Showing papers on "Ribosomal DNA published in 2010"

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

Abstract: The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Abundance of Ribosomal RNA Gene Copies Maintains Genome Integrity

Abstract: 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.

DNA barcoding of arbuscular mycorrhizal fungi

Abstract: Plant beneficial microorganisms, such as arbuscular mycorrhiza fungi (AMF), increasingly attract scientific and agronomic attention due to their capacity to increase nutrient accessibility for plants and to reduce inorganic fertilizer requirements. AMF are thought to form symbioses with most land plants, obtaining carbon from the autotrophic host whilst enhancing uptake of poorly available nutrients. The species of AMF are mainly identified by spore morphology, which is time consuming, requires expertise and is rarely applicable to AMF identification in roots. Molecular tools such as analysis of standardized DNA fragment sequences may allow the recognition of species through a ‘DNA barcode’, which may partly overcome this problem. The focus of this study was to evaluate different regions of widely used rDNA repeats for their use as DNA barcodes for AMF including the small subunit rRNA gene (SSU), the internal transcribed spacer (ITS) and the large subunit rRNA gene (LSU). Closely related species in the genus Ambispora, members of which have dimorphic spores, could not be separated by analysis of the SSU region, but of the ITS region. Consequently, the SSU was not used for subsequent analysis, but a DNA fragment covering a small part of the SSU, the entire ITS region and about 800 bp of the LSU (SSUmCf-LSUmBr fragment) was analysed, providing phylogenetic resolution to species. New AMF specific primers for these potential barcoding regions were developed and can be applied, without amplification of non-target organisms, for AMF species determination, including identification from field and root samples. Analyses based on the application of the SSUmCf-LSUmBr fragment showed that the widely used AMF model organism Glomus sp. DAOM197198 (formerly called Glomus intraradices) is not conspecific with Gl. intraradices. The SSUmCf-LSUmBr fragment clearly provides a much higher species resolution capacity when compared with the formerly preferred ITS and LSU regions. Further study of several groups of AMF species using different regions of the SSUmCf-LSUmBr fragment revealed that only the complete SSUmCf-LSUmBr fragment allowed separation of all analysed species. Based on these results, an extended DNA barcode covering the ITS region and parts of the LSU region is suggested as a DNA barcode for AMF. The complete SSUmCf-LSUmBr fragment sequences can serve as a database backbone for also using smaller rDNA fragments as barcodes. Although the smallest fragment (approximately 400 bp) analysed in this study was not able to discriminate among AMF species completely, such short regions covering the ITS2 or LSU D2 regions, respectively, would most likely be suitable for community analyses with 454 GS-FLX Titanium sequencing, providing that the analyses is based on the longer DNA sequences.

Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects

Abstract: The nuclear ribosomal locus coding for the large subunit is represented in tandem arrays in the plant genome. These consecutive gene blocks, consisting of several regions, are widely applied in plant phylogenetics. The regions coding for the subunits of the rRNA have the lowest rate of evolution. Also the spacer regions like the internal transcribed spacers (ITS) and external transcribed spacers (ETS) are widely utilized in phylogenetics. The fact, that these regions are present in many copies in the plant genome is an advantage for laboratory practice but might be problem for phylogenetic analysis. Beside routine usage, the rDNA regions provide the great potential to study complex evolutionary mechanisms, such as reticulate events or array duplications. The understanding of these processes is based on the observation that the multiple copies of rDNA regions are homogenized through concerted evolution. This phenomenon results to paralogous copies, which can be misleading when incorporated in phylogenetic analyses. The fact that non-functional copies or pseudogenes can coexist with ortholougues in a single individual certainly makes also the analysis difficult. This article summarizes the information about the structure and utility of the phylogenetically informative spacer regions of the rDNA, namely internal- and external transcribed spacer regions as well as the intergenic spacer (IGS).

