani. DNA sequences from the second internal transcribed spacer (ITS2) and a portion of the ribosomal large subunit gene (28S) of the nuclear ribosomal RNA gene repeat, as well as fragments of the mitochondrial cytochrome c oxidase subunit 1 ( cox1) and NADH dehydrogenase subunit 1 ( nad1) genes, all supported this identification. Molecular phylogenetic methods were used for studying the relatedness of these Indian flukes with counterparts from southeast and far-east Asia. Molecular data showed that Indian representatives of the P. westermani complex represent a distinct lineage. It is unclear whether the Indian form can cause disease in humans as some members of the complex do elsewhere.

A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real‐time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus‐specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena/M. laxa/Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No cross‐reactions were observed. The assay was found to have a detection limit of 0·6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real‐time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.

A new role of the rDNA and nucleolus in the nucleus—rDNA instability maintains genome integrity

The nucleolus is a region of the nucleus with high protein density and it acts as a ribosome factory. The nucleolus contains a distinct region of the genome, the ribosomal RNA gene repeats (rDNA) that supply ribosomal RNA (rRNA) molecules. The rDNA is the most-abundant gene and occupies a large part of the genome, for example, there are thousands of rDNA copies in the genomes of plant cells. Therefore, it is natural to suppose that the condition of the rDNA, such as its stability, might affect cellular functions. Here I would like to propose a new model regarding the roles of the rDNA and nucleolus. The key point of this model is that they act to preserve genome stability and trigger aging.

Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data

Repeat families within genomes are often maintained with similar sequences. Traditionally, this has been explained by concerted evolution, where repeats in an array evolve "in concert" with the same sequence via continual turnover of repeats by recombination. Another form of evolution, birth-and-death evolution, can also explain this pattern, although in this case selection is the critical force maintaining the repeats. The level of intragenomic variation is the key difference between these two forms of evolution. The prohibitive size and repetitive nature of large repeat arrays have made determination of the absolute level of intragenomic repeat variability difficult, thus there is little evidence to support concerted evolution over birth-and-death evolution for many large repeat arrays. Here we use whole-genome shotgun sequence data from the genome projects of five fungal species to reveal absolute levels of sequence variation within the ribosomal RNA gene repeats (rDNA). The level of sequence variation is remarkably low. Furthermore, the polymorphisms that are detected are not functionally constrained and seem to exist beneath the level of selection. These results suggest the rDNA is evolving via concerted evolution. Comparisons with a repeat array undergoing birth-and-death evolution provide a clear contrast in the level of repeat array variation between these two forms of evolution, confirming that the rDNA indeed does evolve via concerted evolution. These low levels of intra-genomic variation are consistent with a model of concerted evolution in which homogenization is very rapid and efficiently maintains highly similar repeat arrays.

Strategies to maintain the stability of the ribosomal RNA gene repeats -Collaboration of recombination, cohesion, and condensation-

Ribosomal RNA gene repeats (rDNA) are one of the most characteristic regions in eukaryotic chromosomes. The repeats consist of more than 100 tandem units occupying large part of the chromosome in most of organisms. Cells are known to deal with this "unusual domain" in a unique manner. In this review, I will summarize work on rDNA repeat maintenance, focusing mainly on work done by our group, and show that the maintenance mechanism operates by a collaboration of recombination, sister-chromatid cohesion, and chromatin condensation.

Recombination Regulation by Transcription-Induced Cohesin Dissociation in rDNA Repeats

Organisms maintain ribosomal RNA gene repeats (rDNA) at stable copy numbers by recombination; the loss of repeats results in gene amplification. Here we report a mechanism of amplification regulation. We show that amplification is dependent on transcription from a noncoding bidirectional promoter (E-pro) within the rDNA spacer. E-pro transcription stimulates the dissociation of cohesin, a DNA binding protein complex that suppresses sister-chromatid-based changes in rDNA copy number. This transcription is regulated by the silencing gene, SIR2, and by copy number. Transcription-induced cohesin dissociation may be a general mechanism of recombination regulation.

Specific and sensitive PCR-based detection of Septoria musiva, S. populicola and S. populi the causes of leaf spot and stem canker on poplars

The development of a PCR assay for the detection of the poplar pathogenic fungi Septoria musiva (teleomorph Mycosphaerella populorum), S. populicola (M. populicola) and S. populi (M. populi) is described. Three pairs of species-specific PCR primers were designed using interspecific polymorphisms in the internal transcribed spacer (ITS) of nuclear ribosomal RNA gene (rDNA) repeats. The specificity of the three primer pairs was successfully tested on a collection of 40 S. musiva, 39 S. populicola and six S. populi isolates. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assay was further confirmed with DNA extracted from 12 additional Septoria species and 17 other fungal species obtained from stems or leaves of poplars. Specific amplification of the fragments for S. musiva and S. populicola was sensitive relatively to the technique used, detecting as low as 1 pg template DNA, and 10 pg of DNA of the target species in a background of 1 ng of DNA of the other species. Moreover, using DNA purified directly from disrupted conidia, it was possible to detect with a probability of 90%, using one unique PCR assay, the DNA equivalent of 166 conidia per microl of S. musiva and 156 conidia per microl of S. populicola. The procedures developed in this work can thus be applied for rapid and accurate detection and identification of Septoria species from poplars.