Chromosome spreading of associated transposable elements and ribosomal DNA in the fish Erythrinus erythrinus. Implications for genome change and karyoevolution in fish

Abstract: The fish, Erythrinus erythrinus, shows an interpopulation diversity, with four karyomorphs differing by chromosomal number, chromosomal morphology and heteromorphic sex chromosomes. Karyomorph A has a diploid number of 2n = 54 and does not have differentiated sex chromosomes. Karyomorph D has 2n = 52 chromosomes in females and 2n = 51 in males, and it is most likely derived from karyomorph A by the differentiation of a multiple X1X2Y sex chromosome system. In this study, we analyzed karyomorphs A and D by means of cytogenetic approaches to evaluate their evolutionary relationship. Conspicuous differences in the distribution of the 5S rDNA and Rex3 non-LTR retrotransposon were found between the two karyomorphs, while no changes in the heterochromatin and 18S rDNA patterns were found between them. Rex3 was interstitially dispersed in most chromosomes. It had a compartmentalized distribution in the centromeric regions of only two acrocentric chromosomes in karyomorph A. In comparison, in karyomorph D, Rex3 was found in 22 acrocentric chromosomes in females and 21 in males. All 5S rDNA sites co-localized with Rex3, suggesting that these are associated in the genome. In addition, the origin of the large metacentric Y chromosome in karyomorph D by centric fusion was highlighted by the presence of internal telomeric sites and 5S rDNA/Rex3 sites on this chromosome. We demonstrated that some repetitive DNAs (5S rDNA, Rex3 retroelement and (TTAGGG)n telomeric repeats) were crucial for the evolutionary divergence inside E. erythrinus. These elements were strongly associated with the karyomorphic evolution of this species. Our results indicate that chromosomal rearrangements and genomic modifications were significant events during the course of evolution of this fish. We detected centric fusions that were associated with the differentiation of the multiple sex chromosomes in karyomorph D, as well as a surprising increase of associated 5S rDNA/Rex3 loci, in contrast to karyomorph A. In this sense, E. erythrinus emerges as an excellent model system for better understanding the evolutionary mechanisms underlying the huge genome diversity in fish. This organism can also contribute to understanding vertebrate genome evolution as a whole.

Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences.

Abstract: The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, were performed to assess the efficacy of these sequences for delineating phytoplasma strains within each 16Sr group. The phylogenetic interrelatedness among phytoplasma taxa inferred by secY gene-based phylogeny was nearly congruent with that inferred by 16S rRNA gene-based phylogeny. Phylogenetic analysis based on the secY gene permitted finer differentiation of phytoplasma strains, however. The secY gene-based phylogeny not only readily resolved 16Sr subgroups within a given 16Sr group, but also delineated distinct lineages irresolvable by 16S rRNA gene-based phylogeny. Such high resolving power makes the secY gene a more useful genetic marker than the 16S rRNA gene for finer differentiation of closely related phytoplasma strains based on RFLP analysis with selected restriction enzymes. Such strains were readily identified by collective secY RFLP patterns. The genetic interrelationships among these strains were determined by pattern similarity coefficients, which coincided with delineations by phylogenetic analysis. This study also revealed two heterogeneous spc operons present in the phytoplasma clade. This latter finding may have significant implications for phytoplasma evolution.

Evolutionary dynamics of rDNA clusters on chromosomes of moths and butterflies (Lepidoptera)

Abstract: We examined chromosomal distribution of major ribosomal DNAs (rDNAs), clustered in the nucleolar organizer regions (NORs), in 18 species of moths and butterflies using fluorescence in situ hybridization with a codling moth (Cydia pomonella) 18S rDNA probe. Most species showed one or two rDNA clusters in their haploid karyotype but exceptions with 4-11 clusters also occurred. Our results in a compilation with previous data revealed dynamic evolution of rDNA distribution in Lepidoptera except Noctuoidea, which showed a highly uniform rDNA pattern. In karyotypes with one NOR, interstitial location of rDNA prevailed, whereas two-NOR karyotypes showed mostly terminally located rDNA clusters. A possible origin of the single interstitial NOR by fusion between two NOR-chromosomes with terminal rDNA clusters lacks support in available data. In some species, spreading of rDNA to new, mostly terminal chromosome regions was found. The multiplication of rDNA clusters without alteration of chromosome numbers rules out chromosome fissions as a major mechanism of rDNA expansion. Based on rDNA dynamics in Lepidoptera and considering the role of ordered nuclear architecture in karyotype evolution, we propose ectopic recombination, i.e., homologous recombination between repetitive sequences of non-homologous chromosomes, as a primary motive force in rDNA repatterning.

The obligate endobacteria of arbuscular mycorrhizal fungi are ancient heritable components related to the Mollicutes

Abstract: Arbuscular mycorrhizal fungi (AMF) have been symbionts of land plants for at least 450 Myr. It is known that some AMF host in their cytoplasm Gram-positive endobacteria called bacterium-like organisms (BLOs), of unknown phylogenetic origin. In this study, an extensive inventory of 28 cultured AMF, from diverse evolutionary lineages and four continents, indicated that most of the AMF species investigated possess BLOs. Analyzing the 16S ribosomal DNA (rDNA) as a phylogenetic marker revealed that BLO sequences from divergent lineages all clustered in a well-supported monophyletic clade. Unexpectedly, the cell-walled BLOs were shown to likely represent a sister clade of the Mycoplasmatales and Entomoplasmatales, within the Mollicutes, whose members are lacking cell walls and show symbiotic or parasitic lifestyles. Perhaps BLOs maintained the Gram-positive trait whereas the sister groups lost it. The intracellular location of BLOs was revealed by fluorescent in situ hybridization (FISH), and confirmed by pyrosequencing. BLO DNA could only be amplified from AMF spores and not from spore washings. As highly divergent BLO sequences were found within individual fungal spores, amplicon libraries derived from Glomus etunicatum isolates from different geographic regions were pyrosequenced; they revealed distinct sequence compositions in different isolates. Our results show a vertically inherited, monophyletic and globally distributed lineage of endobacteria thriving in AMF cytoplasm. These bacteria split from their sister groups more than 400 Myr ago, colonizing their fungal hosts already before main AMF lineages separated. The BLO-AMF symbiosis can, therefore, be dated back at least to the time when AMF formed the ancestral symbiosis with emergent land plants.

Sequence-Based Identification of Filamentous Basidiomycetous Fungi from Clinical Specimens: a Cautionary Note

Abstract: The species-level identification of sterile and/or arthroconidium-forming filamentous fungi presumed to be basidiomycetes based upon morphological or physiological features alone is usually not possible due to the limited amount of hyphal differentiation. Therefore, a reliable molecular approach capable of the unambiguous identification of clinical isolates is needed. One hundred sixty-eight presumptive basidiomycetes were screened by sequence analysis of the internal transcribed spacer (ITS) and D1/D2 ribosomal DNA regions in an effort to obtain a species identification. Through the use of this approach, identification of a basidiomycetous fungus to the species level was obtained for 167/168 of the isolates. However, comparison of the BLAST results for each isolate for both regions revealed that only 28.6% (48/168) of the isolates had the same species identification by use of both the ITS and the D1/D2 regions, regardless of the percent identity. At the less stringent genus-only level, the identities for only 48.8% (82/168) of the isolates agreed for both regions. Investigation of the causes for this low level of agreement revealed that 14% of the species lacked an ITS region deposit and 16% lacked a D1/D2 region deposit. Few GenBank deposits were found to be complete for either region, with only 8% of the isolates having a complete ITS region and 10% having a complete D1/D2 region. This study demonstrates that while sequence-based identification is a powerful tool for many fungi, sequence data derived from filamentous basidiomycetes should be interpreted carefully, particularly in the context of missing or incomplete GenBank data, and, whenever possible, should be evaluated in light of compatible morphological features.

The cereal rust mite Abacarus hystrix (Acari: Eriophyoidea) is a complex of species: evidence from mitochondrial and nuclear DNA sequences.

Abstract: The cereal rust mite Abacarus hystrix (Nalepa), a significant pest of grasses, has been regarded as one of a few exceptions among eriophyoid mites with reference to the pattern of host plant utilization. At least 60 grass species have been recorded as its hosts. Thus, the mite has long been considered as a host generalist in which host specialization would not be likely to evolve. However, recent studies have revealed that host-associated specialization is possible in A. hystrix. Here, we aimed to discriminate between the three populations of A. hystrix associated with the different hosts (namely quackgrass, ryegrass and smooth brome) on the basis of mitochondrial (COI) and nuclear (D2 region of 28S rDNA) DNA sequences. The phylogenetic trees obtained with the maximum likelihood analysis of both COI and D2 region data sets showed that host-adapted strains of A. hystrix form distinct clades. Furthermore, on the COI nucleotide tree, the quackgrass- and brome-associated strains were internally divided each into two well-supported monophyletic clusters. The nucleotide D2 region data set tree showed that brome-associated strain is polyphyletic in origin. There is clear co-variation of DNA results with earlier morphological and ecological traits, as well as the results of crossing experiments. We showed that reproductively incompatible strains of A. hystrix exhibit more than 20% sequence divergence in the COI gene and 0.2% sequence divergence in the D2 28S rDNA. Our results did not confirm the placement of three host-associated strains of A. hystrix within one, ostensibly generalist, species.