Identifying gene-independent noncoding functional elements in the yeast ribosomal DNA by phylogenetic footprinting.

Sequences involved in the regulation of genetic and genomic processes are primarily located in noncoding regions. Identifying such cis-acting sequences from sequence data is difficult because their patterns are not readily apparent, and, to date, identification has concentrated on regions associated with gene-coding functions. Here, we used phylogenetic footprinting to look for gene-independent noncoding elements in the ribosomal RNA gene repeats from several Saccharomyces species. Similarity plots of ribosomal intergenic spacer alignments from six closely related Saccharomyces species allowed the identification of previously characterized functional elements, such as the origin-of-replication and replication-fork barrier sites, demonstrating that this method is a powerful predictor of noncoding functional elements. Seventeen previously uncharacterized elements, showing high levels of conservation, were also discovered. The conservation of these elements suggests that they are functional, and we demonstrate the functionality of two classes of these elements, a putative bidirectional noncoding promoter and a series of conserved peaks with matches to the origin-of-replication core consensus. Our results paint a comprehensive picture of the functionality of the Saccharomyces ribosomal intergenic region and demonstrate that functional elements not involved in gene-coding function can be identified by using comparative genomics based on sequence conservation.

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

Isolation and identification of yeasts associated with vineyard and winery by RFLP analysis of ribosomal genes and mitochondrial DNA

Yeast colonies isolated from vineyard and cellar substrates were analysed in the present study. Yeast species assessment was carried out by amplification and digestion of a region of the ribosomal RNA gene repeat unit. Saccharomyces strains were also characterised using mitochondrial DNA restriction analysis. Oxidative basidiomycetous yeasts without enological potential were predominant in the vineyard environment. Yeasts associated with grape skin depend on grape variety, vintage and degree of grape maturation. These species from grape surface constituted the predominant microbiota in must and they developed during the first stages of the process. Yeasts colonies were also isolated and identified from the walls of a fermentation vat some days before the harvest. Contray to what was expected, Saccharomyces cerevisiae was not the major species isolated as Candida sorbosa represented 76% of the species isolated. Saccharomyces strains isolated from the fermentation vat had been previously isolated in wine fermentations in this cellar. Therefore, these strains should be considered as constant residents of this winery.

Cloning and characterisation of a ribosomal RNA gene repeat unit from groundnut

Characterisation of a ribosomal RNA gene repeat unit (rDNA) was carried out on groundnut, Arachis hypogaea L. The rDNA was cloned, a restriction map constructed and the 5.8S and internal transcribed spacer (ITS1 and ITS2) regions were completely sequenced while the 18S and 25S regions were partially sequenced. The restriction map and the sequences were compared with published maps and sequences from other crops. The cloned groundnut rDNA is about 12 kb in size with approximately 6 kb of intergenic spacer (IGS). The unique HindIII site in the IGS and two EcoRI and four BamHI sites in the transcribed region are features it shares with other legumes, while the presence of four of the five XbaI sites in the IGS region is its distinguishing feature.

The impacts of broadcast burning after clear-cutting on the diversity of ectomycorrhizal fungi associated with hybrid spruce seedlings in central British Columbia

Morphological and molecular (polymerase chain reaction – restriction fragment length polymorphism) methods were used to assess ectomycorrhiza (ECM) diversity in naturally regenerating and planted Picea engelmannii Parry ex Engelm. × Picea glauca (Moench) Voss seedlings in two recently clear-cut sites, two clear-cut and broadcast burned sites, and two mature forests (&gt;100 years) in central British Columbia. Based on 24 characterized ECM, burning did not affect overall diversity (Shannon, Simpson, Shannon evenness, and Margalef indices). However, the occurrence and relative abundance of some ECM morphotypes varied significantly. Hebeloma and a Russulaceae type 1 were more abundant and Cenococcum was less abundant in planted seedlings from cut-burned sites compared with those from clear-cut sites. E-strain, MRA, and Amphinema were more abundant in planted seedlings from both cut-burned and clear-cut sites compared with naturally regenerating seedlings from mature sites. ECM diversity of regenerating seedlings was significantly greater in mature forests compared with clear-cut sites and was greater in planted seedlings than naturally regenerating seedlings in clear-cut sites. Molecular analysis of the internal transcribed spacer region of the nuclear-encoded ribosomal RNA gene repeat showed no significant differences among treatments or seedling type. Twenty-two genotypes were identified from eight common morphotypes: Cenococcum (one), Thelephora (two), E-strain, Tuber, Hebeloma, and Russulaceae type 1 (three each), MRA (four), and Amphinema (six). Hebeloma genotypes matched three for Amphinema; genotypes from unidentified lightly colonized tips matched those for E-strain, MRA, and Amphinema-Hebeloma.