History of myxozoan character evolution on the basis of rDNA and EF-2 data

Abstract: Phylogenetic relationships among myxosporeans based on ribosomal DNA data disagree with traditional taxonomic classification: a number of myxosporeans with very similar spore morphology are assigned to the same genera even though they are phylogenetically distantly related. The credibility of rDNA as a suitable marker for Myxozoa is uncertain and needs to be proved. Furthermore, we need to know the history of myxospore evolution to understand the great diversity of modern species. Phylogenetic analysis of elongation factor 2 supports the ribosomal DNA-based reconstruction of myxozoan evolution. We propose that SSU rDNA is a reliable marker for inferring myxozoan relationships, even though SSU rDNA analysis markedly disagrees with the current taxonomy. The analyses of character evolution of 15 morphological and 5 bionomical characters show the evolution of individual characters and uncover the main evolutionary changes in the myxosporean spore morphology and bionomy. Most bionomical and several morphological characters were found to be congruent with the phylogeny. The summary of character analyses leads to the simulation of myxozoan ancestral morphotypes and their evolution to the current species. As such, the ancestor of all myxozoans appears to have infected the renal tubules of freshwater fish, was sphaerosporid in shape, and had a spore with polar capsules that discharged slightly sideways. After the separation of Malacosporea, the spore of the common myxosporean ancestor then changed to the typical sphaerosporid morphotype. This species inhabited the marine environment as a parasite of the gall bladder of marine fish and ultimately separated into the three main myxosporean lineages evident today. Two of these lineages re-entered the freshwater environment, one as a myxosporean with Chloromyxum and another with a primitive sphaerosporid morphotype. The common ancestor of all marine myxosporeans had a ceratomyxid shape of spore. We support rDNA based myxozoan phylogeny by the analysis of a protein coding gene and demonstrate the reliability of rDNA as a marker explaining myxozoan relationships. Our tracing the history of myxozoan character evolution discloses ancestral morphotypes and shows their development over the course of evolution. We point out several myxozoan characters that are to a certain extent congruent with the phylogeny and determined that the discrepancy between phylogeny and current taxonomy based on spore morphology is due to an extreme myxospore plasticity occurring during myxozoan evolution.

Pectobacterium carotovorum subsp. brasiliensis causing blackleg on potatoes in South Africa

Abstract: In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg occurred, causing severe economic losses in commercial potato production fields. Symptoms were initially observed on only one stem per plant, on which the top leaves rolled upwards, wilted and became necrotic. As symptoms progressed to the lower leaves with subsequent leaf desiccation, a light to dark brown discolouration of the vascular system at the stem base developed, followed by external darkening. Under prevailing wet and humid conditions stems became slimy and pale. In the stems, the pith became necrotic and hollow. These symptoms were similar to those described in Brazil, where the causal agent was identified as a new subspecies, Pectobacterium carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical blackleg symptoms were made on CVP medium. Sequences and phylogenetic analysis of the partial 16S–23S rDNA intergenic spacer region indicated that the isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of isolates to reference cultures confirmed the identity of the South African blackleg strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb in South Africa and it appears to be the most important causal agent of blackleg in South Africa. The disease poses a major potential threat to the South African potato industry especially in terms of seed exports, tuber quality and yield.

Species relationships among the wild B genome of Arachis species (section Arachis) based on FISH mapping of rDNA loci and heterochromatin detection: a new proposal for genome arrangement.

Abstract: Arachis hypogaea is an allotetraploid species with low genetic variability. Its closest relatives, all of the genus Arachis, are important sources of alleles for peanut breeding. However, a better understanding of the genome constitution of the species and of the relationships among taxa is needed for the effective use of the secondary gene pool of Arachis. In the present work, we focused on all 11 non-A genome (or B genome sensu lato) species of Arachis recognized so far. Detailed karyotypes were developed by heterochromatin detection and mapping of the 5S and the 18S–25S rRNA using FISH. On the basis of outstanding differences observed in the karyotype structures, we propose segregating the non-A genome taxa into three genomes: B sensu stricto (s.s.), F and K. The B genome s.s. is deprived of centromeric heterochromatin and is homologous to one of the A. hypogaea complements. The other two genomes have centromeric bands on most of the chromosomes, but differ in the amount and distribution of heterochromatin. This organization is supported by previously published data on molecular markers, cross compatibility assays and bivalent formation at meiosis in interspecific hybrids. The geographic structure of the karyotype variability observed also reflects that each genome group may constitute lineages that have evolved through independent evolutionary pathways. In the present study, we confirmed that Arachis ipaensis was the most probable B genome donor for A. hypogaea, and we identified a group of other closely related species. The data provided here will facilitate the identification of the most suitable species for the development of prebreeding materials for further improvement of cultivated peanut.

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

Abstract: 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 Parazoanthidae (Hexacorallia: Zoantharia) DNA taxonomy: description of two new genera

Abstract: The taxonomy of the hexacorallian order Zoantharia is very problematic due to the lack of easily accessible and informative morphological taxonomic characters. This is particularly true in the widespread family Parazoanthidae, members of which use a wide variety of different organisms as substrates. Recently, DNA-based studies have proven to be of great use in clarifying relationships among Parazoanthidae. Here we reconsider Parazoanthidae taxonomy based on analyses of multiple molecular markers [mitochondrial cytochrome oxidase subunit 1 (COI), 16S ribosomal DNA (mt 16S rDNA), and the nuclear internal transcribed spacer region (ITS rDNA)], coupled with ecological and morphological characteristics. Two new genera are described in this study: Hydrozoanthus n. gen. within the new family Hydrozoanthidae, and Antipathozoanthus n. gen in the family Parazoanthidae. The genetic data further suggest that the revised genus Parazoanthus is still polyphyletic and is composed of three distinctive subclades. However, as currently these subclades can essentially be differentiated by genetic data, these subclades should remain within Parazoanthus until further molecular, ecological and morphological studies help to clarify their status and relationships to each other.

Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny?

Abstract: The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.

The bacteriology of pouchitis: a molecular phylogenetic analysis using 16S rRNA gene cloning and sequencing.

Abstract: Objective To identify, compare and contrast the microbiota in patients with and without pouchitis after RPC for UC and FAP.

Short rDNA Barcodes for Species Identification in Foraminifera

Abstract: Ribosomal DNA (rDNA) sequences have been shown to be very useful for identification of microbial eukaryotes. Usually, complete or long partial sequences of the rDNA genes are analysed. However, the development of new massive sequencing technologies producing a large amount of relatively short sequences raises the question about the minimum length of rDNA fragments necessary for species distinction in environmental sampling. To answer this question, we compared six variable regions of the small subunit (SSU) rDNA of foraminifera, known to have rapidly evolving ribosomal genes. For each region, we analysed (1) the sequence divergence between and within foraminiferal morphospecies, (2) the intraspecific polymorphism, and (3) the ability of each region to recognize the phylotypes inferred from analysis of a longer fragment. Our results show that although the variable regions differ considerably between taxonomic groups, most of them perform very well as species identifiers. Taking into account different analyses, the expansion segment of Helix 37 appears to be the best candidate for barcoding foraminifera. We propose that this relatively short region, averaging 50-60 nt in length, could be an ideal barcode for identification of foraminifera in environmental samples using massive sequencing approach.

Dinoflagellate phylogeny as inferred from heat shock protein 90 and ribosomal gene sequences.

Abstract: Background Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages. Methodology/Principal Findings We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved. Conclusions/Significance The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea

Abstract: In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Dicer is associated with ribosomal DNA chromatin in mammalian cells.

Abstract: BACKGROUND: RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines. CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.

Structure and epigenetics of nucleoli in comparison with non-nucleolar compartments.

Abstract: The nucleolus is a nuclear compartment that plays an important role in ribosome biogenesis. Some structural features and epigenetic patterns are shared between nucleolar and non-nucleolar compartments. For example, the location of transcriptionally active mRNA on extended chromatin loop species is similar to that observed for transcriptionally active ribosomal DNA (rDNA) genes on so-called Christmas tree branches. Similarly, nucleolus organizer region-bearing chromosomes located a distance from the nucleolus extend chromatin fibers into the nucleolar compartment. Specific epigenetic events, such as histone acetylation and methylation and DNA methylation, also regulate transcription of both rRNA- and mRNA-encoding loci. Here, we review the epigenetic mechanisms and structural features that regulate transcription of ribosomal and mRNA genes. We focus on similarities in epigenetic and structural regulation of chromatin in nucleoli and the surrounding non-nucleolar region and discuss the role of proteins, such as heterochromatin protein 1, fibrillarin, nucleolin, and upstream binding factor, in rRNA synthesis and processing.

Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology, Pigments, and Ribosomal DNA Gene Sequence

Abstract: The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate-like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension-like furrow. The cingulum is as wide as 0.2-0.3 x cell length and displaced by 0.2-0.3 x cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4-19.3 and 6.1-16.0 microm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17-18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst-nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.

Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae) allotetraploids

Abstract: Tragopogon mirus and T. miscellus are allotetraploids (2n = 24) that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA) following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12) from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA) of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis). We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids. Using Southern blot hybridization and fluorescent in situ hybridization (FISH), we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals) and four lines of synthetic T. miscellus (71 individuals). Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids. Uniparental reductions of homeologous rRNA gene copies occurred in both synthetic and natural populations of Tragopogon allopolyploids. The extent of these rDNA changes was generally higher in natural populations than in the synthetic lines. We hypothesize that locus-specific and chromosomal changes in early generations of allopolyploids may influence patterns of rDNA evolution in later generations.

Repeated Reunions and Splits Feature the Highly Dynamic Evolution of 5S and 35S Ribosomal RNA Genes (rDNA) in the Asteraceae Family

Abstract: In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in ~200 species representing the family diversity and other closely related groups. Dominant linked rDNA genotype was found within three large groups in subfamily Asteroideae: tribe Anthemideae (93% of the studied cases), tribe Gnaphalieae (100%) and in the "Heliantheae alliance" (23%). The remaining five tribes of the Asteroideae displayed canonical non linked arrangement of rDNA, as did the other groups in the Asteraceae. Nevertheless, low copy linked genes were identified among several species that amplified unlinked units. The conserved position of functional 5S insertions downstream from the 26S gene suggests a unique, perhaps retrotransposon-mediated integration event at the base of subfamily Asteroideae. Further evolution likely involved divergence of 26S-5S intergenic spacers, amplification and homogenisation of units across the chromosomes and concomitant elimination of unlinked arrays. However, the opposite trend, from linked towards unlinked arrangement was also surmised in few species indicating possible reversibility of these processes. Our results indicate that nearly 25% of Asteraceae species may have evolved unusual linked arrangement of rRNA genes. Thus, in plants, fundamental changes in intrinsic structure of rDNA units, their copy number and chromosomal organisation may occur within relatively short evolutionary time. We hypothesize that the 5S gene integration within the 35S unit might have repeatedly occurred during plant evolution, and probably once in Asteraceae.

Phylogenetic relationships of sugarcane rust fungi

Abstract: The phylogenetic positions of Puccinia spp. infecting sugarcane (a complex hybrid of Saccharum spp.) were determined using 38 newly generated rust sequences and 26 sequences from GenBank. Rust specimens on sugarcane were collected from 164 locations in 23 countries and identified based on light microscopy. The morphology for all samples matched that of Puccinia kuehnii or P. melanocephala, the orange and brown rust pathogens of sugarcane, respectively. Nuclear ribosomal DNA sequences (rDNA) including portions of the 5.8S rDNA, the complete internal transcribed spacer 2 (ITS2) and 5′ region of the large subunit (nLSU) rDNA were obtained for each species along with 36 additional rust taxa. Despite a shared host, the two Puccinia spp. on sugarcane are not closely related within the Pucciniales. Phylogenetic analyses place P. melanocephala most closely to P. miscanthi, P. nakanishikii, and P. rufipes infecting Miscanthus sinensis, Cymbopogon citratus, and Imperata cylindrica, respectively. Puccinia kuehnii is basal to a clade of Poaceae-infecting rusts including P. agrophila, P. polysora, P. substriata, and Uromyces setariae-italicae infecting Schizachyrium spp., Zea mays, Digitaria spp., and Urochloa mosambicensis, respectively. Light and scanning electron microscopy images highlight morphological differences distinguishing the two sugarcane-infecting species. This study confirms the separation of rust species infecting Poaceae from Cyperaceae- and Juncaceae-infecting rusts and also provides support for the presence of an additional group that includes P. kuehnii and other grass-infecting relatives.