Intersterility group differentiation in Heterobasidion annosum using ribosomal IGS1 region polymorphism

Summary The intergenic spacer region 1 (IGS1) of the nuclear ribosomal RNA gene repeat was examined in three European intersterility groups of the wood-rot fungus Heterobasidion annosum. The IGS1 elements were informative for restriction fragment length polymorphism (RFLP) fingerprinting the fungal intersterility groups following digestion with two different four-cutter restriction endonucleases. The fingerprints of isolates from the same intersterility group from different localities shared some fragments in common, but also showed specific fragments suggesting that RFLP of the IGS1 region might be used in population studies. RFLP patterns between isolates from different intersterility groups differed more markedly. RFLP were also detected in the IGS1 region of North American strains of H. annosum and in the related species Heterobasidion araucariae. The results indicate that ribosomal IGS1 region fingerprinting could be a useful tool to identify the fungal intersterility group in Heterobasidion annosum. L'espaceur intergénique IGS1 de l'ARN ribosomique nucléaire a été examiné chez trois groupes interstériles européens de Heterobasidion annosum. La région IGS1 ont été informative pour l'empreinte RFLP des groupes interstériles du champignon à la suite d'une digestion avec deux enzymes de restriction à site de reconnaissance de quatre bases. Les profils des isolats du même groupe interstérile de différentes localités partageaient quelques fragments en commun, mais montraient aussi des fragments spécifiques ce qui suggère que le polymorphisme de la région IGS1 peut être utilisé pour les études de populations. Les profils RFLP entre isolats de différents groupes interstériles différaient de façon plus importante. Un polymorphisme a aussi été détecté dans la région IGS1 d'isolats nord américains de H. annosum et chez l'espèce voisine Heterobasidion araucariae. Ces résultats indiquent que les empreintes de la région ribosomique IGS1 peuvent être un outil utile pour identifier les groupes interstériles de H. annosum. Bei den drei europäischen Intersterilitätsgruppen des Fäuleerregers Heterobasidion annosum wurde die IGS1-Region des nukleären ribosomalen RNA-Gens untersucht. Die IGS1-Elemente lieferten bei diesem Pilz nach Verdau mit zwei verschiedenen, an 4 Stellen schneidenden Restriktionsendonukleasen Bandenmuster, die für die verschiedenen Intersterilitätsgruppen charakteristisch sind. Die Fingerprints von Isolaten der gleichen Intersterilitätsgruppe von verschiedenen Orten hatten einige gemeinsame Banden, aber es traten auch spezifische Banden auf, was darauf hinweist, dass die RFLP-Analyse der IGS1-Region auch für Populationsstudien benutzt werden könnte. Die RFLP-Muster von Isolaten verschiedener IS-Gruppen unterschieden sich aber deutlicher voneinander. Es wurden auch nordamerikanische Isolate von H. annosum und die verwandte Art Heterobasidion araucariae untersucht. Die Ergebnisse zeigen, dass die ribosomale IGS-Region Fingerprints liefert, welche für die Diagnose der Intersterilitätsgruppen nützlich sein können.

The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

Identification of a approximately 30S size non-ribosomal Saccharomyces cerevisiae RNA that is rapidly labeled on its 3' end by ATP or UTP.

Cell-free extracts prepared from S. cerevisiae cells were incubated in the presence of [alpha-32P]-labeled ATP, CTP, GTP or UTP. An RNA larger than ribosomal 25S RNA with an apparent size of approximately 30S was prominently labeled on its 3' end in the presence of ATP or UTP but not with CTP or GTP. This labeled RNA was not hybrid-selected by cloned yeast ribosomal DNA; in addition, this approximately 30S RNA was not cleaved by RNase H in the presence of complementary deoxyribooligonucleotides to rRNA. These two lines of evidence show that this approximately 30S RNA is not structurally related to ribosomal RNA gene repeat. The cell-free extracts prepared from yeast cells containing temperature-sensitive poly(A) polymerase adenylated this novel yeast RNA at restrictive temperature with efficiency similar to extracts prepared from wild-type yeast cells. These data show that the enzyme responsible for adenylation of this approximately 30S RNA is distinct from mRNA poly(A) polymerase. While the human SRP RNA 3' adenylating enzyme in the HeLa cell extract adenylated human SRP or Alu RNAs, the yeast adenylating enzyme did not adenylate the human SRP or Alu RNAs in vitro; these data indicate species specificity for this adenylating enzyme.